Identification of juvenile hormone III bisepoxide (JHB3), juvenile hormone III and methyl farnesoate secreted by the corpus allatum of Phormia regina (Meigen), in vitro and function of JHB3 either applied alone or as a part of a juvenoid blend

Pergamon

J. Insect Physiol. Vol. 41, No. 6, pp. 473419, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/95 $9.50 + 0.00

0022-1910(94)00134-O

Identification of Juvenile Hormone III Bisepoxide (JHB$, Juvenile Hormone III and Methyl Farnesoate Secreted by the Corpus Allatum of Phormia regina (Meigen), In Vitro and Function of JHB3 Either Applied Alone or as a Part of a Juvenoid Blend CHIH-MING YIN,*? BAI-XIANG ZOU,* MUGENG JIANG,$ THOMAS L. POTTER,@ JOHN G. STOFFOLANO JR*

MEI-FANG

LI,* WENHONG

QIN,*

Received 23 June 1994; revised I November 1994

The corpus allatum of both male and female adult black blow fly, Phormiu regina (Meigen), produced radioisotope-labeled juvenoids when incubated in vitro following a radiochemical assay. The production of juvenoids was greatly enhanced after the flies were fed a protein (liver) meal. These juvenoids were determined as juvenile hormone III bisepoxide (JHB,), juvenile hormone III (JH III) and methyl farnesoate (MF). These three juvenoids were separated and identified by chromatography (TLC and HPLC) using synthesized standards, respectively. Then, JHBJ standard and the biological samples of JHB3 were further identified with PC1 GS-MS using published spectrum data. Bioassay of synthetic JHB, revealed its ability to restore oiigenesis in allatectomized, liver-fed female blow flies. The ability of these three juvenoids and their blends to restore oiigenesis was the rational juvenoid blend (i.e. a mixture of JHB3, JH III and MF according to the percent ratio secreted by the isolated CA of liver-fed females) > JHB, = JH III > MF = the irrational juvenoid blend (i.e. a mixture of an arbitrary ratio of JHB,, JH III and MF). This is the first evidence suggesting that multiple juvenoid production and reception may be a normal event for insects. Allatectomy (JHB,)

Corpus allatum

Juvenoid

blend

Methyl

Diptera

Juvenile hormone III (JH III)

farnesoate

(MF)

Phormia regina

Recent studies from our laboratories have provided information to better understand the interactions among nutrition, endocrines and oiigenesis in the black blow fly, Phormia regina (Meigen). We have shown that after a proteinaceous meal, a hormone from the midgut is released to activate the cephalic endocrine system (Yin and Stoffolano, 1990, 1994; Yin et al., 1993, 1994). The *Department of Entomology, University of Massachusetts, Amherst, 01003-2410,

hormone

III bisepoxide

cephalic neuroendocrine system then activates ecdysteroid biosynthesis within the ovaries (Yin et al., 1990) and juvenile hormone biosynthesis by the corpus allatum (Liu, 1985; Liu et al., 1988; Zou et al., 1989). We have also shown that, in liver-fed flies, biosynthesis of vitellogenin is regulated mainly by ecdysteroids (Yin et al., 1990) whereas uptake of vitellogenin is controlled mainly by juvenile hormone (Yin et al., 1989). In sugar-fed flies, topical application of methoprene does not induce vitellogenin biosynthesis (Stoffolano et al., 1992), indicating that dietary status is of ultimate importance in determining the state of oagenesis. Juvenile hormone biosynthesis can be studied using an in vitro assay. To date, many researchers have adopted this short-term corpus allatum incubation method to examine juvenile hormone biosynthesis and release

INTRODUCTION

MA

Juvenile Oijgenesis

U.S.A.

tTo whom all correspondence should be addressed. JVisiting Scholar from Nanjing Agricultural University, Nanjing, People’s Republic of China. $Massachusetts Spectrometry Facility, University of Massachusetts, Amherst, MA 01003-1410, U.S.A. 413

CHIH-MING

414

in vitro. Two approaches have been used to quantify the juvenile hormone released during this short-term incubation. One approach determines the juvenile hormone produced by the corpus allatum during incubation with a radioimmunoassay for the specific juvenile hormone in question. This approach detects only the juvenile hormone that is specifically recognizable by the radioimmunoassay (Trabalon et al., 1987). The other approach, known as the radiochemical assay, resorts to incubation of the corpus allatum in a suitable medium containing a suitable radioactive precursor of juvenile hormones. This second approach, when used on Phormia regina, provided the first evidence, although contrary to the general belief at the time, that in Diptera (i.e. Phormiu hormone III (JH III) is not the regina) juvenile predominant juvenoid produced by the corpus allatum in vitro. An unknown substance, which may contain up to 10 times or more radioactivity than JH III, was discovered (Liu, 1985; Liu et al., 1988; Zou et al., 1989; Yin, 1994). This predominant unknown is much more polar than JH III as judged from its behavior on TLC and HPLC. It is produced by female adult corpora allata in all the stages (Zou et d., 1989) and we now know from the present study that it is also produced by the male adult corpora allata. A substance similar in polarity to the above-mentioned unknown has been found in the medium when larval ring glands of Drosophila melanogaster are incubated in vitro. This substance has been chemically identified with EI GC-MS as methyl-6,7; 10,ll -bisepoxyfarnesoate or juvenile hormone III bisepoxide by Richard et al. (1989b) and abbreviated as JHB3. Richard et al. (1989b) suggest that the unknown produced by the corpora allata of Phormia regina may also be JHB,. Recently, it has been discovered that corpora allata of another blow fly, Calliphora vomitoria (Cusson et al., 1991; Duve et al., 1992) as well as the corpus allatum of the sheep blow fly, Luciliu cuprina (Lefevere et al., 1993) also produce JHB,. All these results are suggestive that the unknown from Phormia regina may also be JHB3. This present paper shows that the unknown is indeed JHB3, which represents a major product of the corpora allata in both male and female adult Phormia regina in vitro. Evidence also shows that JH III and methyl farnesoate (MF) are also present in the incubation medium. Bioassay of synthetic JHBj revealed its ability to restore oiigenesis in allatectomized, liver-fed females. We compared the efficacy of these three juvenoids as well as juvenoid blends to restore obgenesis in allatectomized, liver-fed females and found evidence that a juvenoid blend at a certain ratio was more effective than individual juvenoids, a situation that is reminiscent of pheromonal blends. MATERIALS

AND METHODS

Maintaining

flies

Phormia previously

regina was reared described (Stoffolano,

and maintained 1974; Zou et

as al.,

YIN et al.

1988). Mature larvae were allowed to crawl out from the medium into sand to pupariate. Puparia were collected daily and adults were allowed to emerge in screened cages. Flies emerging within 12 h were placed in the same age group (day 1). During the first 3 days after emergence, a 4.3% sucrose solution was provided to all flies. The same sugar solution was also provided to the flies following the bout of liver feeding. All flies were kept at 28 + 2°C under a 16 h light, 8 h dark photoregime.

Radiochemical

biosynthesis

of juvenoids

in vitro

Juvenoid biosynthesis and release were obtained using a protocol known as the radiochemical assay (Zou et al., 1989; Yin et al., 1993). Two corpus cardiacumcorpus allatum (CC-CA) complexes were incubated with shaking (300 rpm) at 27°C for 4 h in 50 ~1 of TC199 medium lacking methionine and supplemented with calcium chloride (5 mM) and Ficoll (20 mg/ml). To this medium was added L[3H]methyl-methionine (NEN, final specific radioactivity 198 mCi/mmol; final concentration 160 PM). At the conclusion of the incubation period, CC-CA complexes were removed before the medium was extracted with isooctane. After the extraction isooctane was evaporated under a gentle stream of air, and the products from 10 CC-CA complexes were redissolved in a known volume of isooctane before they were subjected to TLC or HPLC analysis.

TLC and HPLC

analysis of radioisotope-labeledjuvenoids

TLC was performed on flexible Whatman plates (20 x 20 cm, aluminum backing with a 250 pm layer of silica gel, Whatman 4420 222). Sample spotted plates were developed with standards of JHB3, JH III and MF in a glass tank using a solvent mixture of hexane/ethyl acetate/methanol (75:24:1). The radioactivities on the TLC plates were scanned using an automatic TLC-Linear Analyzer from Berthold (Wildbad, Germany). Under the above conditions, the Rf values were averaged to be 0.29, 0.46 and 0.62 for JHB,, JH III and MF, respectively. Average Rf values for radioisotope-labeled juvenoids coincided with the above standard values. HPLC analysis was performed on a Varian 5000 instrument equipped with a C,* reverse-phase column (4 mm i.d. and 250 mm long, Alltech). Fractions were collected at 15 s intervals using a linear gradient of acetonitrile (4&100%) in water at a 1 ml/min flow rate, over a time span of 35 min. JHBS, JH III and MF were eluted completely separated from each other with the peaks of their U.V. absorbance (214 pm) recorded at 11:30 (11 min, 30 s), 20:45 and 30:00, respectively. The elution times coincided with those of the standards. Radioactivity of the fractions was determined by liquid scintillation spectrometry (Rackbeta 1209).

JHB,.

Synthesis standards

and ident$cation

JH III AND

of JHB,,

JH

METHYL

III and MF

(1) Synthesis of MF. Farnesol (96% 2E, 6E from Aldrich) was oxidized to farnesal at room temperature. A solution of farnesol in hexane was slowly added, over a 60 min period, to a stirred suspension of Mn02 (activated first at 110°C in an oven for 24 h). With continuous stirring, the reaction was allowed to progress for 96 h at room temperature. The reaction mixture was then filtered through a pad of celite. The celite pad was washed three times with hexane. All hexane fractions were pooled and the hexane removed under reduced pressure to give a yellow oil. This product was purified by preparative TLC on Whatman plates (20 x 20 cm, glass backing with a 1000 pm layer of silica gel, Whatman 4861 840) using a solvent mixture of ethyl acetate/hexane (20:80). The identity of this compound was confirmed by both IR and NMR spectra. The t,t-farnesal (8.4 g, 35.5 mmol) in methanol (50 ml) was added to a suspension of MnOz (62.2 g, 715.9 mmol) and NaCN (9.9 g, 202.6 mmol), and glacial acetic acid (4.3 g, 71.9 mmol) in methanol (200 ml) at 0°C. The reaction mixture was stirred at room temperature for 24 h. It was filtered through a pad ofcelite and the celite cake was washed with 15 ml of methanol three times. All fractions were combined and the solution was concentrated under reduced pressure. To this concentrate, 75 ml of distilled Hz0 was added. The aqueous mixture was extracted with 50 ml of hexane three times. The hexane was removed to yield a yellow oil (8.2 g). After purification on preparative TLC using a solvent mixture of ethyl acetate/hexane (5:95), 7.5 g MF was obtained. The identity of MF was confirmed by both IR and NMR spectra. (2) Synthesis of JH III. Eight ml of distilled H,O was added to a solution of t,t-methyl farnesoate (1.7 g, 6.6 mmol) in tetrahydrofuran (24 ml). This aqueous solution was chilled to 0°C in an ice bath and stirred during the portionwise addition of 1.3 g of recrystallized white crystals of N-bromosuccinimide over a period of 30 min. After stirring for a further 3.5 h at the same temperature, the solution was concentrated at 40°C under reduced pressure on a rotary evaporator. After 20 ml of brine was added, the mixture was extracted with 30 ml of diethyl ether three times. The solvent was evaporated to produce 2.4 g of crude bromohydrin, which was purified by preparative TLC with ethyl acetate/hexane (15:85) to yield 1.5 g bromohydrin. To a solution of bromohydrin (1.5 g) in 30 ml of dry methanol was added 3.5 g (4 equivalents) of anhydrous potassium carbonate under nitrogen. The mixture was stirred vigorously for 30 min at room temperature. The solid was filtered off under suction and washed with 5 ml of methanol two times. The combined filtrates were concentrated in uacuo on a rotary evaporator and mixed with 20 ml brine. The mixture was extracted with 30 ml diethyl ether/hexane (1: 1) three times to yield an oily residue. After purification on preparative TLC using a developing solvent of ethyl acetate/hexane (20:80), 1.1 g of JH III was obtained. The

FARNESOATE

IN BLOW

FLIES

47.5

identity of JH III was confirmed by both IR and NMR spectra. (3) Synthesis of JHB,. A total of 690.2 mg of m-chloroperbenzoic acid (Aldrich, 50-60% pure, 2 mmol) was added slowly to a solution of 532.7 mg of JH III in 60 ml of dry benzene. The mixture was stirred at room temperature for 24 h. The reaction was washed three times (20 ml each) with 5% NHX-H20, and the water phase was extracted with 20 ml of benzene three times. The combined organic phase was evaporated under reduced pressure on a rotary evaporator. The residue was purified on preparative TLC to give 3 10 mg of JHBX. The identity of JHB, was confirmed by IR, NMR and GC-MS. GC-MS

analysis of JH III bisepoxide

All GC-MS data were obtained using a Hewlett-Packard model 5989A GC-MS system (Hewlett-Packard, Avondale, PA). The fused silica capillary GC column used [HP5 (Hewlett-Packard), 30 m long, 0.25 mm i.d. and 0.25 p film], was directly coupled to the ion source. A guard column (4 m long, 0.53 mm i.d. deactivated fused silica capillary tubing) was attached to the column’s inlet side. It was then connected to the instrument’s pressure-programmable on-column injector. Through use of an appropriate insert, on-column injections using a 26-gauge needle syringe were made. The GC oven temperature profile was as follows: initial temperature 50°C (held for 1 min), increased to 260°C at 20°C per min, held for 1.5 min. Helium carrier gas velocity was fixed at 24 cm/s by operating the pressure-programmable inlet in the vacuum compensation mode. The injection temperature tracked the oven temperature. Mass spectral data were obtained in the electron impact (EI) (70 eV) and positive chemical ionization (PCI) modes. The CI reagent gas was ammonia. Source pressure was 0.87 torr and temperature was 150°C. Bioassay

of JHBl

Newly emerged flies were allatectomized within 4 h of their adult eclosion using a previously published procedure (Thomsen, 1942). The operated flies were allowed to recover on a sugar solution diet for up to 72 h. Liver was provided at 72 h ad lib to allow feeding to repletion (i.e. until the intersegmental membranes between the abdominal segments were fully expanded). Different doses of JHBJ (dissolved in acetone) were topically applied at 5 and 24 h after the onset of liver-feeding. Ovarian development of all the flies was examined 72 h after the liver meal. Stage of development was recorded according to a published system (Adams and Reinecke, 1979). Control flies were treated with acetone only. Treatments of JH III and MF followed the same protocol. To test the efficacy of juvenoid blends, a rational blend was prepared by mixing JHB3, JH III and MF at a ratio of 66:22:12, which resembled the ratio of juvenoids released into incubation medium by the CA from females 24 h after they had fed on liver. An irrational

CHIH-MING

416 TABLE

1. Juvenoids

in vitro, by the corpus P. regina

biosynthesized,

Juvenoids Age and feeding

status

24 h old, sugar-fed

96 h old, 24 h liver-fed

YIN et al. allatum

biosynthesized

(CA) of both sugarin vitro (fmol/h/pair

and liver-fed

CA)

Sex

JHB,

JH III

MF

Total

M

85.4 f 3.4a

4.4 f 1.5a

12.2 * 2.8a

101.1 f 4.9a

F

(84) 81.2 + 9.0a

(4) 20.1 * 4.9a

(12) 18.5 ) 6.5a

(100) 119.9 + 16.8a

M

(68) 907.2 f 116.0b

(17) 162.0 f 6.9b

(15) 63.0 + 9.8a

(100) 1132.2 + 15O.Ob

F

(80) 449.2 Ifr 61.3~

(14) 122.0 f 8.6b

(6) 58.4 k 21.4a

(100) 630.2 f 49.5~

(66)

(22)

(12)

(100)

M = male; F = female; JHB, = juvenile hormone III bisepoxide; JH III = juvenile hormone III; MF = methyl farnesoate. Juvenoid synthesis was the average of three replicates. For each column of data, means f SD followed by a different letter were in a different range (ANOVA, P > 0.05). Radioactivity was measured on a TLC-Linear Analyzer (Berthold). Numbers in parentheses represent the percentage composition of each juvenoid in relation to the total.

juvenoid blend was arbitrarily made by mixing JHB3, JH III and MF at a ratio of 12:22:66, which was not a nature ratio. Blends were also applied twice to test flies with acetone-treated flies as controls. RESULTS

Biosynthesis of JHB,, JH III and MF by corpora allata of liver-fed flies

Radiochemical assay showed that corpora allata from both sugar- and liver-fed blow flies of both sexes produced JHB3, JH III and MF, in uitro. Table 1 summarizes the results. It is clear from Table 1 that liver-fed flies of both sexes produced at least several times more total juvenoids than sugar-fed flies. Also, regardless of sexes and nutritional status, JHB3 was always the predominant species of juvenoid, while JH III and MF were the second and third most abundant species in liver-fed flies. In sugar-fed flies, the amount of JH III and MF was similar. Corpora allata of liver-fed male produced significantly more JHB3 than the female glands. The meaning of this higher male JHB3 production remains unclear. GC-MS

identljication

of JHB, from

biological

samples

A single symmetrical chromatographic peak was obtained for our synthetic JHB, standard. Tabulated data comparing the EI spectra of the synthetic standard and published data (Richard et al., 1989b) are presented in Table 2. In Table 3 the PC1 data obtained from our synthetic standard and the biosynthesized sample are compared. GC retention times of the two coincided. Bioassay

of JHB3, JH III, MF and juvenoid blenlis

Topical application. of JHB3, JH III and MI? to allatectomized (less than 4 h of adult eclosion) females that were liver-fed 72 h after eclosion revealed that all three juvenoids, when applied alone, could promote the oijgenesis abolished by allatectomy (Table 4). It is clear that two applications of either JHB, or JH III at either 50 or 25 pg per allatectomized fly could significantly enhance

the oogenesis from a 3.4 follicle stage index (FSI, which was calculated according to the description in Table 4) in solvent (acetone) treated controls to a 6.7 FSI or more. The differences recorded for the two juvenoids at two different doses were not significant (ANOVA, P < 0.05). In contrast, MF was clearly not as effective as JHB3 or JH III, although flies treated with MF still contained follicles that were significantly more advanced in their development than those in the acetone controls (ANOVA, P > 0.05). Topical application of blends of JHB3, JH III and MF to allatectomized, then liver-fed, females revealed that the rational juvenoid blend was much more effective TABLE

2. EI mass spectral

data *,t,f.

% Base peak mlz 41 42 43 55 59 69 71 81 83 93 108 111 125 135 147 155 165 181 191 207 233 253 281 282

Synthetic

standard

51.4 10.9 100.0 45.4 26.8 28.5 38.8 23.1 26.2 37.8 32.5 29.2 13.3 2.1 3.9 6.3 1.7 0.6 0.8 0.6 0.5 KD KD 0.3

*Literature values (Richard et al., 1989b). t“KD?’ indicates less than detection limit. $m/z (282) = M+.

Literature

values

46 6.4 100 43 19 27 34 18 22 30 23 24 12 3.4 4.7 5.1 2.2 1.1 1.6 2.2 0.9 1.4 1.4 0.5

JHB,, TABLE

JH III AND

3. Ammonia chemical ionization synthetic and biosynthesized

mass JHB,

METHYL

spectral

data

FARNESOATE of

% Base peak mlz

Synthetic

300 283 265 251 301 *The synthetic analyses.

standard*

Biosynthesized

100.0 54.4 24.1 21.4 18.1 standard

sample

100.0 57.1 25.5 21.4 17.9

data represented

the average obtained

from two

than the irrational blend to promote follicle development (Table 5). It required two applications of 6.25 pg of the rational juvenoid blend to advance follicle development from 3.2 FSI (acetone control) to 6.8 FSI. With two doses of 12.5 pg, FSI was advanced to 8.2. The rational blend, at one-quarter of the doses of JHB3 or JH III, could support equally if not more advanced oiigenesis than either JHB, or JH III alone. A dose of 25 pg (applied two times) of the rational blend caused a high mortality. We do not have an explanation for this unexpected toxicity. In contrast, the effectiveness of the irrational bIend was about the same as the effect of MF alone. DISCUSSION

The discovery of the radioactive unknown secreted by the corpora allata of adult female P. regina under the conditions of the radiochemical assay (Liu, 1985; Liu et al., 1988; Zou et al., 1989; Yin, 1994) turns out to be more important than we first expected. Richard et al. (1989b) discovered a similar substance secreted in vitro

TABLE

4. Restoration

of oljgenesis

in allatectomized, liver-fed flies by topical and MF

Total No. of flies

l-3

50 P& 2 x 25 pg, 2 x

31 29

0 0

50 fig, 2 x 25 Pg, 2 x

25 23

0 Sk7

50 Pg, 2 x 25 pg, 2 x

27 23

29 f 35 f

Acetone,

45

54 f

(2 ~1, 2 x )

FLIES

477

from the corpus allatum portion of the ring glands of third instar larvae of Drosophila melanogaster. This substance is JH III-like and was identified as methyl-6,7; lO,l l-bisepoxy-3,7,1 l-trimethyl-(2E)-dodecenoate or juvenile hormone III bisepoxide (JHB,) using thin layer, liquid and gas chromatographies and mass spectrometry. They also find that JHB, is produced by larval ring glands from Sarcophaga bullata, Musca domestica and Calliphora vicina; and is produced by the corpora allata of adult D. melunogaster in addition to JH III. Physiological studies by Richard et al. (1989a,b, 1990) show that JHB3 has JH activity that interferes with normal pupal-adult development, inhibits ecdysteroid synthesis and stimulates vitellogenin synthesis in D. melunogaster, although about a IO-fold amount of JHB, (in comparison to JH III) is required to achieve similar results. Its biological activity, although much lower than that of JH III in D. melunogaster, still points to its hormonal nature, despite the fact that the JH bisepoxide has been considered a metabolite of JH (Ajami and Riddiford, 1973; Yu and Terriere, 1978). Following the above discovery and identification, JHB, has been identified by others, including Cusson et al. (199 1) in adult female Calliphora vomitoria, Lefevere et al. (1993) in larval and female adult Australian sheep blow fly, Lucilia cuprina and now we report its presence in male and female adults of the black blow fly, P. regina. In C. vomitoria, biosynthesis and release of JHB, is regulated by brain allatostatic factor(s), CAMP may not be the intracellular second messenger for the inhibitory factor in this species, and addition of farnesoic acid (i.e. a presumed precursor) does not stimulate an increase in JHB3 production, but instead results in an increase in JH III production (Duve et al., 1992).

Follicle

Dosage

IN BLOW

of JHBI, JH III

stages

45 6-8 (% of flies in each stage category) JHB, treatment 22 f 3 33 f 9 JH III treatment 20 f 6 26 + 2 MF treatment 4 30 + 8 9 3Ok6 Solvent control 4 36 + 8

application

9-10 FSI

38 f 4 35 _+ 3

42 + 2 32 k 7

7.6 k O.la 7.1 _+ 0.P.b

45 + 9 35 * 9

35 * 10 30 + 7

7.4 f 0.3” 6.7 f 0.3b

18 + 5 17* 7

22 * 3 17& 7

5.4 * 0.2 4.9 + 0.y

10 + 8

0

3.4 f 0.2’

FSI = follicle stage index = summation of average follicle stages times the number of flies, divided by the total numberofflies. The average follicle stages used here are 2 for stages’(l-3), 4.5 for (45), 7 for (68) and9.5 for (9-10). The lowest index& @,the highest is 9.5. The percentageof!Iies ineach grotip of foiticle stages (i.e. l-3,4-5, etc.) was the mean f SD of three replicates. Allatectemy was done within 4 h of adult emergence. Flies were fed liver at 72 h of adulthood. Juvenoids were.dissolved in acetone to the concentration used and applied topically at 5 and 24 h after the onset of the liver meal. Follicle development was examined at 72 h after the liver meal. FSI was given as the mean + SD of three replicates. The FSI was in a different range if followed by a different superscript letter (ANOVA, P > 0.05).

CHIH-MING

478 TABLE

5. Restoration

of oligenesis

YIN et al.

in allatectomized, liver-fed and MF blends Follicle

Total No. of flies

Dosage

l-3

flies by topical application

of JHB,, JH III

stages

68 45 (% of flies in each stage category)

FSI

Biologically

25 Pg, 2 x 12.5 /lg, 2 x 6.25 pg, 2 x

12 30 27

25 pg, 2 x 12.5 peg, 2 x

26 26

Acetone,

28

(2 ~1, 2 x )

rational blend 75% mortality 0 13 *4 15 * 5 19 f 8 Biologically irrational blend 23 + 3 35 f 5 34* 11 38 +6 Solvent control 60+ 10 33 * 5

9-10

26 f 4 26 f 4

61 k6 40 * 4

NA 8.2 * 0.3” 6.8 + 0.2b

22 f 9 19 * 7

19 * 7 7+6

5.4 f 0.3’ 4.4 * 0.2d

cl

3.2 k 0.4’

7+6

FSI = follicle stage index, see Table 4 for its calculation. The percentage offlies in each group of follicle stages (i.e. l-3, 45, etc.) was the mean + SD of three replicates. Allatectomy was done within 4 h of adult emergence. Flies were fed liver at 72 h of adulthood. Rational juvenoid blend contained JHB,, JH III and MF in a ratio of66:22: 12, which closely resembled that ofjuvenoids (7 1:20:9) secreted by the CA of female flies liver-fed 24 h ago. Irrational juvenoid blend contained JHB,, JH III and MF dissolved in acetone at an arbitrary ratio of 12:22:66. The blends were applied topically at 5 and 24 h after the onset of the liver meal. Follicle development was examined at 72 h after the liver meal. FSI was given as the mean k SD of three replicates. FSI was in a different range if followed by a different superscript letter (ANOVA, P > 0.05).

The present study provides the strongest indication so far that JHB, is a hormone rather than a metabolite. Data in Table 4 clearly show that the ability of JHB, to restore oogenesis in allatectomized P. regina is equal, if not better, than JH III. Furthermore, the outstanding biological activity exhibited by the rational juvenoid blend and the lack of it by the irrational juvenoid blend (Table 5) are also important findings. First, the result suggests that glandular production of a hormone in multiple forms at a specific ratio may have a special biological meaning. Second, a reception (signal transduction) system must be co-evolved to perceive multiple forms of the hormone. Depending on the number of elements in the hormone forms and their receptors, a wide array of possible combinations may be obtained to fine-tune the endocrine regulation of many different events. We must point out here that since the hormone blends were applied topically, we do not know the true blend ratio nor the true titer present in the hemolymph. More elaborate chemical analyses are needed to provide that kind of information. Our finding, that a hormone blend may be more effective than any of its ingredients alone, may also have some practical implications. In clinical application, hormonal blends may also prove to be more effective in higher animals. In pest management, blends of juvenile hormone analogs (or other insect growth regulators), or pesticides may lower the overall quantity used in the field. Another important contribution from the discovery and identification of JHB, is to foster revision of the widely held concept that Lepidoptera produce homologous JHs and that non-Lepidoptera produce only JH III, despite warnings for caution that are already in the literature (Schooley and Baker, 1985; Baker, 1990). The occurrence of JHBj in Diptera makes clear how far from reality the above general concept may be. JHB3 represents a new category of juvenoids produced by non-

Lepidoptera and opens the question of the possible existence of JHBo, JHB, and JHB, or other related juvenoids in insects as hormones rather than as metabolites of hormones. The fact that the occurrence of JHBX is not solely restricted to higher Diptera has become obvious more recently. JHB3 production has now been observed in an arachnid, i.e. in adult tick, Dermacentor variabilis by Roe et al. (1993) and in the mosquitoes Aedes aegypti, Culex nigripalpus, Anopheles rangeli and Anopheles trinkae by Borovsky et al. (1994). A very close match between the EI spectra of our synthetic standard and published data (Richard et al., 1989b) was observed (Table 2). This match, in combination with CI, IR, NMR and chromatographic data, supports our conclusion that highly purified JHB, was obtained synthetically. With regard to PC1 measurements, molecular ion adducts representing either (M + l)+, m/z = 283, or (M + 18)+, m/z = 300, were observed. The adduct ion, (M + 18)+, was the base peak of the spectrum. Further, the relative abundance of the (A + 1)’ isotope ion, m/z = 301, was 18.1%. Using the formula of McLafferty and Turecek (1993), the theoretical relative abundance for the JHB3-ammonia adduct is 18.0%, Prominent PC1 fragment ions observed were (M-17)+, m/z = 265 and (M-31)+, m/z = 251. Neutral losses of this type are expected for molecules like JHB3. In sum, the characteristics of PC1 spectra proved diagnostic for JHB,. In the identification of the biosynthesized material, the diagnostic features of the PC1 spectrum proved invaluable. As indicated by the Table 3 data, a nearly exact match was observed in the CI spectra of the biosynthesized and synthetic material. We conclude that these data and the fact that GC retention times coincided confirms the identity of the corpus allatum biosynthesized JHB,. Data collected in the EI mode were not useful in this regard. This was due to the fact that even after HPLC

JHB,,

JH III AND

METHYL

FARNESOATE

purification, the biological sample contained materials which in the GC analysis eluted in the same region as JHBs. Like JHBS, EI spectra of these materials were dominated by a high abundance of low mass ions characteristic of hydrocarbons, unsaturated lipids and related substances. Obviously, biological samples from Drosophila melanogaster do not contain interfering materials because the original identification of JHB, used the EI mode of mass spectrometry (Richard et al., 1989b). In our experience, the stability of JHBj in GC analyses is also problematic. When the synthetic standard was analyzed using splitless injection or on columns which had been partially degraded by uses, numerous peaks were observed in the total ion chromatograms (TIC) whose spectra were similar to that of synthesized JHB,. Lefevere et al. (1993) made the same observation and suggested that the compounds they observed were JHB, thermal rearrangement products. We concur with this suggestion and with their recommendation that JHB, analyses are best performed by GC using cool on-column injection with dedicated columns. REFERENCES Adams T. S. and Reinecke J. P. (1979) The reproductive physiology of the screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae). I. Oiigenesis. J. med. Ent. 15,472483. Ajami A. M. and Riddiford L. M. (1973) Comparative metabolism of the Cecropia juvenile hormone. J. Insect Physiol. 19,635~645. Baker F. C. (1990) Techniques for identification and quantification of juvenile hormones and related compounds in arthropods. In Morphogenetic Hormones of Arthropods, Discovery, Synthesis, Metabolism, Evolution, Modes of Action, and Technique (Ed. Gupta A. P.), pp. 389453. Rutgers University Press, New Brunswick. Borovsky D., Carlson D. A., Hancock R. G., Rembold H. and Van Handel E. (1994) De nova biosynthesis of juvenile hormone III and I by the accessory glands of the male mosquito. Insect Biochem. Molec. Biol. 24,437444. Cusson M., Yagi K. J., Ding Q., Duve H., Thorpe A., McNeil J. N. and Tobe S. S. (1991) Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: the search for a unifying arthropod endocrinology. Insect Biochem. 21, 16. Duve H., Thorpe A., Yagi K. J., Yu C. G. and Tobe S. S. (1992) Factors affecting the biosynthesis and release ofjuvenile hormone bisepoxide in the adult blowfly Calliphora vomitoria. J. Insect Physiol. 38, 575-585. Lefevere K. S., Lacey M. J., Smith P. H. and Roberts B. (1993) Identification and quantification ofjuvenile hormone biosynthesized by larval and adult Australian sheep blowfly Lucilia cuprina (Diptera: Calliphoridae). Insect Biochem. Molec. Biol. 23, 713-720. Liu M.-A. (1985) Development and evaluation of an in vitro radiochemical assay for juvenile hormone biosynthesis in the black blowfly, Phormia regina (Meigen). M. SC. thesis, University of Massachusetts, Amherst, MA. Liu M.-A., Jones G. L., Stoffolano J. G. Jr and Yin C.-M. (1988) Conditions for estimation of corpus allatum activity in the blowfly, Phormia regina, in vitro. Physiol. Ent. 13,69-79. McLafferty F. W. and Turecek F. (1993) Interpretation of Mass Spectra, 4th edn, p. 371. University Science Books, Mill Valley, CA. Richard D. S., Applebaum S. W. and Gilbert L. I. (1989a) Developmental regulation of juvenile hormone biosynthesis by the ring gland of Drosophila melanogaster. J. camp. Physiol. B 159, 383-387. Richard D. S., Applebaum S. W. and Gilbert L. I. (1990) Allatostatic regulation ofjuvenile hormone production in vitro by the ring gland of Drosophila melanogaster. Molec. Cell. Endocr. 68, 153-161.

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Acknowledgements-This work was supported by the National Science Foundation (Grants DCB-9104757 and INB-9306650 to C.-M. Yin and J. G. Stoffolano Jr) and the Massachusetts Agricultural Experiment Station (Grants 632 to J. G. Stoffolano Jr and 660 to C.-M. Yin) and published as Contribution No. 3137.