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Effects of juvenile hormone mimics on larval development and metamorphosis of Drosophila melanogaster
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
82, 172-183 (1991)
Effects of Juvenile Hormone Mimics on Larval Development Metamorphosis of Drosophila melanogaster
and
LYNN M. RIDDIFORD* AND MICHAEL ASHBURNER~ *Department
of Zoology, University of Washington, Genetics, Cambridge University, Downing
Seattle, Street,
Washington Cambridge
98195; and 7Department CB2 3EH, England
of
Accepted June 23, 1990 To determine if prolonged larval exposure to juvenile hormone (JH) could influence the decision to metamorphose, Drosophila melanogaster larvae were reared from hatching on medium containing either of the JH mimics, methoprene or 2-[1-methyl-2-(4-phenoxyphenoxy)-ethoxyl-pyridine (S31183). The latter was 23 times more active as a JH mimic in the white puparial assay (ED,, = 0.22 pmole). Larval development and pupariation were unaffected except at 1000 ppm methoprene and 10 ppm or higher S31183 where larval life was prolonged with increased mortality in the second instar. Adult eclosion was prevented by concentrations greater than 1 ppm methoprene and 0.1 ppm S31183. At low concentrations only adult abdominal development was affected, but at the higher concentrations an increasing percentage was blocked at the pupal stage. This latter effect was considerably diminished when the treatment was begun in the mid second instar. The methoprene-resistant mutations, Met’ and Met’, were 10 and 6 times more resistant to S31183 in the white puparial assay and about 20 times more resistant in the larval feeding experiments than the wild-type, indicating that the effects seen are typical of JH. These studies suggest that excess JH may affect adult development of imaginal structures if present at the onset of postembryonic cell proliferation of the imaginal discs or histoblasts. Thus, commitment for adult differentiation must occur early during this proliferative phase. C 1991 Academic Press, Inc.
Insect larval development is controlled by two hormones, ecdysone, which causes molting, and juvenile hormone (JH), which determines the nature of the molt. The decline of JH during the final larval instar is necessary for metamorphosis to occur. In the Hemimetabola, such as the bloodsucking bug Rhodnius profirus (Wigglesworth, 1934, 1940), secretion of JH by the corpora allata ceases at the end of the penultimate instar. In these insects application of JH immediately after the final larval ecdysis causes supernumerary molting (Nijhout and Wheeler, 1982; Riddiford, 1985; Sehnal, 1985). Similarly, the holometabolous Coleoptera and Lepidoptera display supernumerary molting when given JH during the final larval instar. By contrast, supernumerary larval molting is not observed in the higher Diptera
after JH application to last instar larvae. Instead, treated larvae pupariate and the anterior imaginal discs differentiate normally, but differentiation of the adult abdomen may be completely inhibited (Bryant and Sang, 1968; Srivastava and Gilbert, 1968, 1969; Ashburner, 1970; Bhaskaran. 1972; Madhavan, 1973; Postlethwait, 1974; Sehnal and Zdarek, 1976). A supernumerary larval cuticle however could be induced in both Calliphora and Sarcophaga larvae by injection of a high dose of ecdysterone within 6 hr after the last larval ecdysis (Zdarek and Slama, 1972). In Drosophila melanogaster, imaginal discs become competent to metamorphose during the late second instar as assayed by transplantation into a pupariating host (Schubiger, 1974; Gateff and Schneiderman, 1975; Bownes and Roberts, 1979). 172
0016~6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Unlike the imaginal discs, the abdominal histoblasts, which produce the adult abdominal cuticle, do not begin their postembryonic cell proliferation until just after pupariation (Madhavan and Schneiderman, 1977; Roseland and Schneiderman, 1979). Their adult differentiation can be prevented by JH given at this time (Madhavan, 1973; Postlethwait, 1974). To determine whether excessive JH could influence the decision to metamorphose as is typical of other insects, we have continuously exposed D. melanogaster larvae to two potent JH mimics in their food medium. Thus, they received the JH through both the gut and the cuticle. High concentrations of these mimics delayed, but failed to prevent, pupariation of most larvae. Continuous larval exposure to higher concentrations of these mimics caused an increase in the percentage of pupae that failed to differentiate any adult structures, suggesting that early exposure to high amounts of JH may be able to affect imaginal disc differentiation as well as that of the abdominal histoblasts. MATERIALS
AND METHODS
Fly stocks and culfure. Unless noted otherwise, the wild-type Canton S strain of D. melanogaster from the Department of Genetics, University of Cambridge, was used. The Canton S I and II (isogenic lines) and Oregon R stocks were provided respectively by Drs. Barbara Wakimoto and John Palka, Department of Zoology, University of Washington. The mutant stocks )’ w Met (henceforth referred to as Met’) and Met’ were obtained from Dr. Thomas Wilson, University of Vermont. Met’ and Met* are mutant alleles that confer resistance of D. melanogaster to the effects of exogenous JH (Wilson and Fabian, 1986, 1987). The flies were reared at 25” on a 10% yeast-IO% glucose medium containing 0.3% Nipagin to retard mold growth. Juvenile hormone treatments. The juvenile hormone analogs, methoprene [isopropyl-(2E,4E)-I l-methoxy3,7,Il-trimethyl-2,4-dodecadienoate] (both the mixed isomers ZR515 and the natural 7s isomer) (Zoecon Corp.) and S31183 {2-[l-methyl-2-(4-phenoxyphenoxy)-ethoxyl-pyridine} (Sumitomo Chemical Co.) were weighed and dissolved in cyclohexane (Aldrich)
to give a 10 ug/pl stock solution. This was subsequently diluted with acetone (BDH Chemical AnalaR or Aldrich HPLC grade) for topical application. White puparia were placed ventral side down on double-stick tape on a glass slide, and 0.2 pl acetone containing the desired amount of hormone was applied to each within I5 min of pupariation with a IO-p1 Hamilton syringe on a repeating dispenser. The puparia were then placed in a petri dish with a piece of moist Kimwipe at 25” until the controls had emerged. Then the uneclosed puparia were dissected and scored as described below. For the feeding experiments the JH mimic was dissolved in 95% ethanol, then 100 p,l added to 100 ml molten yeast-glucose medium at 50-55” with vigorous stirring. Ten milliliters of medium was then dispensed into 3” shell vials, allowed to solidify overnight, and either used immediately or stored at 2” for up to 3 days. Larvae from 2- to 4-hr egg collections were washed from the surface of the medium with distilled water, placed on Nitex screening, and washed thoroughly of yeast and debris. Then 100 were transferred with a wet brush to each food vial. This standard number of larvae was used throughout to avoid possible effects of larval density on the effectiveness of the JH mimic (Wilson and Chaykin, 1985). The vials were incubated at 25”, 65% relative humidity, and observed daily. Five to seven days after pupariation the noneclosed puparia were removed from the vials, scored. and stored in 70% ethanol containing 1% glycerol. Classification of developmental anomalies. Examination of uneclosed puparia after treatment with the JH mimics revealed a range of developmental anomalies, depending on the dosage and the time of administration. These were scored from 0 to 6 according to the criteria outlined in Table 1. Representatives of categories 3-5 are depicted in Fig. 1. TABLE SCORING
1
SYSTEM FOR JH EFFECTS ADULT DEVELOPMENT
ON
Score
Characteristics
0 1
Normal adult Partially eclosed adult or male with abnormal genitalia rotation Pharate adult, little or no bristle defects on abdomen Pharate adult, cuticular and bristle anomalies on abdomen Pharate adult head and abdomen, little or no adult abdominal development Phanerocephalic pupa, no adult differentiation (stage PS, Bainbridge and Bownes, 1981) Puparium, no head eversion
2 3 4 5 6
174
RIDDIFORD
AND
RESULTS
Relative Activity of the JH Analogs on Adult Abdominal Differentiation Since Postlethwait (1974) showed that the critical period for abdominal histoblast sensitivity to JH was the first few hours after pupariation, we treated white puparia with the JH mimics so as to compare their efficacy. The number of adults failing to eclose was used as a measure of the effectiveness of the compound. Most adults which eclosed appeared normal although some males had failed to rotate their geni-
ASHBURNER
talia. Noneclosed adults showed a range of anomalies from apparently normal abdomens (score 2, Table 1) to ones with defective or missing bristles or with patches of adult cuticle interspersed with patches of pupal cuticle (Fig. lc and d) (score 3, Table 1) as previously described in detail (Madhavan, 1973; Postlethwait, 1974). Figure 2 shows that in prevention of adult emergence, S31183 was 23 times more effective than 7S-methoprene with an ED,, of 0.22 pmol/puparium. Flies bearing either of two alleles of the methoprene-resistant locus Met (Wilson and Fabian, 1986, 1987), Met’ and Met*, proved to be resistant also to S31183 with ED,,‘s of 2.3 and 1.3 pmol respectively (Fig. 2a). Later experiments at the University of Washington indicated that different strains of Drosophila varied in their sensitivity to S31183 as seen in Fig. 2. Oregon R was most sensitive with an ED,, of 0.066 pmol and CSII was least sensitive with an ED,, of 0.53 pmole, an eightfold difference in sensitivity. Thus, Met’ and Met* are 35 and 20-fold more resistant to S31183 than the parental OreR stock. Effect of Continuous Exposure to JH Mimics on Development
FIG. 1. Examples of blocked adult development caused by dietary JH mimics. (a) Pupa (score 5, Table I); (b) pharate adult with normal head and thoracic development but no abdominal development (score 4); (c) and (d) pharate adult with normal head and thoracic development, but mosaic abdominal development (score 3).
When first instar larvae were placed on a diet containing mixed isomers of methoprene (ZR515), 76 + 15% (N = 9 replicates + SD) pupariated at concentrations of 100 ppm and below. This is not significantly different from the controls fed 0.1% ethanol (82 2 17%; N = 17 replicates). At 1 part per thousand, however, only 58 + 17% (N = 6 replicates) pupariated. Most deaths occurred after 1.5-2 days in the second instar, and the larval developmental time of many of the survivors was increased by l-2 days. No abnormally large larvae were observed. By contrast, concentrations of methoprene above 1 ppm were effective in preventing adult eclosion (Fig. 3). The uneclosed
SH
ANI)
Lhmophiia
‘I.7
D6VELOPMEN7
v OreR * CSCom 3 CST
0 cs A hielf A MeP
pmoles
FIG. 2. Dose-response curves for the prevention of adult eclosion of different strains of Drosophilu melanogaster by S31183 applied to the white puparium. N = 25-50 per dose. OreR, Oregon R; CSCam, Canton S, Cambridge stock; CS I and II, Canton S, isogenic lines of Canton S; Met’ and Met’, methoprene-resistant stocks; X, response of CSCam to 7S-methoprene.
adults showed the typical range of abdominal anomalies as seen in animals treated at pupariation, the severity of which depended on the concentration of methoprene (Fig. 3). Also, as the concentration of dietary methoprene increased, an increasing number of pupae were blocked in stage P5 (Bainbridge and Bownes, 1981). These pupae appeared normal with extended legs and wings, but showed no signs of eye or other pigmentation or adult cuticle or chaetae formation (Fig. la). Although development was blocked, these pupae remained
METHOPRENE
viable through the time of expected adult eclosion as judged by the absence of blackening. The latter normally occurs shortly after death and was seen in the few animals which pupariated but failed to show head eversion. This blockage in the pupal stage was not seen after methoprene treatment of white puparia, indicating that it is not due to a toxic effect of the compound but rather required the early exposure to the JH mimic. Similarly, when larvae were fed S31183 from within 4 hr of hatching, there was little
(ZR515)
9
9
‘OOPpm
6
h1000
ppm
3. Effect of dietary methoprene on subsequent pupal-adult development of the Cambridge Canton S strain. Shown are the percentages of the puparia showing scores O-5 (Table 1) as indicated pictorially above. The bars represent the average percentage of puparia formed (*SD) for the number of replicates indicated above the bars. Numbers in parentheses indicate number of different batches of diet used if greater than one. FIG.
176
RIDDIFORD
AND
effect on the percentage that pupariated except at 1 and 10 ppm where only 62 ? 26% and 65 + 22% (N = 16 and 15 replicates, respectively) pupariated. As with methoprene, death occurred primarily among the late second instar larvae. Examination of some of these dead larvae showed no obvious signs of molting in the majority; an occasional one showed double mouth hooks or third instar spiracles, indicating that it was advanced in the second larval molt. Larval development of the survivors was slower by 1 to 2 days, particularly of those fed 10 ppm S31183. The longer time to pupariation did not result in abnormally large larvae. Continuous exposure to as little as 0.1 ppm S31183 was sufficient to affect adult development so that only 69% of those pupariating eclosed (Fig. 4, top). As with methoprene, exposure to higher concentrations of S31183 blocked development before visible adult differentiation (Fig. la) with 72% showing this effect at 10 ppm (Fig. 4, top). Importantly, when mid second instar larvae were transferred to the same S31183-containing diet, only 22% were ar-
ASHBURNER
rested at this pupal stage (Fig. 4, bottom). In this case, adult development of the head and thorax proceeded normally but abdominal defects were prevalent. After feeding on 10 ppm S3 1183 beginning in the mid second instar, 60% showed no adult abdominal differentiation. Effects of S31183 on Methoprene-Resistant
Mutants
The results described above showed that methoprene and S3 1183 had very similar effects on the development of Drosophila although they differed in their potency. Since these two compounds are very different chemically (Hatakoshi and Nakayama, 1987), it is likely that these effects are due to their common action as JH mimics rather than to a nonspecific toxicity. This conclusion was supported by a study of the effects of dietary S31183 on the development of two JH-resistant strains of Drosophila, Met’ and Met’. As seen in Fig. 5a, based on the average developmental score attained, both mutant strains were about 20 times more resistant than the Canton-S strain, a
S31183
loo %
17(4)
9
3(2)
I(l)
16(41
B(4)
50 0 Ll--
loo
2nds
3cL)
4(2)
% 50
FIG. 4. Effect of dietary S31183 on subsequent pupal-adult development of the Cambridge S strain when it was fed beginning in the first instar (top) or in the mid second instar (bottom). are as in Fig. 3.
Canton Details
JH
AND
Drosophila
177
DEVELOPMENT
5-
4z 8 VJ 3Q 5 a
2-
I,a OlOl
d.l ppm
FIG.
resistant average
5a. Relative effectiveness of dietary strains as compared to the Cambridge score L SD calculated for the number
I
I IO
S31183 S31183 fed to first instar larvae on the methopreneCanton S strain. Scoring was based on Table 1 and the of replicates indicated in Figure 5b. A, Met’; A, Met’
value similar to their increased resistance to this compound in the white puparial assay (Fig. 2). Moreover, larval survival of the mutants was not reduced, even in the instances when wild-type larval survival on the same batch of diet was only lO-20%. Figure 5b shows that these strains were
blocked later in development than the Canton S wild-type. Even at 10 ppm S31183, few of the uneclosed adults showed any obvious abnormality in abdominal cuticular development. Interestingly, melanotic pseudotumors were found in 47 -+ 22% (N = 11 replicates) of the Met’ strain reared
FIG. Sb. Effect of dietary S31183 fed throughout larval methoprene-resistant mutants. The data for the Cambridge the top for comparison. Details are as in Fig. 3.
life on pupal-adult Canton S strain
development of the two from Fig. 3 are shown at
178
RIDDIFORD
AND
on the highest concentration whereas only 4 f 3% (N = 13 replicates) of the Met2 strain and less than 1% of the Canton S wild-type developed such pseudotumors after exposure to S31183 diet. Among various wild-type strains, there was a considerable range of sensitivity to S31183 as assessed by the white puparial assay (Fig. 2). To determine whether this differing sensitivity was the same in larval life, the effect of dietary S31183 was tested on Oregon R, the parent strain of the methoprene-resistant mutants, and the isogenic Canton S I. At pupariation the former is 2.6 times more sensitive to S3 1183 than either of the Canton S strains (Fig. 2). In response to dietary S3 1183, both Oregon R and Canton S I showed the same larval survival to pupariation, but Fig. 6 shows that neither had as severe abdominal defects in the adult as those seen in the Cambridge Canton S strain. All were clearly more sensitive than the methoprene-resistant mutant Met’. Thus, larval sensitivity to dietary JH mimics is not necessarily the same as white puparial sensitivity, possibly due to different metabolic factors. In the Oregon R stock melanotic pseudotumors were observed in 20-30% of the treated larvae given 10 ppm or higher (Fig. 6). There was little difference between the parent stock and the resistant mutant in this effect. Such pseudotumors were seen in less than 10% of the Canton S strains at any dose. A comparison of Fig. 6 with Fig. 4 shows that the effects of dietary S31183 on adult development of the Cambridge Canton S strain were not as severe in the study summarized in Fig. 6. Even at 100 ppm, only 34% were blocked in the pupal stage. The only apparent difference is that the experiments reported in Fig. 4 were done in Cambridge between August 1986 and March 1987 and those in Fig. 6 between February and July 1988 in Seattle. The same and a new batch of hormone were used in the Seattle study with no difference in the results.
ASHBURNER
0 O.IXEtOH
IOppm
5OPpn
100ppm
6. Effects of dietary S31183 on adult development of different strains. This study was done at the University of Washington between February and July, 1988. Details are as in Fig. 3 except that the percentage having melanotic pseudotumors is shown in parentheses. All replicates for a particular concentration were done on one batch of diet except for S31183 at 10 and 100 ppm where two different batches were tested. FIG.
Also, the Cambridge Canton S strain still showed the same dose responsivity in the white puparial assay done in Seattle. Besides differences in the sources of the food ingredients, it is possible that different microbial flora are present in the food in the two locations which may in turn influence the effectiveness of the JH mimics. DISCUSSION
These studies show that continuous exposure of Drosophila to excessive juvenile hormone throughout larval life prevents metamorphosis, not at its onset, but only at the final differentiation of the adult structures. Importantly, to have this effect, the excess JH has to be present before the imaginal discs or the histoblasts resume cell division during larval life or at pupariation,
JH
AND
Drosophila
respectively. These studies thus extend the previous findings of others on the effect of JH on abdominal histoblast differentiation (Bryant and Sang, 1968; Srivastava and Gilbert, 1968, 1969; Ashburner, 1970; Bhaskaran, 1972; Madhavan, 1973; Postlethwait, 1974; Sehnal and Zdarek, 1976) to its effect on adult differentiation of imaginal discs. As in other insects, molting and metamorphosis in Drosophila are regulated by ecdysteroids and juvenile hormone. Ecdysteroid peaks are found in the embryo, prior to each larval ecdysis, at pupariation, head eversion, and at the onset of adult development (Kraminsky et al., 1980; Handler, 1982). Juvenile hormone III is present in the late embryo and in decreasing amounts through larval life with an apparent transient peak just prior to pupariation; JH then is absent until adult eclosion (Sliter et al., 1987; Bownes and Rembold, 1987). Recently, Richard et al. (1989a,b) showed that the ring glands of feeding and wandering third instar larvae and of newly pupariated larvae synthesize primarily methyl 6,7; 10,l 1-bisepoxy-farnesoate. This compound proved about ten times less effective than JH III in the white puparial bioassay (Richard ef al., 1989b). Whether younger larval ring glands make this compound and/ or JH III has not been determined although adult corpora allata make both (Richard et al., 1989b). In the present study, both methoprene and S31183 were effective JH mimics in the white puparial assay with the latter being 23 times more active. In mosquitoes and Lepidoptera S31183 is 15 and 320 times more active, respectively, than methoprene (Hatakoshi and Nakayama, 1987; Hatakoshi et al., 1988) due to its resistance to metabolism and to its ready penetrability through the cuticle (Hatakoshi et al., 1987). The effects of these JH mimics in Drosophila were primarily on the abdomen, as previously described for JH I (Postlethwait, 1974) with rotation of the male genitalia be-
DEVELOPMENT
179
ing the most sensitive to disruption followed by inhibition of eclosion. Whether these latter two effects are on the development of the adult nervous system is not known, but new neurons continue to be born in the ventral nervous system during the first 12 to 18 hr after pupariation (Truman and Bate, 1988). When larvae were fed either methoprene or S31183 throughout larval life, relatively low amounts were sufficient to prevent adult eclosion and to disrupt normal adult abdominal development. By contrast, at least loo-fold higher concentration was necessary to prevent normal adult differentiation of the imaginal discs. This high concentration had no apparent effect on disc growth or their pupal morphogenesis since animals blocked in the pupal stage showed normally proportioned pupal appendages. Importantly, this effect on adult differentiation of the imaginal discs was greatly reduced when larvae were fed the JH mimic starting in the mid second instar. In these latter insects, eclosion was blocked and abdominal differentiation was affected, but adult differentiation of the head and thorax proceeded normally. For instance, 78% of larvae fed 10 ppm S31183 beginning in the second instar formed normal adult heads and thoraxes as contrasted to 28% of those fed the same diet throughout larval life. Thus, exposure to high concentrations of JH mimics during the first and early second instars appears critical for the subsequent blockage of adult differentiation of the imaginal discs. That these effects were due to the JH activity of these mimics and not to a toxic action was clearly demonstrated by the normal development of the flies bearing the methoprene-resistant mutation after being fed concentrations which disrupted development of the wild-type. The product of the Met gene has not yet been characterized, but it appears to be involved in JH reception in the target tissue. Although there are only minor differences in penetration, ex-
180
RIDDIFORD
AND
cretion, tissue sequestration, and metabolism of JH III in the Me? mutants, the mutant fat body cytosol has a lo-fold lower binding affinity for JH III than is seen in wild-type (Shemshedini and Wilson, 1990). Based on characterization of the cytosolic binding of JH III in Met heterozygotes, they suggest that the Met+ locus encodes this cytosolic JH-binding protein. This protein is known to be involved in the cellular response to JH, but whether it is a JH receptor is not yet known (Shemshedini et al., 1990). During larval life of D. melanogaster, the imaginal disc cells divide, whereas the abdominal histoblasts remain quiescent until just after pupariation. Madhavan and Schneiderman (1977) showed that the onset of division in the discs begins in the eye disc about 14 hr into the first instar followed within a few hours by division in the wing disc; the antenna, leg, and genital discs begin dividing in the newly molted second instar larva. In the second instar the imaginal discs become competent to metamorphose when transplanted into a metamorphosing host. The eye and the wing discs first show this ability followed by the leg disc (Schubiger, 1974; Gateff and Schneiderman, 1975; Bownes and Roberts, 1979). Importantly, this competence to metamorphose is gained gradually as the disc continues to divide. First, the disc is able to make a thin untanned (possibly pupal) cuticle; then, as it grows larger, it is able to make tanned cuticle and adult-specific structures such as micro- and macrochaetes and eye pigments. Competence to form a completely normal adult structure is not attained until sometime in the third instar after growth is complete (Bryant and Simpson, 1984). The ability of the discs to continue to proliferate during larval life after they first attain competence to metamorphose is probably dependent on the presence of JH, since JH prevents the normal metamorphic response of mature larval discs to 20hydroxyecdysone in vitro (Doctor and Fris-
ASHBURNER
trom, 1985). Yet high exogenous JH in vivo during the third instar has no effect on the metamorphosis of the discs, whereas it can prevent normal adult differentiation of the abdominal histoblasts if given before the onset of their cell division after pupariation (Madhavan, 1973; Postlethwait, 1974). This study shows that high concentrations of dietary JH mimics can affect adult differentiation of imaginal disc-derived structures when fed beginning in the first instar, but not when beginning in the late second instar. Thus, for both the histoblasts and the imaginal discs the abnormally high JH appears to be affecting some process that occurs during the early cell divisions and leads to the acquisition of competence to metamorphose. During these early rounds of proliferative cell division, the imaginal cells must become “committed” to the much later program of expression, and excessive JH blocks this cellular commitment step. This is similar to the role of JH in the prevention of metamorphosis in other insects (Riddiford, 1985) except that in these higher Diptera, commitment to adult differentiation for the proliferating discs appears to be made much earlier in larval life. An alternative explanation which cannot at present be ruled out is that the early exposure to excessive exogenous JH somehow interferes with the normal functioning of the endocrine system after the initiation of metamorphosis (i.e., pupariation). Thus, the animals blocked at the pupal stage could be deficient in ecdysteroid necessary for adult development. This seems unlikely since although these early larvae have been exposed to a higher cumulative dose of the JH mimic, the endocrine system appears to be working normally since these animals form both a normal puparium and a normal pupae. Melanotic pseudotumors occur in some strains of Drosophila, appearing usually in the third larval instar as black masses in the hemocoel (Sparrow, 1978). These pseudotumors are formed by encapsulation of
hemocytes by lamellocytes followed by melanization. Normally lamellocytes, which differentiate from plasmatocytes, are not present until after pupariation. In tumorigenic strains the plasmatocyte-lamellocyte differentiation occurs precociously in the late second or early third larval instar. Juvenile hormone seems to promote this precocious differentiation as various JH analogs either in the diet or topically applied have increased pseudotumor formation in both tumorigenic and nontumorigenic strains (Bryant and Sang, 1969; Madhavan 1972; Wilson and Fabian, 1986; this study). Moreover, Wilson and Fabian (1986) found that methoprene was less effective in promoting tumorigenesis in the methoprene-resistant mutant Met as compared to Met + , again implicating JH in their induction. The role of excess JH in promoting precocious lamellocyte differentiation and thus pseudotumor production is unknown. The hemopoietic lymph gland normally undergoes mitosis throughout larval life until the mid third instar, then begins releasing cells at pupariation and degenerates 12 hr later (Madhavan and Schneiderman, 1977; Srdic and Gloor, 1978). This release of cells seems to be correlated in time with the plasmatocyte-lamellocyte transition (Rizki, 1962). Possibly, prolonged exposure to high JH interferes with the normal developmental progression of the lymph gland itself. These studies have indicated an important role for JH in guiding the postembryonic proliferative cell divisions of imaginal discs and histoblasts and suggest that a carefully regulated JH titer during larval life is necessary both for proliferation and for the commitment to later adult differentiation. The nature of this role remains a challenge for further study. ACKNOWLEDGMENTS This study was performed primarily while L.M.R. was a Visiting Scholar at Cambridge University under
‘h: ausp~c:h :t a !ienior International Fellowship fro is i he Fogarty Cznter of the National Institutes of Hea 1t! I I FOh TWO! X08). The work done later at the Uni\pcr ,ilty of Wabhrnyton was supported by NSF Grail DCBXS-10875 We thank Dr. M. Hatakoshi for t’c S31IP3. Dr. <;erardus Staal for the methoprene. Lb ‘Thomas Wilson for the y w Met’ and Md stocks, ClBarbara Wakimoto for the Canton S I and II isogen$c lines, Dr. .iohn Palka for the Oregon R stock, and D,James Truman for stimulating discussions and critlc:I comments on the manuscript.
REFERENCES Ashburner, M. (1970). Effects ofjuvenile hormone on adult differentiation of Drosophila melanogaster. Nuture 227, 187-189. Bainbridge, S. P., and Bownes, M. (1981). Staging the metamorphosis of Drosophilu melanogaster. .I. Embryoi.
Exp.
Morphol.
66, 57-80.
Bhaskaran, G. (1972). Inhibition of imaginal differentiation in Sarcophaga bullata by juvenile hormone. J. Exp. 2001. 182, 127-142. Bownes. M., and Rembold, H. (1987). The titre of juvenile hormone during the pupal and adult stages of the life cycle of Drosophila melanogaster. Eur. J. Biochem. 164, 709-712. Bownes, M.. and Roberts, S. (1979). Acquisition of differentiative capacity in imaginat wing discs of Drosophilu melanogaster. phol. 49, 103-113.
J. Embryoi.
Exp.
Mor-
Bryant, P. J.. and Sang, J. H. (1968). Drosophila: Lethal derangements of metamorphosis and modifications of gene expression caused by juvenile hormone mimics. Nature 220, 393-394. Bryant, P. J., and Sang, J. H. (1969). Physiological genetics of melanotic tumors in Drosophila me/anogaster. VI. The tumorigenic effects of juvenile hormone-like substances. Genetics 62, 321-336. Bryant, P. J., and Simpson, P. (1984). Intrinsic and extrinsic control of growth in developing organs. Q. Rev.
Biol.
59, 387-415.
Doctor, J. S.. and Fristrom, J. W. (1985). Macromolecular changes in imaginal discs during postembryonic development. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 2, pp. 201-238. Pergamon Press, Oxford. Gateff, E. A., and Schneiderman, H. A. (1975). Developmental capacities of immature eye-antenna] imaginal discs of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 176, 171-189. Handler, A. M. (1982). Ecdysteroid titers during pupal and adult development in Drosophila melanoRaster.
Dev.
Biol.
93, 73-82.
182
RIDDIFORD
AND
Hatakoshi, M., and Nakayama, I. (1987). Juvenile hormone active compounds: Recent researches. Shokubutsu
Boeki
41, 339-347.
Hatakoshi, M., Nakayama, I., and Riddiford, L. M. (1987). Penetration and stability of juvenile hormone analogues in Munduca sexta L. (Lepidoptera: Sphingidae). Appl. Entomol. Zool. 22, 641-644. Hatakoshi, M., Nakayama, I., and Riddiford, L. M. (1988). The induction of an imperfect supemumerary larval moult by juvenile hormone analogues in Manduca
sexta.
J. Insect
Physiol.
34, 373-378.
Kraminsky, G. P., Clark, W. C., Estelle, M. A., Gietz, R. D., Sage, B. A., O’Connor, J. D., and Hodgetts, R. B. (1980). Induction of translatable mRNA for dopa decarboxylase in Drosophila: An early response to ecdysterone. Proc. Natl. Acad. Sci.
USA
17, 41754179.
Madhavan, K. (1972). Induction of melanotic pseudotumors in Drosophila melanogaster by juvenile hormone. Wilhelm Roux’s Archiv. Dev. Biol. 169, 345-349. Madhavan, K. (1973). Morphogenetic effects of juvenile hormone and juvenile hormone mimics on adult development of Drosophila. J. Insect Physiol. 19, 441-453. Madhavan, M. M., and Schneiderman, H. A. (1977). Histological analysis of dynamics of growth of imaginal discs and histoblast nests during the larval development of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 183, 269-305.
ASHBURNER
and L. I. Gilbert, Eds.), Vol. 8, pp. 37-84. Pergamon Press, Oxford. Rizki, T. M. (1962). Experimental analysis of hemocyte morphology in insects. Am. Zool. 2,247-256. Roseland, C. R., and Schneiderman, H. A. (1979). Regulation and metamorphosis of the abdominal histoblasts of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 186, 235-265. Schubiger, G. (1974). Acquisition of differentiative competence in the imaginal leg discs of Drosophila. Wilhelm Roux’s Archiv. Dev. Biol. 174,303311. Sehnal, F. (1985). Growth and life cycles. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 2, pp. l-86. Pergamon Press, Oxford. Sehnal, F., and Zdarek, J. (1976). Action of juvenoids on the metamorphosis of cyclorrhaphous Diptera. J. Znsect
Physiol.
22, 673-682.
Shemshedini, L., Lanoue, M., and Wilson, T. G. (1990). Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster. J. Biol. Chem. 265, 1913-1918. Shemshedini, L., and Wilson, T. G. (1990). Resistance to juvenile hormone and an insect growth regulator in Drosophila is associated with an altered cytosolic juvenile hormone-binding protein. Proc. Natl.
Acad.
Sci.
USA
87, 2072-2076.
383-387. Richard, D. S., Applebaum, S. W., Sliter, T. J., Baker, F. C., Schooley, D. A., Reuter, C. C., Henrich, V. C., and Gilbert, L. I. (1989b). Juvenile hormone bisepoxide biosynthesis in vitro by the ring gland of Drosophila melanogaster: A putative juvenile hormone in the higher Diptera. Proc. Natl. Acad. Sci. USA 86, 1421-1425.
Sliter, T. J., Sedlak, B. J., Baker, F. C., and Schooley, D. A. (1987). Juvenile hormone in Drosophila melanogaster: Identification and titer determination during development. Znsect Biothem. 17, 161-165. Sparrow, J. C. (1978). Melanotic “tumours”. In “The Genetics and Biology of Drosophila” (M. Ashburner and T. R. F. Wright, Eds.), Vol. 2b, pp. 277-3 13. Academic Press, London. Srdic, Z., and Gloor, H. (1978). Le role hematopoietique des glandes lymphatique de Drosophila hydei. Rev. Suisse Zool. 85, 11-19. Srivastava, U. S., and Gilbert, L. I. (1968). Juvenile hormone: Effects on a higher dipteran. Science 101, 61-62. Srivastava, U. S., and Gilbert, L. I. (1969). The influence of juvenile hormone on the metamorphosis of Sarcophaga bullata. J. Znsect Physiol. 15, 177190. Truman, J. W., and Bate, M. (1988). Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster. Dev.
Riddiford, L. M. (1985). Hormone action at the cellular level. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut
Wigglesworth, V. B. (1934). The physiology of ecdysis in Rhodnius prolixus. II. Factors controlling
Nijhout, H. F., and Wheeler, D. E. (1982). Juvenile hormone and the physiological basis of insect polymorphism. Q. Rev. Biol. 57, 109-133. Postlethwait, J. H. (1974). Juvenile hormone and the adult development of Drosophila. Biol. Bull. 147, 119135. Richard, D. S., Applebaum, S. W., and Gilbert, L. I. (1989a). Developmental regulation ofjuvenile hormone biosynthesis by the ring gland of Drosophila
melanogaster.
J.
Comp.
Physiol.
159,
Biol.
125, 145-157.
JH
AND
Drosophila
moulting and metamorphosis. J. Microsc. Sci. 77, 191-222. Wigglesworth, V. B. (1940). The determination of characters at metamorphosis in Rhodnius prolixus (Hemiptera). J. Exp. Biol. 17, 201-222. Wilson, T. G., and Chaykin, D. (1985). Toxicity of methoprene to Drosophila mdanogaster (Diptera: Drosophilidae): A function of larval culture density. J. Econ. Entomol. 78, 1208-1211. Wilson, T. G., and Fabian, J. (1986). A Drosophila
DEVELOPMENT
183
melanogaster mutant resistant to a chemical analog of juvenile hormone. Dev. Biol. 118, 190-201, Wilson, T. G., and Fabian, J. (1987). Selection of methoprene-resistant mutants of Drosophila melanogaster. In “Molecular Entomology” (J. H. Law, Ed.), Vol. 49. pp. 179-188. A. R. Liss, New York. Zdarek, J., and Slama, K. (1972). Supernumerary larval instars in cyclorrhaphous Diptera. Bid. Bull. 142, 350-357.
AND
COMPARATIVE
ENDOCRINOLOGY
82, 172-183 (1991)
Effects of Juvenile Hormone Mimics on Larval Development Metamorphosis of Drosophila melanogaster
and
LYNN M. RIDDIFORD* AND MICHAEL ASHBURNER~ *Department
of Zoology, University of Washington, Genetics, Cambridge University, Downing
Seattle, Street,
Washington Cambridge
98195; and 7Department CB2 3EH, England
of
Accepted June 23, 1990 To determine if prolonged larval exposure to juvenile hormone (JH) could influence the decision to metamorphose, Drosophila melanogaster larvae were reared from hatching on medium containing either of the JH mimics, methoprene or 2-[1-methyl-2-(4-phenoxyphenoxy)-ethoxyl-pyridine (S31183). The latter was 23 times more active as a JH mimic in the white puparial assay (ED,, = 0.22 pmole). Larval development and pupariation were unaffected except at 1000 ppm methoprene and 10 ppm or higher S31183 where larval life was prolonged with increased mortality in the second instar. Adult eclosion was prevented by concentrations greater than 1 ppm methoprene and 0.1 ppm S31183. At low concentrations only adult abdominal development was affected, but at the higher concentrations an increasing percentage was blocked at the pupal stage. This latter effect was considerably diminished when the treatment was begun in the mid second instar. The methoprene-resistant mutations, Met’ and Met’, were 10 and 6 times more resistant to S31183 in the white puparial assay and about 20 times more resistant in the larval feeding experiments than the wild-type, indicating that the effects seen are typical of JH. These studies suggest that excess JH may affect adult development of imaginal structures if present at the onset of postembryonic cell proliferation of the imaginal discs or histoblasts. Thus, commitment for adult differentiation must occur early during this proliferative phase. C 1991 Academic Press, Inc.
Insect larval development is controlled by two hormones, ecdysone, which causes molting, and juvenile hormone (JH), which determines the nature of the molt. The decline of JH during the final larval instar is necessary for metamorphosis to occur. In the Hemimetabola, such as the bloodsucking bug Rhodnius profirus (Wigglesworth, 1934, 1940), secretion of JH by the corpora allata ceases at the end of the penultimate instar. In these insects application of JH immediately after the final larval ecdysis causes supernumerary molting (Nijhout and Wheeler, 1982; Riddiford, 1985; Sehnal, 1985). Similarly, the holometabolous Coleoptera and Lepidoptera display supernumerary molting when given JH during the final larval instar. By contrast, supernumerary larval molting is not observed in the higher Diptera
after JH application to last instar larvae. Instead, treated larvae pupariate and the anterior imaginal discs differentiate normally, but differentiation of the adult abdomen may be completely inhibited (Bryant and Sang, 1968; Srivastava and Gilbert, 1968, 1969; Ashburner, 1970; Bhaskaran. 1972; Madhavan, 1973; Postlethwait, 1974; Sehnal and Zdarek, 1976). A supernumerary larval cuticle however could be induced in both Calliphora and Sarcophaga larvae by injection of a high dose of ecdysterone within 6 hr after the last larval ecdysis (Zdarek and Slama, 1972). In Drosophila melanogaster, imaginal discs become competent to metamorphose during the late second instar as assayed by transplantation into a pupariating host (Schubiger, 1974; Gateff and Schneiderman, 1975; Bownes and Roberts, 1979). 172
0016~6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Unlike the imaginal discs, the abdominal histoblasts, which produce the adult abdominal cuticle, do not begin their postembryonic cell proliferation until just after pupariation (Madhavan and Schneiderman, 1977; Roseland and Schneiderman, 1979). Their adult differentiation can be prevented by JH given at this time (Madhavan, 1973; Postlethwait, 1974). To determine whether excessive JH could influence the decision to metamorphose as is typical of other insects, we have continuously exposed D. melanogaster larvae to two potent JH mimics in their food medium. Thus, they received the JH through both the gut and the cuticle. High concentrations of these mimics delayed, but failed to prevent, pupariation of most larvae. Continuous larval exposure to higher concentrations of these mimics caused an increase in the percentage of pupae that failed to differentiate any adult structures, suggesting that early exposure to high amounts of JH may be able to affect imaginal disc differentiation as well as that of the abdominal histoblasts. MATERIALS
AND METHODS
Fly stocks and culfure. Unless noted otherwise, the wild-type Canton S strain of D. melanogaster from the Department of Genetics, University of Cambridge, was used. The Canton S I and II (isogenic lines) and Oregon R stocks were provided respectively by Drs. Barbara Wakimoto and John Palka, Department of Zoology, University of Washington. The mutant stocks )’ w Met (henceforth referred to as Met’) and Met’ were obtained from Dr. Thomas Wilson, University of Vermont. Met’ and Met* are mutant alleles that confer resistance of D. melanogaster to the effects of exogenous JH (Wilson and Fabian, 1986, 1987). The flies were reared at 25” on a 10% yeast-IO% glucose medium containing 0.3% Nipagin to retard mold growth. Juvenile hormone treatments. The juvenile hormone analogs, methoprene [isopropyl-(2E,4E)-I l-methoxy3,7,Il-trimethyl-2,4-dodecadienoate] (both the mixed isomers ZR515 and the natural 7s isomer) (Zoecon Corp.) and S31183 {2-[l-methyl-2-(4-phenoxyphenoxy)-ethoxyl-pyridine} (Sumitomo Chemical Co.) were weighed and dissolved in cyclohexane (Aldrich)
to give a 10 ug/pl stock solution. This was subsequently diluted with acetone (BDH Chemical AnalaR or Aldrich HPLC grade) for topical application. White puparia were placed ventral side down on double-stick tape on a glass slide, and 0.2 pl acetone containing the desired amount of hormone was applied to each within I5 min of pupariation with a IO-p1 Hamilton syringe on a repeating dispenser. The puparia were then placed in a petri dish with a piece of moist Kimwipe at 25” until the controls had emerged. Then the uneclosed puparia were dissected and scored as described below. For the feeding experiments the JH mimic was dissolved in 95% ethanol, then 100 p,l added to 100 ml molten yeast-glucose medium at 50-55” with vigorous stirring. Ten milliliters of medium was then dispensed into 3” shell vials, allowed to solidify overnight, and either used immediately or stored at 2” for up to 3 days. Larvae from 2- to 4-hr egg collections were washed from the surface of the medium with distilled water, placed on Nitex screening, and washed thoroughly of yeast and debris. Then 100 were transferred with a wet brush to each food vial. This standard number of larvae was used throughout to avoid possible effects of larval density on the effectiveness of the JH mimic (Wilson and Chaykin, 1985). The vials were incubated at 25”, 65% relative humidity, and observed daily. Five to seven days after pupariation the noneclosed puparia were removed from the vials, scored. and stored in 70% ethanol containing 1% glycerol. Classification of developmental anomalies. Examination of uneclosed puparia after treatment with the JH mimics revealed a range of developmental anomalies, depending on the dosage and the time of administration. These were scored from 0 to 6 according to the criteria outlined in Table 1. Representatives of categories 3-5 are depicted in Fig. 1. TABLE SCORING
1
SYSTEM FOR JH EFFECTS ADULT DEVELOPMENT
ON
Score
Characteristics
0 1
Normal adult Partially eclosed adult or male with abnormal genitalia rotation Pharate adult, little or no bristle defects on abdomen Pharate adult, cuticular and bristle anomalies on abdomen Pharate adult head and abdomen, little or no adult abdominal development Phanerocephalic pupa, no adult differentiation (stage PS, Bainbridge and Bownes, 1981) Puparium, no head eversion
2 3 4 5 6
174
RIDDIFORD
AND
RESULTS
Relative Activity of the JH Analogs on Adult Abdominal Differentiation Since Postlethwait (1974) showed that the critical period for abdominal histoblast sensitivity to JH was the first few hours after pupariation, we treated white puparia with the JH mimics so as to compare their efficacy. The number of adults failing to eclose was used as a measure of the effectiveness of the compound. Most adults which eclosed appeared normal although some males had failed to rotate their geni-
ASHBURNER
talia. Noneclosed adults showed a range of anomalies from apparently normal abdomens (score 2, Table 1) to ones with defective or missing bristles or with patches of adult cuticle interspersed with patches of pupal cuticle (Fig. lc and d) (score 3, Table 1) as previously described in detail (Madhavan, 1973; Postlethwait, 1974). Figure 2 shows that in prevention of adult emergence, S31183 was 23 times more effective than 7S-methoprene with an ED,, of 0.22 pmol/puparium. Flies bearing either of two alleles of the methoprene-resistant locus Met (Wilson and Fabian, 1986, 1987), Met’ and Met*, proved to be resistant also to S31183 with ED,,‘s of 2.3 and 1.3 pmol respectively (Fig. 2a). Later experiments at the University of Washington indicated that different strains of Drosophila varied in their sensitivity to S31183 as seen in Fig. 2. Oregon R was most sensitive with an ED,, of 0.066 pmol and CSII was least sensitive with an ED,, of 0.53 pmole, an eightfold difference in sensitivity. Thus, Met’ and Met* are 35 and 20-fold more resistant to S31183 than the parental OreR stock. Effect of Continuous Exposure to JH Mimics on Development
FIG. 1. Examples of blocked adult development caused by dietary JH mimics. (a) Pupa (score 5, Table I); (b) pharate adult with normal head and thoracic development but no abdominal development (score 4); (c) and (d) pharate adult with normal head and thoracic development, but mosaic abdominal development (score 3).
When first instar larvae were placed on a diet containing mixed isomers of methoprene (ZR515), 76 + 15% (N = 9 replicates + SD) pupariated at concentrations of 100 ppm and below. This is not significantly different from the controls fed 0.1% ethanol (82 2 17%; N = 17 replicates). At 1 part per thousand, however, only 58 + 17% (N = 6 replicates) pupariated. Most deaths occurred after 1.5-2 days in the second instar, and the larval developmental time of many of the survivors was increased by l-2 days. No abnormally large larvae were observed. By contrast, concentrations of methoprene above 1 ppm were effective in preventing adult eclosion (Fig. 3). The uneclosed
SH
ANI)
Lhmophiia
‘I.7
D6VELOPMEN7
v OreR * CSCom 3 CST
0 cs A hielf A MeP
pmoles
FIG. 2. Dose-response curves for the prevention of adult eclosion of different strains of Drosophilu melanogaster by S31183 applied to the white puparium. N = 25-50 per dose. OreR, Oregon R; CSCam, Canton S, Cambridge stock; CS I and II, Canton S, isogenic lines of Canton S; Met’ and Met’, methoprene-resistant stocks; X, response of CSCam to 7S-methoprene.
adults showed the typical range of abdominal anomalies as seen in animals treated at pupariation, the severity of which depended on the concentration of methoprene (Fig. 3). Also, as the concentration of dietary methoprene increased, an increasing number of pupae were blocked in stage P5 (Bainbridge and Bownes, 1981). These pupae appeared normal with extended legs and wings, but showed no signs of eye or other pigmentation or adult cuticle or chaetae formation (Fig. la). Although development was blocked, these pupae remained
METHOPRENE
viable through the time of expected adult eclosion as judged by the absence of blackening. The latter normally occurs shortly after death and was seen in the few animals which pupariated but failed to show head eversion. This blockage in the pupal stage was not seen after methoprene treatment of white puparia, indicating that it is not due to a toxic effect of the compound but rather required the early exposure to the JH mimic. Similarly, when larvae were fed S31183 from within 4 hr of hatching, there was little
(ZR515)
9
9
‘OOPpm
6
h1000
ppm
3. Effect of dietary methoprene on subsequent pupal-adult development of the Cambridge Canton S strain. Shown are the percentages of the puparia showing scores O-5 (Table 1) as indicated pictorially above. The bars represent the average percentage of puparia formed (*SD) for the number of replicates indicated above the bars. Numbers in parentheses indicate number of different batches of diet used if greater than one. FIG.
176
RIDDIFORD
AND
effect on the percentage that pupariated except at 1 and 10 ppm where only 62 ? 26% and 65 + 22% (N = 16 and 15 replicates, respectively) pupariated. As with methoprene, death occurred primarily among the late second instar larvae. Examination of some of these dead larvae showed no obvious signs of molting in the majority; an occasional one showed double mouth hooks or third instar spiracles, indicating that it was advanced in the second larval molt. Larval development of the survivors was slower by 1 to 2 days, particularly of those fed 10 ppm S31183. The longer time to pupariation did not result in abnormally large larvae. Continuous exposure to as little as 0.1 ppm S31183 was sufficient to affect adult development so that only 69% of those pupariating eclosed (Fig. 4, top). As with methoprene, exposure to higher concentrations of S31183 blocked development before visible adult differentiation (Fig. la) with 72% showing this effect at 10 ppm (Fig. 4, top). Importantly, when mid second instar larvae were transferred to the same S31183-containing diet, only 22% were ar-
ASHBURNER
rested at this pupal stage (Fig. 4, bottom). In this case, adult development of the head and thorax proceeded normally but abdominal defects were prevalent. After feeding on 10 ppm S3 1183 beginning in the mid second instar, 60% showed no adult abdominal differentiation. Effects of S31183 on Methoprene-Resistant
Mutants
The results described above showed that methoprene and S3 1183 had very similar effects on the development of Drosophila although they differed in their potency. Since these two compounds are very different chemically (Hatakoshi and Nakayama, 1987), it is likely that these effects are due to their common action as JH mimics rather than to a nonspecific toxicity. This conclusion was supported by a study of the effects of dietary S31183 on the development of two JH-resistant strains of Drosophila, Met’ and Met’. As seen in Fig. 5a, based on the average developmental score attained, both mutant strains were about 20 times more resistant than the Canton-S strain, a
S31183
loo %
17(4)
9
3(2)
I(l)
16(41
B(4)
50 0 Ll--
loo
2nds
3cL)
4(2)
% 50
FIG. 4. Effect of dietary S31183 on subsequent pupal-adult development of the Cambridge S strain when it was fed beginning in the first instar (top) or in the mid second instar (bottom). are as in Fig. 3.
Canton Details
JH
AND
Drosophila
177
DEVELOPMENT
5-
4z 8 VJ 3Q 5 a
2-
I,a OlOl
d.l ppm
FIG.
resistant average
5a. Relative effectiveness of dietary strains as compared to the Cambridge score L SD calculated for the number
I
I IO
S31183 S31183 fed to first instar larvae on the methopreneCanton S strain. Scoring was based on Table 1 and the of replicates indicated in Figure 5b. A, Met’; A, Met’
value similar to their increased resistance to this compound in the white puparial assay (Fig. 2). Moreover, larval survival of the mutants was not reduced, even in the instances when wild-type larval survival on the same batch of diet was only lO-20%. Figure 5b shows that these strains were
blocked later in development than the Canton S wild-type. Even at 10 ppm S31183, few of the uneclosed adults showed any obvious abnormality in abdominal cuticular development. Interestingly, melanotic pseudotumors were found in 47 -+ 22% (N = 11 replicates) of the Met’ strain reared
FIG. Sb. Effect of dietary S31183 fed throughout larval methoprene-resistant mutants. The data for the Cambridge the top for comparison. Details are as in Fig. 3.
life on pupal-adult Canton S strain
development of the two from Fig. 3 are shown at
178
RIDDIFORD
AND
on the highest concentration whereas only 4 f 3% (N = 13 replicates) of the Met2 strain and less than 1% of the Canton S wild-type developed such pseudotumors after exposure to S31183 diet. Among various wild-type strains, there was a considerable range of sensitivity to S31183 as assessed by the white puparial assay (Fig. 2). To determine whether this differing sensitivity was the same in larval life, the effect of dietary S31183 was tested on Oregon R, the parent strain of the methoprene-resistant mutants, and the isogenic Canton S I. At pupariation the former is 2.6 times more sensitive to S3 1183 than either of the Canton S strains (Fig. 2). In response to dietary S3 1183, both Oregon R and Canton S I showed the same larval survival to pupariation, but Fig. 6 shows that neither had as severe abdominal defects in the adult as those seen in the Cambridge Canton S strain. All were clearly more sensitive than the methoprene-resistant mutant Met’. Thus, larval sensitivity to dietary JH mimics is not necessarily the same as white puparial sensitivity, possibly due to different metabolic factors. In the Oregon R stock melanotic pseudotumors were observed in 20-30% of the treated larvae given 10 ppm or higher (Fig. 6). There was little difference between the parent stock and the resistant mutant in this effect. Such pseudotumors were seen in less than 10% of the Canton S strains at any dose. A comparison of Fig. 6 with Fig. 4 shows that the effects of dietary S31183 on adult development of the Cambridge Canton S strain were not as severe in the study summarized in Fig. 6. Even at 100 ppm, only 34% were blocked in the pupal stage. The only apparent difference is that the experiments reported in Fig. 4 were done in Cambridge between August 1986 and March 1987 and those in Fig. 6 between February and July 1988 in Seattle. The same and a new batch of hormone were used in the Seattle study with no difference in the results.
ASHBURNER
0 O.IXEtOH
IOppm
5OPpn
100ppm
6. Effects of dietary S31183 on adult development of different strains. This study was done at the University of Washington between February and July, 1988. Details are as in Fig. 3 except that the percentage having melanotic pseudotumors is shown in parentheses. All replicates for a particular concentration were done on one batch of diet except for S31183 at 10 and 100 ppm where two different batches were tested. FIG.
Also, the Cambridge Canton S strain still showed the same dose responsivity in the white puparial assay done in Seattle. Besides differences in the sources of the food ingredients, it is possible that different microbial flora are present in the food in the two locations which may in turn influence the effectiveness of the JH mimics. DISCUSSION
These studies show that continuous exposure of Drosophila to excessive juvenile hormone throughout larval life prevents metamorphosis, not at its onset, but only at the final differentiation of the adult structures. Importantly, to have this effect, the excess JH has to be present before the imaginal discs or the histoblasts resume cell division during larval life or at pupariation,
JH
AND
Drosophila
respectively. These studies thus extend the previous findings of others on the effect of JH on abdominal histoblast differentiation (Bryant and Sang, 1968; Srivastava and Gilbert, 1968, 1969; Ashburner, 1970; Bhaskaran, 1972; Madhavan, 1973; Postlethwait, 1974; Sehnal and Zdarek, 1976) to its effect on adult differentiation of imaginal discs. As in other insects, molting and metamorphosis in Drosophila are regulated by ecdysteroids and juvenile hormone. Ecdysteroid peaks are found in the embryo, prior to each larval ecdysis, at pupariation, head eversion, and at the onset of adult development (Kraminsky et al., 1980; Handler, 1982). Juvenile hormone III is present in the late embryo and in decreasing amounts through larval life with an apparent transient peak just prior to pupariation; JH then is absent until adult eclosion (Sliter et al., 1987; Bownes and Rembold, 1987). Recently, Richard et al. (1989a,b) showed that the ring glands of feeding and wandering third instar larvae and of newly pupariated larvae synthesize primarily methyl 6,7; 10,l 1-bisepoxy-farnesoate. This compound proved about ten times less effective than JH III in the white puparial bioassay (Richard ef al., 1989b). Whether younger larval ring glands make this compound and/ or JH III has not been determined although adult corpora allata make both (Richard et al., 1989b). In the present study, both methoprene and S31183 were effective JH mimics in the white puparial assay with the latter being 23 times more active. In mosquitoes and Lepidoptera S31183 is 15 and 320 times more active, respectively, than methoprene (Hatakoshi and Nakayama, 1987; Hatakoshi et al., 1988) due to its resistance to metabolism and to its ready penetrability through the cuticle (Hatakoshi et al., 1987). The effects of these JH mimics in Drosophila were primarily on the abdomen, as previously described for JH I (Postlethwait, 1974) with rotation of the male genitalia be-
DEVELOPMENT
179
ing the most sensitive to disruption followed by inhibition of eclosion. Whether these latter two effects are on the development of the adult nervous system is not known, but new neurons continue to be born in the ventral nervous system during the first 12 to 18 hr after pupariation (Truman and Bate, 1988). When larvae were fed either methoprene or S31183 throughout larval life, relatively low amounts were sufficient to prevent adult eclosion and to disrupt normal adult abdominal development. By contrast, at least loo-fold higher concentration was necessary to prevent normal adult differentiation of the imaginal discs. This high concentration had no apparent effect on disc growth or their pupal morphogenesis since animals blocked in the pupal stage showed normally proportioned pupal appendages. Importantly, this effect on adult differentiation of the imaginal discs was greatly reduced when larvae were fed the JH mimic starting in the mid second instar. In these latter insects, eclosion was blocked and abdominal differentiation was affected, but adult differentiation of the head and thorax proceeded normally. For instance, 78% of larvae fed 10 ppm S31183 beginning in the second instar formed normal adult heads and thoraxes as contrasted to 28% of those fed the same diet throughout larval life. Thus, exposure to high concentrations of JH mimics during the first and early second instars appears critical for the subsequent blockage of adult differentiation of the imaginal discs. That these effects were due to the JH activity of these mimics and not to a toxic action was clearly demonstrated by the normal development of the flies bearing the methoprene-resistant mutation after being fed concentrations which disrupted development of the wild-type. The product of the Met gene has not yet been characterized, but it appears to be involved in JH reception in the target tissue. Although there are only minor differences in penetration, ex-
180
RIDDIFORD
AND
cretion, tissue sequestration, and metabolism of JH III in the Me? mutants, the mutant fat body cytosol has a lo-fold lower binding affinity for JH III than is seen in wild-type (Shemshedini and Wilson, 1990). Based on characterization of the cytosolic binding of JH III in Met heterozygotes, they suggest that the Met+ locus encodes this cytosolic JH-binding protein. This protein is known to be involved in the cellular response to JH, but whether it is a JH receptor is not yet known (Shemshedini et al., 1990). During larval life of D. melanogaster, the imaginal disc cells divide, whereas the abdominal histoblasts remain quiescent until just after pupariation. Madhavan and Schneiderman (1977) showed that the onset of division in the discs begins in the eye disc about 14 hr into the first instar followed within a few hours by division in the wing disc; the antenna, leg, and genital discs begin dividing in the newly molted second instar larva. In the second instar the imaginal discs become competent to metamorphose when transplanted into a metamorphosing host. The eye and the wing discs first show this ability followed by the leg disc (Schubiger, 1974; Gateff and Schneiderman, 1975; Bownes and Roberts, 1979). Importantly, this competence to metamorphose is gained gradually as the disc continues to divide. First, the disc is able to make a thin untanned (possibly pupal) cuticle; then, as it grows larger, it is able to make tanned cuticle and adult-specific structures such as micro- and macrochaetes and eye pigments. Competence to form a completely normal adult structure is not attained until sometime in the third instar after growth is complete (Bryant and Simpson, 1984). The ability of the discs to continue to proliferate during larval life after they first attain competence to metamorphose is probably dependent on the presence of JH, since JH prevents the normal metamorphic response of mature larval discs to 20hydroxyecdysone in vitro (Doctor and Fris-
ASHBURNER
trom, 1985). Yet high exogenous JH in vivo during the third instar has no effect on the metamorphosis of the discs, whereas it can prevent normal adult differentiation of the abdominal histoblasts if given before the onset of their cell division after pupariation (Madhavan, 1973; Postlethwait, 1974). This study shows that high concentrations of dietary JH mimics can affect adult differentiation of imaginal disc-derived structures when fed beginning in the first instar, but not when beginning in the late second instar. Thus, for both the histoblasts and the imaginal discs the abnormally high JH appears to be affecting some process that occurs during the early cell divisions and leads to the acquisition of competence to metamorphose. During these early rounds of proliferative cell division, the imaginal cells must become “committed” to the much later program of expression, and excessive JH blocks this cellular commitment step. This is similar to the role of JH in the prevention of metamorphosis in other insects (Riddiford, 1985) except that in these higher Diptera, commitment to adult differentiation for the proliferating discs appears to be made much earlier in larval life. An alternative explanation which cannot at present be ruled out is that the early exposure to excessive exogenous JH somehow interferes with the normal functioning of the endocrine system after the initiation of metamorphosis (i.e., pupariation). Thus, the animals blocked at the pupal stage could be deficient in ecdysteroid necessary for adult development. This seems unlikely since although these early larvae have been exposed to a higher cumulative dose of the JH mimic, the endocrine system appears to be working normally since these animals form both a normal puparium and a normal pupae. Melanotic pseudotumors occur in some strains of Drosophila, appearing usually in the third larval instar as black masses in the hemocoel (Sparrow, 1978). These pseudotumors are formed by encapsulation of
hemocytes by lamellocytes followed by melanization. Normally lamellocytes, which differentiate from plasmatocytes, are not present until after pupariation. In tumorigenic strains the plasmatocyte-lamellocyte differentiation occurs precociously in the late second or early third larval instar. Juvenile hormone seems to promote this precocious differentiation as various JH analogs either in the diet or topically applied have increased pseudotumor formation in both tumorigenic and nontumorigenic strains (Bryant and Sang, 1969; Madhavan 1972; Wilson and Fabian, 1986; this study). Moreover, Wilson and Fabian (1986) found that methoprene was less effective in promoting tumorigenesis in the methoprene-resistant mutant Met as compared to Met + , again implicating JH in their induction. The role of excess JH in promoting precocious lamellocyte differentiation and thus pseudotumor production is unknown. The hemopoietic lymph gland normally undergoes mitosis throughout larval life until the mid third instar, then begins releasing cells at pupariation and degenerates 12 hr later (Madhavan and Schneiderman, 1977; Srdic and Gloor, 1978). This release of cells seems to be correlated in time with the plasmatocyte-lamellocyte transition (Rizki, 1962). Possibly, prolonged exposure to high JH interferes with the normal developmental progression of the lymph gland itself. These studies have indicated an important role for JH in guiding the postembryonic proliferative cell divisions of imaginal discs and histoblasts and suggest that a carefully regulated JH titer during larval life is necessary both for proliferation and for the commitment to later adult differentiation. The nature of this role remains a challenge for further study. ACKNOWLEDGMENTS This study was performed primarily while L.M.R. was a Visiting Scholar at Cambridge University under
‘h: ausp~c:h :t a !ienior International Fellowship fro is i he Fogarty Cznter of the National Institutes of Hea 1t! I I FOh TWO! X08). The work done later at the Uni\pcr ,ilty of Wabhrnyton was supported by NSF Grail DCBXS-10875 We thank Dr. M. Hatakoshi for t’c S31IP3. Dr. <;erardus Staal for the methoprene. Lb ‘Thomas Wilson for the y w Met’ and Md stocks, ClBarbara Wakimoto for the Canton S I and II isogen$c lines, Dr. .iohn Palka for the Oregon R stock, and D,James Truman for stimulating discussions and critlc:I comments on the manuscript.
REFERENCES Ashburner, M. (1970). Effects ofjuvenile hormone on adult differentiation of Drosophila melanogaster. Nuture 227, 187-189. Bainbridge, S. P., and Bownes, M. (1981). Staging the metamorphosis of Drosophilu melanogaster. .I. Embryoi.
Exp.
Morphol.
66, 57-80.
Bhaskaran, G. (1972). Inhibition of imaginal differentiation in Sarcophaga bullata by juvenile hormone. J. Exp. 2001. 182, 127-142. Bownes. M., and Rembold, H. (1987). The titre of juvenile hormone during the pupal and adult stages of the life cycle of Drosophila melanogaster. Eur. J. Biochem. 164, 709-712. Bownes, M.. and Roberts, S. (1979). Acquisition of differentiative capacity in imaginat wing discs of Drosophilu melanogaster. phol. 49, 103-113.
J. Embryoi.
Exp.
Mor-
Bryant, P. J.. and Sang, J. H. (1968). Drosophila: Lethal derangements of metamorphosis and modifications of gene expression caused by juvenile hormone mimics. Nature 220, 393-394. Bryant, P. J., and Sang, J. H. (1969). Physiological genetics of melanotic tumors in Drosophila me/anogaster. VI. The tumorigenic effects of juvenile hormone-like substances. Genetics 62, 321-336. Bryant, P. J., and Simpson, P. (1984). Intrinsic and extrinsic control of growth in developing organs. Q. Rev.
Biol.
59, 387-415.
Doctor, J. S.. and Fristrom, J. W. (1985). Macromolecular changes in imaginal discs during postembryonic development. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 2, pp. 201-238. Pergamon Press, Oxford. Gateff, E. A., and Schneiderman, H. A. (1975). Developmental capacities of immature eye-antenna] imaginal discs of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 176, 171-189. Handler, A. M. (1982). Ecdysteroid titers during pupal and adult development in Drosophila melanoRaster.
Dev.
Biol.
93, 73-82.
182
RIDDIFORD
AND
Hatakoshi, M., and Nakayama, I. (1987). Juvenile hormone active compounds: Recent researches. Shokubutsu
Boeki
41, 339-347.
Hatakoshi, M., Nakayama, I., and Riddiford, L. M. (1987). Penetration and stability of juvenile hormone analogues in Munduca sexta L. (Lepidoptera: Sphingidae). Appl. Entomol. Zool. 22, 641-644. Hatakoshi, M., Nakayama, I., and Riddiford, L. M. (1988). The induction of an imperfect supemumerary larval moult by juvenile hormone analogues in Manduca
sexta.
J. Insect
Physiol.
34, 373-378.
Kraminsky, G. P., Clark, W. C., Estelle, M. A., Gietz, R. D., Sage, B. A., O’Connor, J. D., and Hodgetts, R. B. (1980). Induction of translatable mRNA for dopa decarboxylase in Drosophila: An early response to ecdysterone. Proc. Natl. Acad. Sci.
USA
17, 41754179.
Madhavan, K. (1972). Induction of melanotic pseudotumors in Drosophila melanogaster by juvenile hormone. Wilhelm Roux’s Archiv. Dev. Biol. 169, 345-349. Madhavan, K. (1973). Morphogenetic effects of juvenile hormone and juvenile hormone mimics on adult development of Drosophila. J. Insect Physiol. 19, 441-453. Madhavan, M. M., and Schneiderman, H. A. (1977). Histological analysis of dynamics of growth of imaginal discs and histoblast nests during the larval development of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 183, 269-305.
ASHBURNER
and L. I. Gilbert, Eds.), Vol. 8, pp. 37-84. Pergamon Press, Oxford. Rizki, T. M. (1962). Experimental analysis of hemocyte morphology in insects. Am. Zool. 2,247-256. Roseland, C. R., and Schneiderman, H. A. (1979). Regulation and metamorphosis of the abdominal histoblasts of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 186, 235-265. Schubiger, G. (1974). Acquisition of differentiative competence in the imaginal leg discs of Drosophila. Wilhelm Roux’s Archiv. Dev. Biol. 174,303311. Sehnal, F. (1985). Growth and life cycles. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 2, pp. l-86. Pergamon Press, Oxford. Sehnal, F., and Zdarek, J. (1976). Action of juvenoids on the metamorphosis of cyclorrhaphous Diptera. J. Znsect
Physiol.
22, 673-682.
Shemshedini, L., Lanoue, M., and Wilson, T. G. (1990). Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster. J. Biol. Chem. 265, 1913-1918. Shemshedini, L., and Wilson, T. G. (1990). Resistance to juvenile hormone and an insect growth regulator in Drosophila is associated with an altered cytosolic juvenile hormone-binding protein. Proc. Natl.
Acad.
Sci.
USA
87, 2072-2076.
383-387. Richard, D. S., Applebaum, S. W., Sliter, T. J., Baker, F. C., Schooley, D. A., Reuter, C. C., Henrich, V. C., and Gilbert, L. I. (1989b). Juvenile hormone bisepoxide biosynthesis in vitro by the ring gland of Drosophila melanogaster: A putative juvenile hormone in the higher Diptera. Proc. Natl. Acad. Sci. USA 86, 1421-1425.
Sliter, T. J., Sedlak, B. J., Baker, F. C., and Schooley, D. A. (1987). Juvenile hormone in Drosophila melanogaster: Identification and titer determination during development. Znsect Biothem. 17, 161-165. Sparrow, J. C. (1978). Melanotic “tumours”. In “The Genetics and Biology of Drosophila” (M. Ashburner and T. R. F. Wright, Eds.), Vol. 2b, pp. 277-3 13. Academic Press, London. Srdic, Z., and Gloor, H. (1978). Le role hematopoietique des glandes lymphatique de Drosophila hydei. Rev. Suisse Zool. 85, 11-19. Srivastava, U. S., and Gilbert, L. I. (1968). Juvenile hormone: Effects on a higher dipteran. Science 101, 61-62. Srivastava, U. S., and Gilbert, L. I. (1969). The influence of juvenile hormone on the metamorphosis of Sarcophaga bullata. J. Znsect Physiol. 15, 177190. Truman, J. W., and Bate, M. (1988). Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster. Dev.
Riddiford, L. M. (1985). Hormone action at the cellular level. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut
Wigglesworth, V. B. (1934). The physiology of ecdysis in Rhodnius prolixus. II. Factors controlling
Nijhout, H. F., and Wheeler, D. E. (1982). Juvenile hormone and the physiological basis of insect polymorphism. Q. Rev. Biol. 57, 109-133. Postlethwait, J. H. (1974). Juvenile hormone and the adult development of Drosophila. Biol. Bull. 147, 119135. Richard, D. S., Applebaum, S. W., and Gilbert, L. I. (1989a). Developmental regulation ofjuvenile hormone biosynthesis by the ring gland of Drosophila
melanogaster.
J.
Comp.
Physiol.
159,
Biol.
125, 145-157.
JH
AND
Drosophila
moulting and metamorphosis. J. Microsc. Sci. 77, 191-222. Wigglesworth, V. B. (1940). The determination of characters at metamorphosis in Rhodnius prolixus (Hemiptera). J. Exp. Biol. 17, 201-222. Wilson, T. G., and Chaykin, D. (1985). Toxicity of methoprene to Drosophila mdanogaster (Diptera: Drosophilidae): A function of larval culture density. J. Econ. Entomol. 78, 1208-1211. Wilson, T. G., and Fabian, J. (1986). A Drosophila
DEVELOPMENT
183
melanogaster mutant resistant to a chemical analog of juvenile hormone. Dev. Biol. 118, 190-201, Wilson, T. G., and Fabian, J. (1987). Selection of methoprene-resistant mutants of Drosophila melanogaster. In “Molecular Entomology” (J. H. Law, Ed.), Vol. 49. pp. 179-188. A. R. Liss, New York. Zdarek, J., and Slama, K. (1972). Supernumerary larval instars in cyclorrhaphous Diptera. Bid. Bull. 142, 350-357.