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Blood meal and cytochrome P-450 monooxygenases in the northern house mosquito, Culex pipiens
PESTICIDE
BIOCHEMISTRY
AND PHYSIOLOGY
25,
407-413 (1986)
Blood Meal and Cytochrome P-450 Monooxygenases in the Northern House Mosquito, Culex pipiens G. D. BALDRIDGE* Departments
of *Entomology
and
fAAgricu/tural
AND R. FEYEREISEN*+ Chemistry,
Oregon
State
University,
Corvallis,
Oregon
97331
Received May 2, 1985; accepted July 18. 1985 Conditions for the measurement of aldrin epoxidation by microsomes prepared from abdominal tissues (fat body + integument) of adult female Culex pipiens were characterized. The enzyme activity had a pH optimum of 7.2 and an apparent K,,, of 3.4 JLM. Aldrin epoxidation and NADPHcytochrome c reductase had similar patterns of inhibition by a rabbit antiserum to house fly NADPH-cytochrome P-450 reductase, thus implicating cytochrome P-450 monooxygenase(s) in the epoxidation of aldrin. Low (71 pmol/mg protein) levels of cytochrome P-450 were detected in abdominal tissue microsomes. In non-blood-fed insects, aldrin epoxidation and NADPH-cytochrome c reductase activities did not change between Day 1 and Day 12 after adult emergence. except for a small peak on Day 2. In insects fed a blood meal on Day 6 after emergence both activities increased (two- to threefold) to a plateau maintained between 2 and 4 days after the blood meal. Aldrin epoxidation and NADPH-cytochrome c reductase activities decreased to normal values between 4 and 6 days after the blood meal. Q 1986 Academic Press. Inc. INTRODUCTION
Mosquitoes have been called man’s worst enemy (1) because of their role in the transmission of diseases such as malaria, yellow fever, filariasis, dengue, encephalitis, etc. Despite early successes of vector control by the use of insecticide chemicals, there is an alarming proliferation of resistance problems in mosquitoes (2). Further use of chemicals for mosquito control and the prevention or management of resistance requires some knowledge of how mosquitoes deal with toxicants. However,
(5) showed that metabolic detoxication was an important mechanism of resistance to methoprene in Culex pipiens pipiens, but failed to detect any aldrin epoxidation activity in larval homogenates. To our knowledge, no in vitro study of cytochrome P-450 monooxygenases has been reported from any adult mosquito species, even though adult mosquitoes are a target of chemical control measures. This study was undertaken as a first step to characterize the cytochrome P-450 monooxygenase system in adult Culex pipiens, and to de-
our understanding of mosquitodetoxica- scribethe effectsof a bloodmealon this tion capabilities is very limited and the cytochrome P-450 monooxygenase system (mixed-function oxidases, polysubstrate monooxygenases) of mosquitoes is virtually unexplored by in vitro approaches. Shrivastava et al. (3, 4) studied a larval enzyme in Culex pipiens fatigans. They showed that cytochrome P-450 monooxygenases play a major role in the detoxication of carbamate insecticides in a propoxur-resistant strain. Brown and Hooper ’ To whom correspondence
should be addressed.
enzyme system. MATERIALS
AND METHODS
Insects. An anautogenous strain of Clalex pipiens L. (Diptera, Culicidae) collected in
1978 from a log-pond at Philomath, Oregon, was used to establish a colony. Insects were maintained at 26°C under a 16-hr light: 8-hr dark photoperiod. Adults were fed a 10% sucrose solution ad h&turn and live Quail were used to provide blood meals. mY females synchronized to
407 0048-3575186 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
408
BALDRIDGE
AND
within 24 hr of adult emergence or within 2 hr of a blood meal were used in this study. Preparation of microsomes. Two methods were used to prepare microsomes. A differential centrifugation method was used for large-scale preparation of microsomes from abdominal tissues and a sucrose density gradient preparation of microsomes was used for establishing a developmental profile of cytochrome P-450 monooxygenases. For the first method, 100 to 200 abdomens were dissected from adult females taken 72 hr after a blood meal. The midguts and ovaries were removed and the abdomens (i.e., essentially integument, muscle, and fat body) were homogenized in 20 ml MOPS (morpholinopropanesulfonate) buffer, 50 m&I, pH 7.2 containing 1 mM EDTA and 0.4 mM freshly prepared PMSF (phenylmethylsulfonylfluoride), with 10 strokes of a motor-driven Teflon/ glass homogenizer. The homogenate was centrifuged sequentially at IOOOg for 15 min, 10,OOOgfor 15 min and 105,OOOgfor 65 min. The final pellet was resuspended in 30% sucrose in 50 mM MOPS buffer, pH 7.2, containing 1 mM EDTA. All procedures were performed at 0-4°C. For the second method (6), from 10 to 3.5 abdomens (depending on age) prepared as described above were homogenized in 0.3 ml of MOPS buffer, pH 7.2, containing 1 mM EDTA and 0.4 r&I PMSE The homogenate was centrifuged at IOOOgfor 10 min and the supernatant was removed with a finely drawn Pasteur pipet to prevent contamination by the floating lipid layer. Supernatant (made up to 0.35 ml) containing no more than 1 mg protein was loaded on a 5 ml linear 45 to 15% sucrose gradient and centrifuged at 65,000 rpm in a Beckman VTi 80 rotor for 20 min (o*t = 3.7 X 1O’O rad*.sec-I). The bottom 2.0 ml of the gradient was discarded and the middle 2.1 ml (which contained the majority of the NADPH-cytochrome c reductase activity) were used as “microsomal” enzyme source. Details and justification of this procedure are given elsewhere (6). Sucrose was prepared in MOPS/EDTA buffer and
FEYEREISEN
protein levels were determined by the Bradford method (7) using y-globulins as standard. Enzyme assays. NADPH cytochrome c reductase activity (8) was measured spectrophotometrically at 30°C in an Aminco DW2a instrument. Reduction of cytochrome c was measured at 550 nm in the split beam mode in 1 ml cuvettes each containing 50 FM cytochrome c. I mM EDTA. 5 mM NaCN and enzyme in 50 mM MOPS buffer, pH 7.2. The reaction was started bq the addition of 50 ~1 of an NADPH (final concentration 150 @I) regenerating system (10 pmol glucose 6-phosphate, and 1.5 units glucose-6-phosphate dehydrogenase). Aldrin epoxidation was measured at 30°C in a final volume of 350 ~1 containing 50 mM MOPS buffer, pH 7.2 with 1 mM EDTA, enzyme and an NADPH (150 pJI,I regenerating system. The reaction was started by the addition of the substrate, aldrin, at a final concentration of 20 p,,M. The reaction was stopped with 1 M HCI after 20 min unless noted otherwise and the product, dieldrin, was extracted with 100 ~1 of hexane and analyzed by electron capture gas chromatography (9) using endrin as internal standard. Cytochrome P-450 determination. A “microsomal” pellet prepared by the differential centrifugation procedure (see above) was resuspended in 50 mM MOPS buffer, pH 7.2, containing 1 mM EDTA and 10% sucrose. Cytochrome P-450 level was measured according to Omura and Sato (IO) in an Aminco DW2a spectrophotometer thermostatted at 12°C. using I-ml cuvettes. RESULTS
Aldrin Epoxidation Activity In vitro conditions for the measurement of aldrin epoxidation activity were determined using “microsomes” prepared by a differential centrifugation procedure. This reaction was chosen because the oxygenated product, dieldrin, could be detected by gas chromatography with electron-cap-
CYTOCHROME
P-450
MONOOXYGENASES
ture detection, an easy and sensitive method suitable for the low activities expected from mosquito abdominal tissue. Other sensitive assays, such as the O-demethylation of methoxyresorufin or alkoxycoumarins (7-methoxy-,7-ethoxy-, and 7-methoxy-4-methylcoumarin) were also tried. However, the extremely low specific activities obtained in preliminary experiments precluded work on a small number of insects and thus a full characterization of those activities was not attempted in this study. The effect of “microsomal” protein concentration on aldrin epoxidation activity is shown on Fig. la. A clear dependence of product formation on enzyme concentration in the assay was observed. In view of the deviation in linearity at high concentrations, protein levels were kept below 0.06 mg/ml in all subsequent experiments. Figure lb shows that a linear dependence of dieldrin formation on incubation time was maintained for at least 20 min of incubation. Linearity of product formation with increasing enzyme concentration (up to at least 0.06 mg/ml) and with increasing incu-
Time , min
pg Protein/ml
PH
l/S
(pd
FIG. 1. Characterization of aldrin epoxidation activity from Culex pipiens abdominal tissues. “Microsomes” were prepared from adult females taken 72 hr after a blood meal. (a) Effect of “microsomal” protein concentration; (b) effect of incubation time; (c) effect of incubation pH; (d) effect of substrate concentration. Each point is the mean of duplicate incubations.
IN
Culex pipiens
409
bation time (up to 30 min) was also observed when using “microsomes” prepared by the sucrose density-gradient method (results not shown). Figure Ic shows that the enzymatic epoxidation of aldrin was pH dependent. Maximal activity was observed at pH 7.2 with a sharp decrease at lower pH values. Aldrin epoxidation was also dependent on the substrate concentration as shown on Fig. Id. The Lineweaver-Burk double reciprocal plot of the results (9 = 0.9802) indicated that a simple Michaelis-Menten behavior was a valid first approximation of the enzyme kinetics. The apparent K, of aldrin epoxidation was 3.4 FM and a V,,, of 38 pm01 * min-’ * mg protein was calculated. These results led us to adopt the assay conditions that were used in the further characterization of aldrin epoxidation in mosquito abdominal tissue. Aldrin Epoxidation Catalyzed by Cytochrome P-450 Monooxyygenase(s)
Because aldrin epoxidation activity was found in fractions enriched in NADPH-cytochrome c reductase activity (6) and was dependent on the presence of NADPH in the incubation, an involvement of microsomal cytochrome P-450 monooxygenase(s) was suggested. Rabbit antibodies to house fly NADPH cytochrome P-450 reductase (11) were used to demonstrate the involvement of NADPH-cytochrome P-450 reductase in the aldrin epoxidation reaction. When mosquito abdominal “microsomes” were preincubated in the presence of rabbit antibodies (IgG fraction) to house fly NADPH-cytochrome P-450 reductase and subsequently assayed for aldrin epoxidation activity, an inhibition of dieldrin production was observed (Fig. 2). This inhibition was dependent on the ratio of IgG to “microsomal” protein and aldrin epoxidation was fully inhibited at a ratio of 0.6. In contrast, only 30.1% inhibition of aldrin epoxidation was observed at the highest level of control (non-immune) IgG (ratio of 1.0). The inhibition of aldrin epoxidation closely paralleled the inhibition of
410
BALDRIDGE
NADPH-cytochrome (Fig. 2).
c reductase
AND
activity
I LA-=-l 0.002 AU.
Cytochrome P-450 in Abdominal ’ ‘Microsomes”
Mosquitoes taken 96 hr after a blood meal were used to prepare “microsomes,” and the method of Omura and Sato (10) was used to measure levels of cytochrome P-450. A representative spectrum (Fig. 3) shows that only low amounts of cytochrome P-450 could be detected. The carbon monoxide-difference spectrum of reduced ‘Imicrosomes” slowly developed a peak at 450.4 nm. A calculated level of 71 pmol/mg protein was obtained using the extinction coefficient for cytochrome P-450 in rabbit liver microsomes (91 cm-’ * mM-I). Figure 3 also shows that a significant degradation of cytochrome P-450 to cytochrome P-420 had occurred. Developmental Profile of Aldrin Epoxidation and NADPH-Cytochrome c Reductase and Effects of a Blood Meal
“Microsomes” were obtained by a sucrose density gradient procedure which allowed the rapid preparation of a large number of samples. For each sample, 25 to 35 non-blood-fed mosquitoes, or 12 to 25
0
FEYEREISEN
0.5 mg WJ 1 mg
1.0 protein
FIG. 2. Inhibition of aldrin epoxidation (0) and NADPH-cytochrome c reductase (m) by an ZgG fraction from rabbit antiserum raised against house fry NADPH-cytochrome P-450 reductase (II). Each point is the mean of duplicate incubations. (0, 0) Control, non-immune ZgG fraction.
8 2t I
v
400
450
500
Wavelength bml FIG. 3. Carbon monoxide difference spectrum of dithionite-reduced “microsomes” from Culex pipiens abdominal tissues. The absorbance peak (450.4 nm, determined manually) was maximal 20 min after saturation with carbon monoxide. Protein level was 0.59 mglml.
blood-fed mosquitoes were used. In each case the ovaries and gut were removed from the dissected abdomens. Aldrin epoxidation and NADPH-cytochrome c reductase activities were determined on precisely timed non-blood-fed and blood-fed mosquitoes and expressed as specific activities per insect-equivalent. Figure 4 shows that a small but significant peak in both enzyme activities was observed on Day 2 after adult emergence in non-blood fed insects. A relatively stable level of activities was maintained from Day 3 through Day 12. Figure 4 also shows the pattern of activities in mosquitoes fed a blood meal on Day 6 after adult emergence. A rapid increase in both aldrin epoxidation and NADPH-cytochrome c reductase activities was observed during the first 48 hr following the blood meal. Between 48 and 96 hr after a blood meal the activities were maintained at a plateau which was about threefold (aldrin epoxidation) and twofold (NADPHcytochrome c reductase) higher than the values for non-blood-fed insects. Both activities then declined to values typical of non-blood-fed insects by 6 days after the blood meal. When the enzyme activities were expressed per milligram protein, an identical pattern of change was observed, but the amplitude of the changes was lower
CYTOCHROME
P-450
MONOOXYGENASES
IN
L L
2
4 Days
IO ad
I2
ent&flC~
FIG. 4. Developmental proj2e of aldrin epoxidation (m), NADPH-cytochrome c reductase activity (0). from the abdominal tissues (fat body) of non blood-fed (-) and blood fed (- . - * -) adult female Culex pipiens. Each point represents the mean ? SEM of 4 to 7 experiments. A blood meal was given on Day 6 after adult emergence.
because the specific activities were shown to be lower than controls (non-blood-fed) until 36 hr after the blood meal. A very rapid increase in “microsomal” protein level was observed between 0 and 24 hr after a blood meal, this level then slowly decreased as aldrin epoxidation and NADPH-cytochrome c reductase reached their peaks (Fig. 5). “Microsomal” protein levels were still higher at 6 days after a blood meal when compared to non-bloodfed insects. DISCUSSION
The tissue used for this study was a compromise between a single, defined tissue, and a mixture of all abdominal tissues as has been done repeatedly in studies of dipteran cytochrome P-450 monooxygenase systems. By removing the ovaries and the gut tissues, we removed the source of large variations in the relative contribution of different tissues to the microsomal preparations. Although we have tried to characterize cytochrome P-450 monooxygenases in dissected Malpighian tubules or midgut, practical problems such as time of dissec-
2
C&x
4 Days
411
pipiens
6 after
0
IO
12
J
smarV
5. Development profile of “microsomal” protein levels from the abdominal tissues Vat hudy) of non blood-fed (-) and blood ,fed (-. - * -) adult female Culex pipiens. Data from the experiments shown on Fig. 4. FIG.
tion and number of insects became limiting factors in a precise and detailed study of biochemical changes following a blood meal. A reliable method for a rapid and reproducible dissection of midgut tissue free of blood meal contaminants is still needed. The integument + fat body preparation has precedents in the literature (12) and the tissue used in the present study can be considered as essentially the fat body for the purpose of this discussion. Levels of cytochrome P-450 were measured at 94 hr after a blood meal, which corresponded to a maximum in the activities of aldrin epoxidation and NADPH-cytochrome c reductase. When compared to values obtained with microsomes obtained from whole abdomens of other adult Diptera of various ages, the specific activity of the Cufex pipiens cytochrome P-450 (71 pmol/mg protein) appears to be about half that of house flies (13) and approximately equal to that of blow flies (14, 15) and flesh flies (15). However, such a comparison is difficult because gut tissues (usually rich sources of cytochrome P-450 monooxygenases)) were excluded form our study. Cytochrome P-450 levels of CL&X pipiens appear quite low when compared to values reported from the fat body of other insects
412
BALDRIDGE
AND FEYEREISEN
(84 pmol/mg in Spodoptera eridania larvae, 16, 200-400 pmol/mg in Locusta migratoria, 17) particularly if one assumes that in non-blood-fed insects the levels would be even lower. The levels of activity measured for aldrin epoxidation are also lower than in any other insect as reviewed by Yu et al. (18). Obviously such absolute comparisons have only a limited value, because cytochrome P-450 levels or monooxygenase activities depend on age and tissue involved. In addition, aldrin epoxidation may be catalyzed by one or more cytochrome P-450 isozymes and it is not known how well changes in aldrin epoxidation reflect changes in other monooxygenase activities. In anautogenous mosquitoes such as the Culex pipiens strain used in this study, a blood meal is the necessary requisite for the completion of oocyte growth and egg laying. In unfed females, the length of the follicles increases from 20 to 90 km (resting stage) during the first 3 days after adult emergence. No further growth is observed until after a blood meal, Ovarian follicles grow from the resting stage to about 600 pm between 0 and 60 hr after a blood meal, and egg-laying occurs between 72 and 96 hr after the blood meal. The peak activity of cytochrome P-450 monooxygenases as estimated here by aldrin epoxidation and NADPH-cytochrome c reductase is therefore concomitant with the final phase of vitellogenesis, chorionation, and oviposition. However, it occurs after the peak level of “microsomal” protein (possibly related to massive vitellogenin synthesis by the fat body) observed 24 hr after the blood meal. The decline in activities occurs after egg laying. In non-blood-fed insects, the small peak in activity noted on Day 2 after emergence is concomitant with the previtellogenie growth of oocytes to the resting stage. It would be of interest to determine whether changes in cytochrome P-450 monooxygenases are in any way related to the gonotrophic cycle of the mosquito and its hormonal regulation. In Culex pipiens, a
peak of ecdysteroids (400 fmol20-hydroxyecdysone eq/insect) is observed at 36 hr after a blood meal (unpublished results) and therefore precedes the maximum level of cytochrome P-450 monooxygenases. When the procedures to measure cytochrome P-450 monooxygenase activities in mosquito midgut become available, it will be possible to compare the timing of cytochrome P-450 monooxygenase changes in midgut and fat body after a blood meal. It was somewhat surprising to find that fat body enzymes reached a maximum only 48 hr after the blood meal, whereas microsomal protein levels had already peaked at 24 hr. The importance of insect enzyme systems capable of detoxifying potentially toxic plant chemicals has been documented in detail and it is hypothesized that such enzyme systems (cytochrome P-450 monooxygenases in particular) may serve as defensive countermeasures in the “coevolutionary arms race” between plants and phytophagous insects (19-21). In view of the enormous importance for mankind of a more recent coevolution, that of hematophagous insects and their vertebrate hosts, it is astonishing how little is known about the biochemical adaptations of hematophagous insects to the vast array of potentially toxic constituents of a blood meal. To our knowledge this is the first study of the changes in cytochrome P-450 monooxygenases in response to a blood meal. Because blood-fed mosquitoes are epidemiologically more important than non-bloodfed mosquitoes, it will now be necessary to assess the toxicological significance of our results in view of some reports on the differences in insecticide tolerance between blood-fed and non-blood-fed mosquitoes cm. ACKNOWLEDGMENTS This work was supported by NIH Grant AI 19192 to R. F. Oregon Agricultural Experiment Station Technical Paper No. 7552.
CYTOCHROME
P-450
MONOOXYGENASES
REFERENCES
1. J. D. Gillett, The mosquito: still man’s worst enemy, Amer. Scientisr 61, 430 (1973). 2. G. P Georghiou and R. B. Mellon, Pesticide resistance in time and space, in “Pest resistance to pesticides” (G. P. Georghiou and T. Saito, Eds.), pp. l-46, Plenum Press, New York, 1983. 3. S. P. Shrivastava, G. P. Georghiou, R. L. Metcalf and T. R. Fukuto, Carbamate resistance in mosquitos. The metabolism of propoxur by susceptible and resistant larvae of Culex pipiensfatigans, Bull. W.H.O. 42, 931 (1970). 4. S. P. Shrivastava, G. P. Georghiou and T. R. Fukuto, Metabolism of N-methylcarbamate insecticides by mosquito larval enzyme system requiring NADPH,, En?. exp. & appl. 14, 333-348 (1971). 5. T. M. Brown and G. H. S. Hooper, Metabolic detoxication as a mechanism of methoprene resistance in Culex pipiens pipiens. Pest. Biochem. Physiol.
12, 79 (1979).
6. R. Feyereisen. G. D. Baldridge, and D. E. Farnsworth, A rapid method for preparing insect microsomes, Comp. Biochem. Physiol. 82B, 559 (1985). 7. M. M. Bradford, A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Analyt. Biochem. 12, 248 (1976). 8. C H. Williams and H. Kamin, Microsomal triphosphopyridine nucleotide cytochrome c reductase: isolation, characterization, and kinetic studies, J. Biol. Chem. 237, 587 (1962). 9. R. Feyereisen, Polysubstrate monooxygenases (cytochrome P-450) in larvae of susceptible and resistant strains of house flies, Pesric. Biothem.
Physiol.
19, 262 (1983).
10. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370 (1964). 11. R. Feyereisen and D. R. Vincent, Characterization of antibodies to house fly NADPH-cytochrome P-450 reductase. Insect biochem. 14, 163 (1984).
IN
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12. H. H. Hagedorn, The control of vitellogenesis in the mosquito, Aedes aegypti, Amer. Zool. 14. 1207 (1974). 13. R. H. Stanton. F. W. Plapp, Jr.. R. A. White, and M. Agosin, Induction of multiple cytochrome P-450 species in housefly microsomes-SDS-gel electrophoresis studies, Comp. Biochrm. Physiol. 61B, 297 (1978). 14. H. A. Rose and L. C. Terriere. Microsomal oxidase activity of three blowfly species and its induction by phenobarbital and B-naphthoflavone, Pestic. Biochem. Physiol. 14, 275 (1980). 15. L. C. Terriere and S. J. Yu. Cytochrome P-450 in insects. 2. Multiple forms in the flesh fly (Sarcophaga bullara. Parker), and the blow fly (Phormia regina. Meigen). Pestic. Biochcm Physiol.
12, 249 (1979).
16. L. B. Brattsten. S. L. Price, and C. A. Gunderson, Microsomal oxidases in midgut and fat body tissues of a broadly herbivorous insect larva, Spodoptera eridania Cramer (Noctuidae). Comp. Biochem. Ph.vsio/. 66C, 23 I (1980).
17. R. Feyereisen and F. Durst, Development of microsomal cytochrome P-450 monooxygenases during the last larval instar of the locust. Locusta migraforia: Correlation with the hemolymph 20-hydroxyecdysone titer. Mo/. Cell. Endocrinol. 20, IS7 (1980). 18. S. J. Yu. F. A. Robinson, and J. L. Nation. Detoxication capacity in the honey bee, Apis me/lifera L.. Prstic. Biochem. Physio/. 22, 360 (1984). 19. R. 1. Krieger. P. P. Feeny. and C. F. Wilkinson. Detoxication enzymes in the guts of caterpillars: An evolutionary answer to plant defenses? Science 172, 579 (197 I ). 20. L. B. Brattsten, Ecological significance of mixedfunction oxidations. Drug Memb. Rels. 10. 35 11979).
21. E. Hodgson. The significance of cytochrome P-450 in insects, Insect Biochem. 13, 237 (1983).
22. A. W. A. Brown and R. Pal, “Insecticide Resistance in Arthropods,“ W.H.O. Geneva. 1971.
BIOCHEMISTRY
AND PHYSIOLOGY
25,
407-413 (1986)
Blood Meal and Cytochrome P-450 Monooxygenases in the Northern House Mosquito, Culex pipiens G. D. BALDRIDGE* Departments
of *Entomology
and
fAAgricu/tural
AND R. FEYEREISEN*+ Chemistry,
Oregon
State
University,
Corvallis,
Oregon
97331
Received May 2, 1985; accepted July 18. 1985 Conditions for the measurement of aldrin epoxidation by microsomes prepared from abdominal tissues (fat body + integument) of adult female Culex pipiens were characterized. The enzyme activity had a pH optimum of 7.2 and an apparent K,,, of 3.4 JLM. Aldrin epoxidation and NADPHcytochrome c reductase had similar patterns of inhibition by a rabbit antiserum to house fly NADPH-cytochrome P-450 reductase, thus implicating cytochrome P-450 monooxygenase(s) in the epoxidation of aldrin. Low (71 pmol/mg protein) levels of cytochrome P-450 were detected in abdominal tissue microsomes. In non-blood-fed insects, aldrin epoxidation and NADPH-cytochrome c reductase activities did not change between Day 1 and Day 12 after adult emergence. except for a small peak on Day 2. In insects fed a blood meal on Day 6 after emergence both activities increased (two- to threefold) to a plateau maintained between 2 and 4 days after the blood meal. Aldrin epoxidation and NADPH-cytochrome c reductase activities decreased to normal values between 4 and 6 days after the blood meal. Q 1986 Academic Press. Inc. INTRODUCTION
Mosquitoes have been called man’s worst enemy (1) because of their role in the transmission of diseases such as malaria, yellow fever, filariasis, dengue, encephalitis, etc. Despite early successes of vector control by the use of insecticide chemicals, there is an alarming proliferation of resistance problems in mosquitoes (2). Further use of chemicals for mosquito control and the prevention or management of resistance requires some knowledge of how mosquitoes deal with toxicants. However,
(5) showed that metabolic detoxication was an important mechanism of resistance to methoprene in Culex pipiens pipiens, but failed to detect any aldrin epoxidation activity in larval homogenates. To our knowledge, no in vitro study of cytochrome P-450 monooxygenases has been reported from any adult mosquito species, even though adult mosquitoes are a target of chemical control measures. This study was undertaken as a first step to characterize the cytochrome P-450 monooxygenase system in adult Culex pipiens, and to de-
our understanding of mosquitodetoxica- scribethe effectsof a bloodmealon this tion capabilities is very limited and the cytochrome P-450 monooxygenase system (mixed-function oxidases, polysubstrate monooxygenases) of mosquitoes is virtually unexplored by in vitro approaches. Shrivastava et al. (3, 4) studied a larval enzyme in Culex pipiens fatigans. They showed that cytochrome P-450 monooxygenases play a major role in the detoxication of carbamate insecticides in a propoxur-resistant strain. Brown and Hooper ’ To whom correspondence
should be addressed.
enzyme system. MATERIALS
AND METHODS
Insects. An anautogenous strain of Clalex pipiens L. (Diptera, Culicidae) collected in
1978 from a log-pond at Philomath, Oregon, was used to establish a colony. Insects were maintained at 26°C under a 16-hr light: 8-hr dark photoperiod. Adults were fed a 10% sucrose solution ad h&turn and live Quail were used to provide blood meals. mY females synchronized to
407 0048-3575186 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
408
BALDRIDGE
AND
within 24 hr of adult emergence or within 2 hr of a blood meal were used in this study. Preparation of microsomes. Two methods were used to prepare microsomes. A differential centrifugation method was used for large-scale preparation of microsomes from abdominal tissues and a sucrose density gradient preparation of microsomes was used for establishing a developmental profile of cytochrome P-450 monooxygenases. For the first method, 100 to 200 abdomens were dissected from adult females taken 72 hr after a blood meal. The midguts and ovaries were removed and the abdomens (i.e., essentially integument, muscle, and fat body) were homogenized in 20 ml MOPS (morpholinopropanesulfonate) buffer, 50 m&I, pH 7.2 containing 1 mM EDTA and 0.4 mM freshly prepared PMSF (phenylmethylsulfonylfluoride), with 10 strokes of a motor-driven Teflon/ glass homogenizer. The homogenate was centrifuged sequentially at IOOOg for 15 min, 10,OOOgfor 15 min and 105,OOOgfor 65 min. The final pellet was resuspended in 30% sucrose in 50 mM MOPS buffer, pH 7.2, containing 1 mM EDTA. All procedures were performed at 0-4°C. For the second method (6), from 10 to 3.5 abdomens (depending on age) prepared as described above were homogenized in 0.3 ml of MOPS buffer, pH 7.2, containing 1 mM EDTA and 0.4 r&I PMSE The homogenate was centrifuged at IOOOgfor 10 min and the supernatant was removed with a finely drawn Pasteur pipet to prevent contamination by the floating lipid layer. Supernatant (made up to 0.35 ml) containing no more than 1 mg protein was loaded on a 5 ml linear 45 to 15% sucrose gradient and centrifuged at 65,000 rpm in a Beckman VTi 80 rotor for 20 min (o*t = 3.7 X 1O’O rad*.sec-I). The bottom 2.0 ml of the gradient was discarded and the middle 2.1 ml (which contained the majority of the NADPH-cytochrome c reductase activity) were used as “microsomal” enzyme source. Details and justification of this procedure are given elsewhere (6). Sucrose was prepared in MOPS/EDTA buffer and
FEYEREISEN
protein levels were determined by the Bradford method (7) using y-globulins as standard. Enzyme assays. NADPH cytochrome c reductase activity (8) was measured spectrophotometrically at 30°C in an Aminco DW2a instrument. Reduction of cytochrome c was measured at 550 nm in the split beam mode in 1 ml cuvettes each containing 50 FM cytochrome c. I mM EDTA. 5 mM NaCN and enzyme in 50 mM MOPS buffer, pH 7.2. The reaction was started bq the addition of 50 ~1 of an NADPH (final concentration 150 @I) regenerating system (10 pmol glucose 6-phosphate, and 1.5 units glucose-6-phosphate dehydrogenase). Aldrin epoxidation was measured at 30°C in a final volume of 350 ~1 containing 50 mM MOPS buffer, pH 7.2 with 1 mM EDTA, enzyme and an NADPH (150 pJI,I regenerating system. The reaction was started by the addition of the substrate, aldrin, at a final concentration of 20 p,,M. The reaction was stopped with 1 M HCI after 20 min unless noted otherwise and the product, dieldrin, was extracted with 100 ~1 of hexane and analyzed by electron capture gas chromatography (9) using endrin as internal standard. Cytochrome P-450 determination. A “microsomal” pellet prepared by the differential centrifugation procedure (see above) was resuspended in 50 mM MOPS buffer, pH 7.2, containing 1 mM EDTA and 10% sucrose. Cytochrome P-450 level was measured according to Omura and Sato (IO) in an Aminco DW2a spectrophotometer thermostatted at 12°C. using I-ml cuvettes. RESULTS
Aldrin Epoxidation Activity In vitro conditions for the measurement of aldrin epoxidation activity were determined using “microsomes” prepared by a differential centrifugation procedure. This reaction was chosen because the oxygenated product, dieldrin, could be detected by gas chromatography with electron-cap-
CYTOCHROME
P-450
MONOOXYGENASES
ture detection, an easy and sensitive method suitable for the low activities expected from mosquito abdominal tissue. Other sensitive assays, such as the O-demethylation of methoxyresorufin or alkoxycoumarins (7-methoxy-,7-ethoxy-, and 7-methoxy-4-methylcoumarin) were also tried. However, the extremely low specific activities obtained in preliminary experiments precluded work on a small number of insects and thus a full characterization of those activities was not attempted in this study. The effect of “microsomal” protein concentration on aldrin epoxidation activity is shown on Fig. la. A clear dependence of product formation on enzyme concentration in the assay was observed. In view of the deviation in linearity at high concentrations, protein levels were kept below 0.06 mg/ml in all subsequent experiments. Figure lb shows that a linear dependence of dieldrin formation on incubation time was maintained for at least 20 min of incubation. Linearity of product formation with increasing enzyme concentration (up to at least 0.06 mg/ml) and with increasing incu-
Time , min
pg Protein/ml
PH
l/S
(pd
FIG. 1. Characterization of aldrin epoxidation activity from Culex pipiens abdominal tissues. “Microsomes” were prepared from adult females taken 72 hr after a blood meal. (a) Effect of “microsomal” protein concentration; (b) effect of incubation time; (c) effect of incubation pH; (d) effect of substrate concentration. Each point is the mean of duplicate incubations.
IN
Culex pipiens
409
bation time (up to 30 min) was also observed when using “microsomes” prepared by the sucrose density-gradient method (results not shown). Figure Ic shows that the enzymatic epoxidation of aldrin was pH dependent. Maximal activity was observed at pH 7.2 with a sharp decrease at lower pH values. Aldrin epoxidation was also dependent on the substrate concentration as shown on Fig. Id. The Lineweaver-Burk double reciprocal plot of the results (9 = 0.9802) indicated that a simple Michaelis-Menten behavior was a valid first approximation of the enzyme kinetics. The apparent K, of aldrin epoxidation was 3.4 FM and a V,,, of 38 pm01 * min-’ * mg protein was calculated. These results led us to adopt the assay conditions that were used in the further characterization of aldrin epoxidation in mosquito abdominal tissue. Aldrin Epoxidation Catalyzed by Cytochrome P-450 Monooxyygenase(s)
Because aldrin epoxidation activity was found in fractions enriched in NADPH-cytochrome c reductase activity (6) and was dependent on the presence of NADPH in the incubation, an involvement of microsomal cytochrome P-450 monooxygenase(s) was suggested. Rabbit antibodies to house fly NADPH cytochrome P-450 reductase (11) were used to demonstrate the involvement of NADPH-cytochrome P-450 reductase in the aldrin epoxidation reaction. When mosquito abdominal “microsomes” were preincubated in the presence of rabbit antibodies (IgG fraction) to house fly NADPH-cytochrome P-450 reductase and subsequently assayed for aldrin epoxidation activity, an inhibition of dieldrin production was observed (Fig. 2). This inhibition was dependent on the ratio of IgG to “microsomal” protein and aldrin epoxidation was fully inhibited at a ratio of 0.6. In contrast, only 30.1% inhibition of aldrin epoxidation was observed at the highest level of control (non-immune) IgG (ratio of 1.0). The inhibition of aldrin epoxidation closely paralleled the inhibition of
410
BALDRIDGE
NADPH-cytochrome (Fig. 2).
c reductase
AND
activity
I LA-=-l 0.002 AU.
Cytochrome P-450 in Abdominal ’ ‘Microsomes”
Mosquitoes taken 96 hr after a blood meal were used to prepare “microsomes,” and the method of Omura and Sato (10) was used to measure levels of cytochrome P-450. A representative spectrum (Fig. 3) shows that only low amounts of cytochrome P-450 could be detected. The carbon monoxide-difference spectrum of reduced ‘Imicrosomes” slowly developed a peak at 450.4 nm. A calculated level of 71 pmol/mg protein was obtained using the extinction coefficient for cytochrome P-450 in rabbit liver microsomes (91 cm-’ * mM-I). Figure 3 also shows that a significant degradation of cytochrome P-450 to cytochrome P-420 had occurred. Developmental Profile of Aldrin Epoxidation and NADPH-Cytochrome c Reductase and Effects of a Blood Meal
“Microsomes” were obtained by a sucrose density gradient procedure which allowed the rapid preparation of a large number of samples. For each sample, 25 to 35 non-blood-fed mosquitoes, or 12 to 25
0
FEYEREISEN
0.5 mg WJ 1 mg
1.0 protein
FIG. 2. Inhibition of aldrin epoxidation (0) and NADPH-cytochrome c reductase (m) by an ZgG fraction from rabbit antiserum raised against house fry NADPH-cytochrome P-450 reductase (II). Each point is the mean of duplicate incubations. (0, 0) Control, non-immune ZgG fraction.
8 2t I
v
400
450
500
Wavelength bml FIG. 3. Carbon monoxide difference spectrum of dithionite-reduced “microsomes” from Culex pipiens abdominal tissues. The absorbance peak (450.4 nm, determined manually) was maximal 20 min after saturation with carbon monoxide. Protein level was 0.59 mglml.
blood-fed mosquitoes were used. In each case the ovaries and gut were removed from the dissected abdomens. Aldrin epoxidation and NADPH-cytochrome c reductase activities were determined on precisely timed non-blood-fed and blood-fed mosquitoes and expressed as specific activities per insect-equivalent. Figure 4 shows that a small but significant peak in both enzyme activities was observed on Day 2 after adult emergence in non-blood fed insects. A relatively stable level of activities was maintained from Day 3 through Day 12. Figure 4 also shows the pattern of activities in mosquitoes fed a blood meal on Day 6 after adult emergence. A rapid increase in both aldrin epoxidation and NADPH-cytochrome c reductase activities was observed during the first 48 hr following the blood meal. Between 48 and 96 hr after a blood meal the activities were maintained at a plateau which was about threefold (aldrin epoxidation) and twofold (NADPHcytochrome c reductase) higher than the values for non-blood-fed insects. Both activities then declined to values typical of non-blood-fed insects by 6 days after the blood meal. When the enzyme activities were expressed per milligram protein, an identical pattern of change was observed, but the amplitude of the changes was lower
CYTOCHROME
P-450
MONOOXYGENASES
IN
L L
2
4 Days
IO ad
I2
ent&flC~
FIG. 4. Developmental proj2e of aldrin epoxidation (m), NADPH-cytochrome c reductase activity (0). from the abdominal tissues (fat body) of non blood-fed (-) and blood fed (- . - * -) adult female Culex pipiens. Each point represents the mean ? SEM of 4 to 7 experiments. A blood meal was given on Day 6 after adult emergence.
because the specific activities were shown to be lower than controls (non-blood-fed) until 36 hr after the blood meal. A very rapid increase in “microsomal” protein level was observed between 0 and 24 hr after a blood meal, this level then slowly decreased as aldrin epoxidation and NADPH-cytochrome c reductase reached their peaks (Fig. 5). “Microsomal” protein levels were still higher at 6 days after a blood meal when compared to non-bloodfed insects. DISCUSSION
The tissue used for this study was a compromise between a single, defined tissue, and a mixture of all abdominal tissues as has been done repeatedly in studies of dipteran cytochrome P-450 monooxygenase systems. By removing the ovaries and the gut tissues, we removed the source of large variations in the relative contribution of different tissues to the microsomal preparations. Although we have tried to characterize cytochrome P-450 monooxygenases in dissected Malpighian tubules or midgut, practical problems such as time of dissec-
2
C&x
4 Days
411
pipiens
6 after
0
IO
12
J
smarV
5. Development profile of “microsomal” protein levels from the abdominal tissues Vat hudy) of non blood-fed (-) and blood ,fed (-. - * -) adult female Culex pipiens. Data from the experiments shown on Fig. 4. FIG.
tion and number of insects became limiting factors in a precise and detailed study of biochemical changes following a blood meal. A reliable method for a rapid and reproducible dissection of midgut tissue free of blood meal contaminants is still needed. The integument + fat body preparation has precedents in the literature (12) and the tissue used in the present study can be considered as essentially the fat body for the purpose of this discussion. Levels of cytochrome P-450 were measured at 94 hr after a blood meal, which corresponded to a maximum in the activities of aldrin epoxidation and NADPH-cytochrome c reductase. When compared to values obtained with microsomes obtained from whole abdomens of other adult Diptera of various ages, the specific activity of the Cufex pipiens cytochrome P-450 (71 pmol/mg protein) appears to be about half that of house flies (13) and approximately equal to that of blow flies (14, 15) and flesh flies (15). However, such a comparison is difficult because gut tissues (usually rich sources of cytochrome P-450 monooxygenases)) were excluded form our study. Cytochrome P-450 levels of CL&X pipiens appear quite low when compared to values reported from the fat body of other insects
412
BALDRIDGE
AND FEYEREISEN
(84 pmol/mg in Spodoptera eridania larvae, 16, 200-400 pmol/mg in Locusta migratoria, 17) particularly if one assumes that in non-blood-fed insects the levels would be even lower. The levels of activity measured for aldrin epoxidation are also lower than in any other insect as reviewed by Yu et al. (18). Obviously such absolute comparisons have only a limited value, because cytochrome P-450 levels or monooxygenase activities depend on age and tissue involved. In addition, aldrin epoxidation may be catalyzed by one or more cytochrome P-450 isozymes and it is not known how well changes in aldrin epoxidation reflect changes in other monooxygenase activities. In anautogenous mosquitoes such as the Culex pipiens strain used in this study, a blood meal is the necessary requisite for the completion of oocyte growth and egg laying. In unfed females, the length of the follicles increases from 20 to 90 km (resting stage) during the first 3 days after adult emergence. No further growth is observed until after a blood meal, Ovarian follicles grow from the resting stage to about 600 pm between 0 and 60 hr after a blood meal, and egg-laying occurs between 72 and 96 hr after the blood meal. The peak activity of cytochrome P-450 monooxygenases as estimated here by aldrin epoxidation and NADPH-cytochrome c reductase is therefore concomitant with the final phase of vitellogenesis, chorionation, and oviposition. However, it occurs after the peak level of “microsomal” protein (possibly related to massive vitellogenin synthesis by the fat body) observed 24 hr after the blood meal. The decline in activities occurs after egg laying. In non-blood-fed insects, the small peak in activity noted on Day 2 after emergence is concomitant with the previtellogenie growth of oocytes to the resting stage. It would be of interest to determine whether changes in cytochrome P-450 monooxygenases are in any way related to the gonotrophic cycle of the mosquito and its hormonal regulation. In Culex pipiens, a
peak of ecdysteroids (400 fmol20-hydroxyecdysone eq/insect) is observed at 36 hr after a blood meal (unpublished results) and therefore precedes the maximum level of cytochrome P-450 monooxygenases. When the procedures to measure cytochrome P-450 monooxygenase activities in mosquito midgut become available, it will be possible to compare the timing of cytochrome P-450 monooxygenase changes in midgut and fat body after a blood meal. It was somewhat surprising to find that fat body enzymes reached a maximum only 48 hr after the blood meal, whereas microsomal protein levels had already peaked at 24 hr. The importance of insect enzyme systems capable of detoxifying potentially toxic plant chemicals has been documented in detail and it is hypothesized that such enzyme systems (cytochrome P-450 monooxygenases in particular) may serve as defensive countermeasures in the “coevolutionary arms race” between plants and phytophagous insects (19-21). In view of the enormous importance for mankind of a more recent coevolution, that of hematophagous insects and their vertebrate hosts, it is astonishing how little is known about the biochemical adaptations of hematophagous insects to the vast array of potentially toxic constituents of a blood meal. To our knowledge this is the first study of the changes in cytochrome P-450 monooxygenases in response to a blood meal. Because blood-fed mosquitoes are epidemiologically more important than non-bloodfed mosquitoes, it will now be necessary to assess the toxicological significance of our results in view of some reports on the differences in insecticide tolerance between blood-fed and non-blood-fed mosquitoes cm. ACKNOWLEDGMENTS This work was supported by NIH Grant AI 19192 to R. F. Oregon Agricultural Experiment Station Technical Paper No. 7552.
CYTOCHROME
P-450
MONOOXYGENASES
REFERENCES
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Physiol.
19, 262 (1983).
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12, 249 (1979).
16. L. B. Brattsten. S. L. Price, and C. A. Gunderson, Microsomal oxidases in midgut and fat body tissues of a broadly herbivorous insect larva, Spodoptera eridania Cramer (Noctuidae). Comp. Biochem. Ph.vsio/. 66C, 23 I (1980).
17. R. Feyereisen and F. Durst, Development of microsomal cytochrome P-450 monooxygenases during the last larval instar of the locust. Locusta migraforia: Correlation with the hemolymph 20-hydroxyecdysone titer. Mo/. Cell. Endocrinol. 20, IS7 (1980). 18. S. J. Yu. F. A. Robinson, and J. L. Nation. Detoxication capacity in the honey bee, Apis me/lifera L.. Prstic. Biochem. Physio/. 22, 360 (1984). 19. R. 1. Krieger. P. P. Feeny. and C. F. Wilkinson. Detoxication enzymes in the guts of caterpillars: An evolutionary answer to plant defenses? Science 172, 579 (197 I ). 20. L. B. Brattsten, Ecological significance of mixedfunction oxidations. Drug Memb. Rels. 10. 35 11979).
21. E. Hodgson. The significance of cytochrome P-450 in insects, Insect Biochem. 13, 237 (1983).
22. A. W. A. Brown and R. Pal, “Insecticide Resistance in Arthropods,“ W.H.O. Geneva. 1971.