Effects of azadirachtin on oogenesis in Aedes aegypti

Physiological Entomology (1988) 13,177-184

Effects of azadirachtin on oogenesis in Aedes aegypti

CAROLE T. LUDLUM" and KLAUS-PETER SIEBER Department of Entomology, Cornell University, Ithaca, New York

ABSTRACT. Azadirachtin in blood fed to adult female Aedes aegypti through an artificial membrane does not cause feeding inhibition over a wide dose range (0-200 ng/female), and high doses of ingested azadirachtin fail to inhibit or delay oviposition. However, significant, tran- sient retardation of oocyte growth is observed for up to 72 h after feeding. Immature oocytes are observed in 86% of azadirachtin-fed females decapitated 10 h after a blood meal, whereas 96% of decapitated control females contain maturing oocytes. This suggests that azadirachtin delays the release of one or more factors from the head that regulate oogenesis. We propose that adult females overcome the effect of azadirachtin by rapid metabolism rather than by excretion of the compound, since by 2 h after a blood meal, only 0.1% of ingested azadirachtin was recovered from excreta and 5% recovered from the body.

Key words. Aedes aegypti, azadirachtin, insect hormones, neem, oogenesis, ovarian development, vitellogenin.

Introduction

Of the phagodeterrent and growth-regulating compounds identified from the neem tree (Azadirachta indica A. Juss), azadirachtin has been shown to be the most effective (Rembold, 1984). Its effects on feeding, development and reproduction have been reported in a variety of insect species (Butterworth & Morgan, 1968; Schmutterer et al., 1981). Azadirachtin appears to interefere with insect ecdysteroid levels (Rembold & Sieber, 1981; Sieber & Rembold, 1983) and presumably acts on the neuroen-

* Present address: Department of Entomology, University of California, Davis, California 95616, U.S.A.

Correspondence: Dr Klaus-Peter Sieber, Depart- ment of Entomology, University of Maryland, College Park, Maryland 20742, U.S.A.

docrine control of development (Sieber & Rem- bold, 1983; Dorn et al., 1986), although other modes of action, such as competition by azadirachtin for ecdysteroid binding sites (Leuschner, 1972; Redfern et al., 1982), have been suggested.

In this study we investigated the mode of action of azadirachtin by examining its influence on development in the mosquito Aedes aegypti. A.aegypti is particularly well suited for such work because its endocrinology and reproduc- tive physiology have been thoroughly explored (see review by Hagedorn, 1985). Since the onset of ovarian development in A . aegypti is triggered by a blood meal, it is possible to study short-term physiological effects of biologically active sub- stances, such as azadirachtin, on a precise time scale. Furthermore, the availability of numerous in vitro assays involving A.aegypti allowed us to

177

178 Carole T. Ludlum and Klaus-Peter Sieber

isolate and examine oogenesis-related phenomena that may be disrupted by azadirachtin. Here we present further evidence of how this promising insect growth regulator influences the neuroendocrine control of development.

Materials and Methods

Animals

Aedes aegypti (L.) derived from the Rock strain were reared at 27°C and 75% relative humidity under LD 16:s h. Larvae were fed a mixture (1:l:l w/w) of ground rat chow, yeast hydrolysate, and lactalbumin (Baker et al., 1983), and adults were fed 3% sucrose; 3-5-day- old females were used for all assays. For egg counts, females were placed in cages with moist paper towelling. Eggs were removed and counted 96 h after the blood meal.

Azadirachtin and method of application

Azadirachtin (a gift of H. Rembold, F.R.G.) was isolated from neem seeds by extraction with methanol and purified as described by Rembold e.t al. (1980). Aliquots of azadirachtin in 50% ethanol were dried under nitrogen, redissolved in 1Opl water, and mixed with 49Opl bovine blood. Aqueous dilution of blood was 7% in all treatments, including control blood, which contained no azadirachtin. To increase avidity, females were starved c . 15 h prior to the meal. They were then allowed to feed to repletion through an artificial membrane on blood kept at 37°C. Cold-anaesthetized females were weighed before and after feeding to determine the volume of the blood meal. All animals that consumed less than 2 pl blood were discarded.

Oocyte measurements and decapitation

Blood-fed, undecapitated females were dis- sected and the yolks of their oocytes measured to quantify the response to blood with and without azadirachtin. The role of the head in this response was examined via oocyte maturation experiments after Greenplate et al. (1985) and Lea et al. (1978). In these experiments, azadirachtin-fed and control females were decapitated at different times after the blood meal. 48 h after feeding the insects were dis-

sected and their oocytes placed into two groups: those measuring 100,um or more, defined as maturing, and those measuring less than 100 pm, defined as arrested. The developmental stage of oocytes was also noted using the criteria described by Clements & Boocock (1984).

Rocket immunoelectrophoresis

Whole animals and ovaries were assayed for vitellogenin 48 h after a blood meal using the rocket electrophoresis technique developed by Laurel1 (1966) and used by Hagedorn et al, (1978), except that Gell-Bond agarose gel sup- port medium (FMC) was used on slides. Groups of ten animals and of twenty whole, saline-rinsed ovaries were homogenized in 1 ml of 0.05 M Tris-OH buffer, pH 8.0, containing 0.4 M NaCl. Sodium azide (2%) and 0.1 M diisopropyl fluorophosphate were added to inhibit bacterial growth and enzymatic degradation. For quantitative assessment, heights of electrophoretic peaks were divided by the mean height of peaks obtained, in the same experiment, from an ovarian extract of blood-fed, control females. Thus results were expressed as a percentage of controls.

Fat body incubation

The ability of azadirachtin to inhibit vitellogenin synthesis by fat bodies was tested in vitro by the method of Hagedorn & Fallon (1973). Fat bodies from fifteen females were incubated in 100 pl of medium containing M 20-hydroxyecdysone (Rhoto) and 10-4-10-7 M azadirachtin (two replicates per treatment). Controls contained no azadirachtin. Medium was removed from incubation dishes after a 24 h incubation period and stored at -80°C prior to rocket immunoelectrophoresis.

Ovary incubation and radioimmunoassay (RIA)

Ovarian synthesis of ecdysteroids was assayed after Hagedorn et al. (1979) by incubating ovaries for 6 h at 27°C with active head extract and azadirachtin (0-5 ,ug/incubation). Each treatment consisted of two replicates of ten pairs of ovaries each. A RIA described by Borst & O’Connor (1974) was used to assay for ecdysteroids in the incubation medium as described by Greenplate et al. (1985). Anti-

Azadirachtin and oogenesis in A.aegypti 179

ecdysteroid antiserum was a gift of J. Koolman (Marburg, F.R.G.). Tritiated ecdysone was obtained from ICN (50 Ci/mmol), and ecdysone (Rhoto) was used for the standard curve. No cross-reactivity was found between ecdysone and azadirachtin. Samples were counted in a Beckman LS315OP scintillation counter.

Retention of azadirachtin: excreta and whole- body extracts

To determine whether females retain azadirachtin after the blood meal, two groups of 100 females were placed in separate, siliconized, 250 ml beakers closed with nylon netting. Blood containing 0 and 100 ng/pl azadirachtin, respectively, was provided for 0.5 hand then removed. Animals were left in the beakers for an addi- tional 1.5 h and then removed. Methylene chloride, methanol and water (2:l:l v/v) were added in turn to the beakers, which were swirled gently after the addition of each solvent. The resulting extract of excreta was transferred to a culture tube, and two phases were allowed to form. The methylene chloride phase, which contained all of the azadirachtin as measured by HPLC, was washed twice with aqueous methanol (50%), dried under nitrogen, and redissolved in 500 pul methanol.

Whole-body extracts were obtained by homo- genizing twenty-five blood-fed, azadirachtin- treated females in 500 pl methylene chloride and 250 pl methanol, to which 250 pl water was subsequently added. The homogenate was transferred to a glass culture tube and centrifuged, the supernatant removed, and the pellet re-homogenized in 1 ml of methylene chloride, methanol and water. Subsequent extraction was identical to that of excreta samples.

For the determination of extraction yield, a known amount of azadirachtin (20pg) was added to an extract of blood-fed control females (n= 100) and subsequently quantified by HPLC. Loss of azadirachtin during extraction was included in all calculations of metabolism and excretion rates.

High-performance liquid chromatography (HPLC)

Extracts were dried under nitrogen, redissolved in 40% aqueous acetonitrile, and

subjected to HPLC on a reversed-phase, 3 0 ~ 0 . 7 5 cm i.d. pBondapak C18 column (Waters Asssoc.). A Waters Assoc. 6000A pump, a Rheodyne 7215 injector, a Waters Assoc. Model 660 solvent programmer and a Schoeffel Instrument Corp. GM 770 Mono- chromator multi-wavelength UV detector at 228 nm were used for all HPLC separations. The amount of azadirachtin in samples was calculated by determining the area of chromatograph peaks.

Statistical analysis

The Minitab statistical computing program (Pennsylvania State University, 1981) was used for statistical analyses of data. Probability levels of 0.05 or less were used for accepting a difference as statistically significant.

Results

Oogenesis and related effects

Oocyte development in A . aegypti was found to be suppressed after blood meals in which females consumed more than 20 ng azadirachtin/pl blood, as examined 48 h later (Fig. 1). Calculations from blood meals admin- istered in six separate assays indicated that females given blood containing 100 ng azadirachtin/pl consumed slightly more blood than control females (1.75k0.19 and 1.90k0.18 pl, respectively), but the difference was not significant (two-tailed test, P>0.25). Fig. 1 shows that oocyte length in females treated with more than 50 ng/pl azadirachtin was reduced by about 30% compared to control females at 48 h. This is confirmed in Fig. 2, which also shows that despite the initial suppres- sion of oocyte development among azadirachtin- treated females, both azadirachtin-treated and control females completed oogenesis 64-72 h after feeding. All azadirachtin-treated females deposited viable eggs that developed to normal adults, and a two-tailed test showed no significant difference between the number of eggs deposited by azadirachtin-treated and control females (P>O.25).

Strikingly different results arose when azadirachtin-treated females were decapitated 10 h after feeding and examined 38 h later to determine if azadirachtin interferes with the


410 r

\

0- 0 5 20 50 75 100 150

A Z A D I R A C H T I N (NG/)JL B L O O D )

FIG. 1 . Oocyte length in relation to azadirachtin in the blood meal 48 h after feeding. Each point represents the mean length (fSEM) of ten oocytes from each of seven to ten females.

release of head factors controlling oogenesis. Of twenty-eight control females, twenty-seven contained maturing oocytes (100 to c . 300 pm) when examined 38 h later. However, only four of twenty-eight females fed 100 ng azadirachtin/pl blood contained maturing oocytes (=lo0 pm), a highly significant difference (chi-square test, P<0.005). Yolks of arrested oocytes from decapitated, azadirachtin-fed females measured 40-90 pm, showing development beyond the resting stage (<30pm, stage IIa and IIb) to

I270 I- W z w J

w I- >

0 90

0

HOURS POST BLOOD MEAL

FIG. 2. Oocyte length in control (W) and azadirachtin- fed (0) females (100 ng azadirachtin/pl blood) @90 h after a blood meal. Each point represents the mean (fSEM) of ten oocytes from each of ten females.

stage I11 only (<lo0 pm) (Christophers, 1911; Clements & Boocock, 1984).

Results of in vitro assays showed that the synthesis of ecdysone in response to head extract (Table 1) and synthesis of vitellogenin in response to 20-hydroxyecdysone (Table 2) was

TABLE 1. Ecdysteroids produced by ovaries incubated with active head extract and azadirachtin, and detected by radioimmunoassay.

Azadirachtin Ecdysteroids* (ng per dish) (PP)

0 75 10 97 100 87

loo0 83 SO00 112

=5.234, R2=0.636. Regression equation : Y = 84.3 + 0.0053X. F, ,3

*Each figure represents the mean of two replicates.

TABLE 2. Vitellogenin synthesized by fat bodies in azadirachtin and 1 x 10-6 M ecdysone, and detected by rocket immunoelectrophoresis. Percentages with the same letter are not significantly different at the 5% level using orthogonal contrasts.

Azadirachtin Vitellogenin' (% of control (+SEM))

0 (control) 1 x 10-7 M

1 x 10-5 M 1x10-4 M

1 X M

100.0 f7.51 68.5 f 1.8b 64.0k5.0b

117.0f 15.9' 82 .O * 10.6ab

Regression equation: Y=19.5+0.75X. F,,,,=0.371,

*Each percentage is the mean of four replicates. R2-0.021.

TABLE 3. Comparison of whole-body and ovarian vitellogenin from 4-day-old, blood-fed females, detected by rocket immunoelectrophoresis and expressed as percentage of control (ovarian extract from undecapitated females fed blood without azadirachtin).

Azadirachtin Ovarian* Whole-body* (ng/jA blood)

0 100.0+6.3 91.6L4.4 50 88.3 f 1.5 93.2k4.8 100 86.9 f 6 . 7 91.6 f 2.8 f 91.7rt4.8 94.2f 4.0

Regression equations: Whole-body vitellogenin: Y=27.9+0.02X. FI,,,=1.475, R'=0.066. Ovarian vitellogenin: Y= 30.1 -0.04X. F,,,,=3.881, R2=0.177.

*Means (f SEM) obtained from three experiments of two replicates each.


not significantly influenced by azadirachtin (least squares regression, F1,3=5.234 and F1,17=0.371, respectively). Azadirachtin exerted no effect on vitellogenin content in vivo as shown by rocket immunoelectrophoresis of whole-body and ovarian extracts (Table 3; least squares regression, F1,*,=1.475 and Fl,18=3.881r respectively). However, the vitellogenin content of whole-body extracts tended to be greater for treated females than for controls, while the reverse was true of vitellogenin in ovarian extracts.

I: z W N N I

w u 2 a m U 0 v) m a

0 2 4 6 8 10 12

RETENTION TIME ( M I N I

FIG. 3. Chromatographs of urine and whole-body extracts prepared 2 h after a blood meal and subjected to HPLC. Arrows indicate elution of azadirachtin. (A) Whole-body extract from azadirachtin-fed females; (B) whole-body extract from control females; ( C ) urine extract from azadirachtin-fed females; (D) urine extract from control females.

HPLC

Samples of extracts from excreta and whole bodies were subjected to HPLC analysis and separated on an isocratic gradient. Within 2 h of the blood meal, most of the ingested azadirachtin had disappeared. Only 5% was recovered from the body, as calculated by comparison of peak areas from both azadirachtin standards and extract samples (Fig. 3). Minute amounts of azadirachtin (0.1%) were found in the excreta of females fed azadirachtin.

Discussion

In the adult female mosquito, azadirachtin fails to halt ovarian development permanently when fed in doses of up to about 200 ng/female. Azadirachtin, which has been cited as a potent antifeedant for several insect species (Butter- worth & Morgan, 1968; Ruscoe, 1972; Schmut- terer & Rembold, 1980; Sieber & Rembold, 1983), did not disrupt or inhibit feeding in adult A.aegypti at concentrations at least as high as 100 ng/pl blood. Thus it was possible to study the effects of azadirachtin on oocyte development without interference from antifeedant effects, which are thought to induce a suppres- sion of ovarian development in many insect species, including A.aegypti (Lea et at . , 1978). Although we found a delay in the stages of oogenesis subsequent to the blood meal (Fig. 2), azadirachtin failed to slow the timing of oviposition in A.aegypti. This absence of lasting effect at high doses of azadirachtin differs considerably from reports for most insect species, where the compound exhibits strong antifeedant effects as well as growth disruption when applied in low doses (Rembold & Sieber, 1981; Schmutterer et al . , 1981; Garcia & Rembold, 1984). In this con- text we note the observations of Schulz (1981) and Schulz & Schluter (1984), who after feeding neem extract to adult Epilachna varivestis found severe ovarian damage, permanent stoppage of oogenesis, and inhibited ability of ovaries to sequester vitellogenin. Interestingly, azadirachtin is highly effective in disrupting moults of larval A.aegypti when added to the rearing medium (Ludlum & Sieber, unpublished; Zebitz, 1984).

By investigating the action of azadirachtin on oogenesis in vitro as well as in vivo we were able to pinpoint the head as the target site of


azadirachtin. Decapitation experiments have shown that the head is necessary for 8 h after a blood meal if oocyte development is to follow (Gillett, 1957), a time period in which egg development neurosecretory hormone (EDNH) is released into the haemolymph (Gillett, 1956; Lea, 1972; Greenplate et al., 1985). EDNH stimulates ovarian ecdysone synthesis and so indirectly controls vitellogenin synthesis by the fat body (Hagedorn et al . , 1978,1979; Hanaoka & Hagedorn, 1980). Thus the ability of A.uegypti to mature oocytes after a blood meal increases with the time intervening between blood meal and decapitation (Gillett, 1957; Greenplate et al., 1985). In agreement with these findings, 96% of control females that we decapitated 10 h after a blood meal developed mature oocytes. The presence of arrested oocytes in most azadirachtin-treated females decapitated at 10 h (i.e. at least 2 h after the release of EDNH in controls) strongly suggests interference by azadirachtin with EDNH release. Another sign of interference in decapitated, azadirachtin-treated females was the length of maturing oocytes. Such oocytes, unlike those of azadirachtin-fed, undecapitated females (Figs. 1 and 2), never surpassed 100 pm. Development of oocytes beyond stage IIIa (100 pm) to stage V (2200 pm) is thought to be EDNH-dependent (Greenplate et al., 1985).

Our studies in vitro indicated that azadirachtin blocks the release of EDNH rather than other regulatory steps of oogenesis. First, ovaries stimulated by active head extract to synthesize ecdysone in vitro failed to change their synthetic activity when various concentrations of azadirachtin were added to the incubation medium, demonstrating that EDNH, once released into the haemolymph, acts on its target site in the presence of azadirachtin. A similar effect has been shown in two species: Locusta tzigratoria, where azadirachtin was unable to inhibit moulting when applied to nymphs after the time of release of prothoracicotrophic hormone (PTTH) but before synthesis of ecdysone (Sieber, 1982); and larval Bombyx mori, where prothoracic glands incubated with PTTH and azadirachtin secreted the same quantity of ecdysone as glands incubated with PTTH only (Koul et ul., 1987). Second, isolated fat bodies of A.aegypti in the presence of azadirachtin were stimulated by 20-hydroxyecdysone to synthesize vitellogenin (Table 2). Thus azadirachtin

appears not to compete with 20-hydroxyecdysone for 20-hydroxyecdysone receptors in the fat body, a conclusion also reached by Sieber & Rembold (1983).

We have noted that undecapitated, azadirachtin-treated A . uegypti produced normal eggs in the same time as control females (Fig. 2 ) . Clearly, such females resume egg development after a short period of azadirachtin-induced interference. The absence of a regression rela- tionship between azadirachtin dose and vitellogenin levels in whole-body extracts 48 h after a blood meal (Table 3) also points to renewed egg development in treated females. However, the trend towards lower vitellogenin in ovarian extracts of treated females, as compared to whole-animal extracts, is suggestive and may indicate a phenomenon that appears not to be mediated by EDNH. Greenplate et al. (1985) showed that females of A.aegypti require the head for up to 24 h after a blood meal to develop normal oocytes while only 12 h were needed to produce normal levels of ecdysteroids. Those results suggested the exis- tence of head factors besides EDNH that are active in oocyte growth after the blood meal. Here, the trend towards diminished uptake of vitellogenin by ovaries may reflect action by azadirachtin on one or more factors controlling the uptake mechanism itself.

The apparent ability of A.aegypti to resume the release of brain factors offers an interesting point of comparison with other insect species. For example, Sieber & Rembold (1983) reported that the inhibition of development shown by azadirachtin-treated L.migratoria was also eventually reversed. But while L. migrutoria required up to 40 days after treatment for endocrine-controlled development to continue. adult A.aegypti treated here with far higher doses of azadirachtin (2.3-45.5 ,ug/g fresh weight v . 0.6-6.0pg/g fresh weight for L. migratoria (Sieber & Rembold, 1983)) over- came apparent neurohormonal inhibition within hours. Female A.aegypti thus appear to possess mechanisms that inactivate ingested azadirachtin at a rate and with an efficiency so far undescribed in insect systems. Excretion does not account for this rapid recovery: whereas L.migratoriu excretes 40% of unmetabolized azadirachtin within a few hours of application (Rembold et al . , 1984), azadirachtin could barely be detected in the


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excreta of A.aegypti 2 h after feeding. Detoxica- tion of azadirachtin is presumably achieved through rapid breakdown because only 5% of the ingested compound is detectable in the body 2 h after a blood meal. Examination of the time- course of ingested azadirachtin in A.aegypti will clarify the fate of this compound (Sieber & Ludlum, in preparation).

The evidence presented here confirms the neuroendocrine system as the main target of azadirachtin, and suggests that the long-lasting inhibitory effects of azadirachtin in L.migratoria and other insect species arise from the absence of efficient mechanisms for excretion and/or metabolism of this compound. We demonstrate here that insect species capable of tolerating high doses of azadirachtin are suitable for investigations on the physiological effects of azadirachtin. Since the potential practical value of azadirachtin lies in its selective ability to disrupt the feeding and growth of insect species, it is essential to continue investigating the mechanisms by which species like A.aegypti detoxify azadirachtin.

Acknowledgments

We are grateful to S. S. Duffey, G.W. Felton and C . H. Judson for helpful criticism and sup- port, and to H. H . Hagedorn for laboratory space. This research was funded under Project No. 420 of the New York State Experiment Sta- tion, Cornell University.

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Carole T. Ludlum and Klaus-Peter Sieber

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Accepted 21 November 1987

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Effects of azadirachtin on oogenesis in Aedes aegypti