Juvenile Hormone Regulation of Reproductionin the Cyclorrhaphous Diptera withEmphasis on OogenesisChih-Ming Yin* and John G. Stoffolano, Jr.Department of Entomology, University of Massachusetts, Amherst
Within the Diptera, the division or infraorder Cyclorrhapha [= Muscomorpha]is the largest (80,000+ species) group. At the taxonomic level, it has also beenone of the most controversial collections of Diptera. This diverse group datesback to the Cretaceous (ca. 100 Myr or between 65 and 135 Myr) and hasundergone rapid proliferation and diversification (Beverley and Wilson, 1984;Wiegmann et al., 1993). In fact, the range of both larval and adult nicheswithin the Cyclorrhapha is greater than within all the other flies.
Dedication: To Dr. Thomas J. Kelly, whose recent death left a research void in helping us under-stand the endocrinology of oogenesis in the Diptera.
Acknowledgment: The authors wish to thank B.-X. Zou for his years of assistance. This paper ispublished as Contribution No. 3192 of the Massachusetts Agricultural Experiment Station.
Contract grant sponsor: National Science Foundation, contract grant number DCB-9104757 andIBN-9306650; contract grant sponsor: Massachusetts Agriculture Experiment Station, contractgrant number Hatch 743 and Hatch 632.
*Correspondence to: Dr. Chih-Ming Yin, Department of Entomology, University of Massachu-setts, Amherst, MA 01003-2410.
Received 15 August 1996; Accepted 14 February 1997.
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Research at the molecular level is starting to examine the ways in whichmembers of this group share common biochemical or physiological mecha-nisms. The use of specific cyclorrhaphan proteins to examine phylogenic re-lationships has provided some clues. Examination of cytochrome C in adiverse group of cyclorrhaphous flies suggests that Lucilia cuprina may becloser to Drosophila than to Musca domestica (Inoue et al., 1986). Martinez andBownes (1992) demonstrated that the specificity of the mechanism involvedin yolk protein uptake was highly conserved for 13 different Drosophila sp.and five other different species, which included Calliphoridae, Sarcophagidae,and Muscidae. It has been suggested that species showing differences in thespecificity in the mechanism involving yolk protein uptake probably sepa-rated at least 36, and probably more than 80, million years ago (Beverley andWilson, 1984). This time frame overlaps the proposed 100 million years forthe evolution of this group.
Thus, we are left with the glaring fact that for a group as diverse as thecyclorrhaphous Diptera a common hormonal mechanism for controlling re-production probably does not exist. If anything within the group that mightlead to a commonly shared hormonal mechanism, it might be the adultlifestyle and nutritional impact on the neuroendocrine system. At the sametime, however, some specific events, such as specificity of yolk protein up-take, may be highly conserved and shared within the group.
JUVENILE HORMONES IN THE CYCLORRHAPHOUS DIPTERA
The complexity of the physicochemical methods required for juvenile hor-mone (JH*) determination restricts the identification of JHs to a relativelysmall number of insects that include some cyclorrhaphous flies. Even withinthis limited group of flies, inconsistencies exist as to what type of JH is present.For example, earlier studies report the presence of JH I, II, and III in tissueextracts from adults and larvae (Schooley et al., 1976), or just JH I, but not IIand III (Girard et al., 1976) from adults of the house fly, M. domestica. Laterstudies with improved methods, however, failed to find any known JH inadult M. domestica (Baker, 1990; Baker et al., 1990).
More recently, a combination of various chromatographic analyses and theradiochemical assay has been used to study the JH products from the cor-pora allata (CA) or the ring glands in vitro. This approach led to the identifi-cation (Richard et al., 1989) of a new JH, JH III bisepoxide or JHB3 (see reviewby Yin, 1994). Table 1 summarizes the different types of JHs biosynthesizedby different stages of various cyclorrhaphous flies and the physicochemicalmethods used for their identification.
The more consistent JH identification from the in vitro studies may resultfrom the lesser degree of “contamination” in the preparations. Earlier in vivostudies analyzed whole-body extracts or tissue extracts that contained a large
array of tissue lipids and of considerable quantity, whereas the recent invitro studies analyzed JH extracts from the tissue culture media (e.g.,TC199 or Eagle’s minimum essential medium, etc.) that had a much lowerlipid content.
It is interesting to note that in Phormia regina, the production of JHB3, JHIII, and methyl farnesoate by the CA appears to have functional significance.When tested singly, using a bioassay to check their ability to restore oogen-esis in allatectomized flies, the potency is JHB3=JH III > methyl farnesoate.However, a blend of JHB3, JH III, and methyl farnesoate at a ratio of 66:22:12(i.e., the ratio that these juvenoids are produced by liver-fed females) is aboutfour-fold more effective than JHB3 or JH III alone (Yin et al., 1995). In thefuture, we may have to consider the physiological effect of the juvenile hor-mones with a more holistic view than ever before.
Of all the cyclorrhaphous flies studied to date, few species have been mod-eled with respect to how JHs influence reproduction in general. Recent in-vestigations on JHs in P. regina have permitted a general model to bedeveloped.
JUVENILE HORMONE INVOLVEMENT IN REPRODUCTION OFPHORMIA REGINA
In adult P. regina, JH produced by the CA has been shown to influence theontogeny of mating behavior in both sexes and development of the fat body,ovaries, and the accessory reproductive glands (ARG) in both sexes (see Fig.1). Rather than elaborate here on the specifics of how JH is involved in thesefour areas, details will be provided within the context of the appropriate sec-tion of this paper. One obvious effect of high JH levels is on maturation ofthe ovaries. In sugar-fed females with low levels of JH, the ovaries remainsmall and the surrounding ovarian muscle sheaths are not stretched (Fig. 2,1–2). In contrast, in those females with high JH levels (i.e., protein-fed), theovaries increase significantly in size and the ovarian muscle sheaths becomegreatly stretched to accommodate ca. 150 eggs/ovary (Fig. 2, 3–4).
OVERALL EFFECT OF JUVENILE HORMONES ON REPRODUCTION
One must be rather selective in choosing a focal point for a discussion onreproduction, since reproduction in any system includes so many diverseevents (i.e., starting from sexual maturation to egg laying). The shortcomingof this narrow approach, however, is that it often leaves the reader with anincomplete picture. To avoid this, we provide a short account of most eventsreported to be influenced by JH, while keeping a focus on one specific event(i.e., oogenesis). We hope that such a treatment will help others focus onproblems needing clarification and will stimulate thinking about those areasof JH research that need further exploration and refinement.
The role of JHs on insect reproduction in general was reviewed by Koeppeet al. (1985). At that time, the key insect model systems investigated weremainly lepidopterans, cockroaches, locusts, and the hemipteran, Rhodnius pro-lixus. Limited information was available on a few cyclorrhaphous flies (i.e.,
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M. domestica, Calliphora erythrocephala, and Drosophila melanogaster). In fact, ofthe 194 references which listed the insect being studied within the title of thepaper, only 11.9% were cyclorrhaphous flies (Koeppe et al., 1985). Raabe (1986)also reviewed insect reproduction in general, while one year later Kelly et al.(1987) reviewed the impact of JH only on ovarian maturation. Since thesethree reviews, new techniques and approaches have produced a new bodyof information on the involvement of JH in adult reproduction of this groupof flies, especially the black blowfly, P. regina. We have made an attempt inthis review to show why and how this particular species is a better modelthan some of the other flies when it comes to certain studies involving link-ing JH with a reproductive event. Events prior to adult eclosion are not dis-cussed in this review.
Maturation of Adults and Their Reproductive Structures
Since Roubaud (1929) first coined the term “autogeny” for mosquitoes, thisterm, and its counterpart, “anautogeny,” have been extended to include eventhe cyclorrhaphous Diptera. Also, these terms initially were meant only tocover the adults‘ ability to produce eggs without a blood meal (i.e., autog-eny), compared to those that needed at least one (i.e., anautogeny). Whetheran insect is autogenous or not also has a bearing on its ability to mate. Thus,autogenous dipterans in this review usually do not need an exogenous pro-tein meal for mating and successful sperm transfer, whereas anautogenousflies do. This physiological condition has nutritional, genetic, and endocrinecomponents. Consequently, the complex interactions among these compo-
Fig. 1. The role of the CA and its products, JH’s (heavy dark arrows), on various aspects of thereproductive biology of cyclorrhaphous flies (photo of CA taken from Yin et al., 1989).
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nents can greatly affect how one interprets experiments aimed at separatingnutritional, endocrine, and genetic control mechanisms.
Many events occur during adult maturation, but we have chosen to focusour attention on the following three: JH-regulated disappearance of pupalfat body, gonad development, and ARG development.
Pupal Fat Body
Pupal and adult fat bodies are found to co-exist in the newly emerged adultsof cyclorrhaphous flies of the following genera: Sarcophaga (Day, 1943), Calli-phora (Thomsen, 1952), Protophormia (Harlow, 1956), Drosophila (Butterworth,1972), Musca (Adams and Nelson, 1970), and Phormia (Stoffolano, 1974). Pu-
Fig. 2. Previtellogenic ovary (1) from an adult, sugar-fed P. regina, also showing the relaxed orunstretched ovarian muscle sheath surrounding these ovaries (2). In comparison, the ovary of aprotein-fed female showing mature eggs (3) and the stretched ovarian muscle sheath (4) (takenfrom Mazzini et al., 1987).
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Fig. 3. Hormone profiles showing the total hemolymph ecdysteroids (filled circles) and JH IIIbiosynthesis (open circles) during the 1st gonotrophic cycle of P. regina. Note that JH is notdetected until ca. 4 h after ecdysteroids are detected and the JH biosynthesis peaks 24 h afterthe highest quantity of ecdysteroids was detected (taken from Yin and Stoffolano, 1990).
pal fat body comes from larval fat body, which separates into individual cellsduring the pupal stage. In Drosophila, approximately 1,000 of these separatedpupal fat body cells survive the metamorphosis and persist into the adultstage (Butterworth, 1972). In the fleshfly, Neobellieria (Sarcophaga) bullata, pu-pal fat body never completely disappears if the fly is allatectomized immedi-ately after adult emergence (Day, 1943). Results consistent with the aboveobservation are reported for D. melanogaster (Postlethwait and Jones, 1978),M. domestica (Sakurai, 1977), and P. regina (Qin, 1996). With a combination ofneck-ligation, allatectomy, and brain implantation, Qin (1996) found that pu-pal fat body disappearance in adult female P. regina is controlled by the brainvia the CA. In contrast, Adams and Nelson (1970) have maintained that dis-appearance of pupal fat body in M. domestica is related to ovary or egg de-velopment, rather than to the CA or JH. In mutant apterous4 (a non-vitellogenic,JH-deficient mutant, which dies precociously), the fat body fails to undergocomplete cytolysis (Butterworth, 1972; Richard et al., 1993) strongly suggest-ing that JH plays a role in normal fat body cytolysis. However, other Droso-phila mutants (such as ap56f and ap78jts), which are also JH-deficient, have anormal fat body cytolysis, suggesting that JH may not play a role (Richard etal., 1993). Since fat body cytolysis is controlled at many different levels, therole of JH in ap56f and ap78jts may be superseded by a factor(s) that exerts itsregulation more downstream than JH.
Pupal fat body histolysis should provide raw materials for other bodilyfunctions, including reproduction. To date, the significance of pupal fatbody disappearance compared to other events in the adult fly has notbeen determined.
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Gonad Development
In general, spermatogenesis in adult insects has not been shown to be in-fluenced by any hormone (Engelmann, 1970; Dumser, 1980). This also seemsto be the case for females, even though Gwadz and Spielman (1973) in Aedesaegypti and Readio and Meola (1985) in Culex pipiens showed that pre-vitello-genic growth (i.e., size) of the secondary follicles in these mosquitoes is JH-dependent. Other than the report of Adams (1974) for M. domestica, we knowof no other studies showing a JH influence on pre-vitellogenic gonad devel-opment in the cyclorrhaphous flies. In P. regina, we suggest that such a JH-dependent mechanism, if it exists, is not operative at high JH titers becauseJHB3 and JH III are synthesized at only low levels until a protein meal istaken (Yin et al., 1995). This is one example where P. regina is a better systemto study than autogenous species and species where it is difficult to separate
Fig. 4. A TEM section (1) showing the follicle cell / nurse cell interface of an ovary from asugar-fed fly. Note the thick and wrinkled ovariole muscle sheath (OS) enveloping the folliclesand the arrow pointing to the close apposition of the adjacent follicle cells. BL = basementlamina; FC = follicle cells; GA = golgi apparatus; N = follicle cell nucleus. Below is a schematicshowing the TEM, but this time showing the ovariole muscle sheath (OVS) and the oolemma(OL). A TEM section (2) showing the follicle cell / nurse cell interface of an ovary from a pro-tein-fed fly. Note the large arrow pointing to the follicular extracellular space in the TEM. Alsonote the extracellular spaces in the schematic below the TEM. OC = oocyte; L = lipid droplets;NC = nurse cells; Y = yolk spheres; OMS = ovarian muscle sheath. (TEM taken from Mazzini etal., 1987; schematic courtesy of Dr. F. Giorgi).
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nutritional from endocrine control. This aspect of reproductive biology of P.regina needs to be examined.
Accessory Reproductive Glands
The ARG secretions of male cyclorrhaphous dipterans have been shown tohave a dual role (i.e., affecting female behavior as well as performing func-tions directly related to sperm maintenance, viability, and transfer) (Leopold,1976; Chen, 1984; Happ, 1992). These glands are paired in most members ofthis group but some calyptrate muscoid flies lack distinct glands (Hori, 1960).The evolutionary significance of this has not been addressed.
Even though the initial research on the role of the secretions from theseglands in male Diptera was originally done using M. domestica (Leopold, 1976),this model system, which lacks distinct glands, was readily abandoned whenit came time to investigate the biochemistry and molecular biology of theseglands. D. melanogaster, the workhorse of molecular genetics and biology, tookits place (Chen, 1984). To date, most research on the ARG has focused onthose in males, not in females (Kaulenas, 1992).
Thomsen (1942) provided the first evidence that the CA was involved inaccessory gland development. Since then, a few other indirect reports existon JH involvement in ARG development (Stein et al., 1984); but mainly, “Verylittle information is available concerning the role of JH in the development ofmale accessory glands” (Koeppe et al., 1985). Overall, it is believed that JH isessential for these glands to become competent and functional in the adultsof both sexes. Whether they have a direct effect on gland biosynthesis, influ-ence fat body biosynthesis of essential amino acids (Gillott and Friedel, 1977),or act directly on the membranes of the glands, which in turn influence up-take of essential blood-borne factors from the hemolymph (Yamamoto et al.,1988), remains to be demonstrated. Even though previous investigators im-plicated the role of JH on the ARG by removal and/or implants of the CA,the only reference showing direct JH effect on the glands is that of Yama-moto and colleagues. Yamamoto et al. (1988), using in vitro experiments,showed that either JH III or the JH analog hydroprene stimulated proteinsynthesis by the ARGs. In P. regina, Stoffolano (1974) indirectly showed thatin sugar-fed flies, which have low levels of JH compared to protein-fed flies,the ARGs need higher levels of JH to develop.
One known function of the male’s ARG secretion on female behavior, insome species, is the inhibition of sexual receptivity following mating. As wewill discuss next, JH has been shown to play a major role in the ontogeny ofsexual behavior in both sexes of many fly species. Thus, the inhibition offemales to mate following a successful mating, and until the eggs are laid, isoften attributed to the inhibition of JH biosynthesis. However, in P. regina JHbiosynthesis remains high until the eggs are laid.
ONTOGENY OF SEXUAL BEHAVIOR
In addition to a proteinaceous meal for initiation of oogenesis, anautogenousflies also require exogenous protein for ontogenesis of mating behavior. An
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extensive literature exists on the role of JH in mating behavior of insects,which also includes the cyclorrhaphous flies.
Since both castrated male and ovariectomized female Diptera mate, it wasassumed that mating behavior is not controlled by development of the go-nads, but by some other factor(s) (Drosophila—Geigy, 1931; M. domestica—LaBrecque et al., 1962; Scatophaga stercoraria—Foster, 1967). Thus, the generalrule seems to hold, with some exceptions, that the presence of the ovaries (orovarian ecdysteroids), in most adult insects, is not involved in the appear-ance of sexual behavior (Truman and Riddiford, 1974).
Initially, Day (1943) noted that the CA was not necessary for mating tooccur in Lucilia and Sarcophaga. Later, however, the CA or its product, JH,has been shown to be involved in the onset of mating behavior in thefollowing flies: M. domestica (Adams and Hintz, 1969); Glossina morsitans andG. austeni (Gillot and Langley, 1981); D. melanogaster (Manning, 1967;Bouletreau-Merle, 1973; Ringo et al., 1991); Calliphora vomitoria (Trabalon andCampan, 1984, 1985).
Unpublished results from our laboratory on P. regina show that allatectomyperformed within 4 h of adult emergence prevented males from developingsexual aggressiveness and females from becoming receptive. When allatecto-mized flies of either sex were treated with methoprene, they exhibited nor-mal sexual behavior. These results are similar to those of Adams and Hintz(1969) for M. domestica females and the report of De Clerck and De Loof (1983)on male N. bullata. The dependence of adult flies on JH for sexual maturationconfirm previous studies that have shown that development of the ARGs arealso influenced by this hormone. Certainly, one cannot imagine successfulmating taking place without fully mature ARG.
MATE LOCATION AND PHEROMONE PRODUCTION
Most dipterans do not use long-range pheromones to bring the sexes to-gether. Instead, they rely on specific aggregation sites (i.e., hosts, feces, promi-nent landmarks, etc.). P. regina males, for example, use various animal fecesto find females (Stoffolano et al., 1990). Females frequenting these sites aregenerally unmated and with undeveloped ovaries, while females at carcasseshave mated and are ready to lay eggs. Thus, a male waiting at fresh fecesstands a good chance of encountering an unmated and receptive female. Atthe aggregation site, males use vision to locate a prospective mate.
Most cyclorrhaphous males will fly toward any moving object if it is thecorrect size. Once contact is made, the male makes a decision as to whetherhe has caught the correct species, and sometimes even the correct sex. Thisdecision is based on the cuticular hydrocarbons of the captured fly. Thesecuticular, contact pheromones are usually perceived by the male’s tarsi, andnot by the olfactory system (e.g., antenna or palps).
A species recognition and copulation pheromone has recently been identi-fied from both sexes of P. regina (Stoffolano, Yin, Tillman, and Blomquist,unpublished). Unlike other dipterans, this cuticular hydrocarbon is not usedto distinguish between the sexes. When a male captures a conspecific female,both fall in tandem to the ground where the recognition of the cuticular hy-
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drocarbon should initiate copulatory attempts by the male. If the female electsto mate with the male, she extrudes her ovipositor and copulation takes place.
In the Diptera, sex pheromone production may be under hormonal con-trol, but the hormone is not JH, but rather the ecdysteroids (Blomquist et al.,1987, 1993; Trabalon et al., 1994; Wicker and Jallon, 1995).
MATING BEHAVIOR AND COPULATION
Once mating behavior in both sexes of these dipterans has been “turnedon,” JH does not appear to have any further effect. Other chemicals, espe-cially peptide hormones from the male’s ARG, are involved in “turning off”mating behavior in the females of some species. The role of the male‘s sexpeptide is best understood in D. melanogaster, where Moshitzky et al. (1996)demonstrate that the male‘s sex peptide activates JH biosynthesis in the cor-pus allatum of the female. JH has not been reported to influence the devel-opment of the aedeagus in any way (Koeppe et al., 1985), nor has it beenshown to influence the process of copulation.
OVARIAN COMPETENCE
Unlike in A. aegypti (Shapiro and Hagedorn, 1982), no evidence exists show-ing that the competence of the ovaries of cyclorrhaphous flies is under thecontrol of the brain ovarian ecdysteroidogenic hormone (= egg developmentneurosecretory hormone). Also, no one has published that this competence isdependent on the ovaries first being exposed to JH. Adams (1974), however,shows that in allatectomized M. domestica, the germarial cyst did not emergeto form the stage 2 follicle of the antepenultimate cycle.
Examination of JH and ecdysteroid profiles for P. regina, prior to andafter a protein meal (Fig. 3), suggests that the ovaries do not need to beexposed to JH prior to releasing the ecdysteroids necessary to begin vitel-logenin (Vg) production by the fat body. This is based on the fact that a highlevel of JH production after a protein meal does not begin until about 4 hafter ecdysteroids are present. The possible effect of low levels of JH beforethe protein meal on the ovaries, and subsequent ecdysteroidogenesis, remainsto be demonstrated.
EGG LAYING OR LARVIPOSITIONING
No known reports showing the involvement of JH on either egg laying orlarviposition have been published.
OOGENESIS
Fat Body Competence
Except under certain experimental conditions, fat body cells in adult maleinsects do not produce Vg, despite the presence of the gene, as well as thehormones (i.e., JH and ecdysteroids). This sexual disparity in gene expres-
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sion by the fat body also exists in cyclorrhaphous flies. Even when the hu-moral and hormonal milieu are made identical through parabiosis of maleand female P. regina, the male fails to sustain vitellogenesis (Belzer, 1987).The mechanism leading to this sex-specific inability of gene expression inmale cyclorrhaphous flies remains largely unknown, except in the case of D.melanogaster. Sex differentiation in this species is determined by the ratio of Xchromosomes to autosomes, which in turn switches on a cascade of regula-tory genes including sex-lethal, transformer, transformer-2, and doublesex. Themanner in which the regulatory genes in this pathway are expressed deter-mines whether or not the yolk protein genes are transcribed. In females, thispathway affects only the yolk protein gene (yp) in the fat body and not in theovary (Bownes et al., 1990).
In female, anautogenous cyclorrhaphous flies, competence of fat body cellsto produce Vg depends on a number of events, including the acquisition ofan adequate protein meal and the activation of the CA (Qin et al., 1995). In P.regina, Orr (1964a, 1964b) has documented an indirect role by which JH af-fects egg development by regulating fat body lipid metabolism. Allatectomybefore the ingestion of an adequate protein meal resulted in fat body hyper-trophy in test flies that produced little Vg. We have confirmed Orr’s resultson P. regina, and further tested the role of JH in fat body hypertrophy byusing JH therapy on allatectomized (within 4 h of adult emergence) and liver-fed (at 72 h of adult age) flies. Results show that methoprene treatment canrestore Vg biosynthesis and prevent fat body hypertrophy in allatectomizedflies only if it is applied before 48 h after the liver feeding. After the hyper-trophied fat body is well developed, it becomes insensitive to methoprene(Qin et al., 1995). It is clear that the CA is required to capacitate the fat bodyto turn into a vitellogenic mode of biosynthesis after the protein meal.
Biosynthesis of Vitellogenin
The exact role of JH in regulating Vg biosynthesis in the Drosophilidae isnot clear, even though it has been implicated in regulating yolk protein geneexpression (Jowett and Postlethwait, 1980; Bownes et al., 1987). In theMuscoidea (Muscomorpha), however, JH or its analogues have not beenshown to trigger vitellogenin biosynthesis (Huybrechts and De Loof, 1982;Stoffolano et al., 1992).
Endocrine control of dipteran vitellogenesis (Davey, 1994), and specificallyD. melanogaster and blow flies, has been recently reviewed (Kelly, 1994; Yinand Stoffolano, 1994). Some of the following discussion will focus on aspectsnot emphasized by these reviews.
Like other insects, fat body is the major tissue producing Vg in cyclorrhaphousflies. However, in species such as D. melanogaster and M. domestica, the ovarianfollicle cells also produce a significant amount of Vg (Brennan and Mahowald,1982; Agui et al., 1985). In an even more extreme case, Vg synthesis occurssolely in the ovarian tissues of the stable fly, Stomoxys calcitrans (Chen et al.,1987). Yolk protein of this group of flies may also have different numbers ofsubunits. Three subunits, designated YP1, YP2, and YP3, are identified forthe yolk protein of D. melanogaster, together with three corresponding YP genesyp1, yp2, and yp3, located on the X chromosome (Barnett et al., 1980). Each
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gene is transcribed as individual mRNA. Three subunits have been reportedfor C. erythrocephala, Lucilia cuprina, M. domestica, Protophormia terrae-novae,and N. bullata (see Zou et al., 1988). However, four subunits have been re-ported for P. regina (Zou et al., 1988), five for M. domestica (Bianchi et al.,1985), and six for S. calcitrans (Chen et al., 1987). We believe the YP number,as determined by protein analysis, should be considered tentative until theircorresponding genes or mRNAs are identified and characterized.
The role of the CA, or JH, in the control of Vg biosynthesis has been stud-ied at both the tissue/organismal and biochemical/molecular levels. SinceJH is merely a necessary, but not a sufficient, condition required for the con-trol of Vg biosynthesis, a straightforward comparison of results from differ-ent cyclorrhaphous flies may be impossible and meaningless. This may bedue to lack of standardization of various factors. Some of these factors listedin various studies may include nutritional requirements (i.e., autogeny vs.anautogeny), nutritional status (i.e., carbohydrate vs. protein feeding, andpartial vs. replete feeding, etc.), and age (which will alter the temporal rela-tionship between JH and developmental status of fat body and ovary, andbetween JH and other neural or endocrine milieu). The following discussionwill try to find some general common roles that JH plays in Vg biosynthesisof cyclorrhaphous flies.
Through extirpation and implantation of the CA, and JH (analog) therapy,it was shown that the CA is required at strategic times of the ovarian cycle.During the first ovarian cycle, allatectomy may or may not affect egg matu-ration, depending on whether the surgical operation is conducted prior to orafter a protein meal in anautogenous flies, such as C. erythrocephala and P.regina (Thomsen, 1940, 1942; Strangways-Dixon, 1961a, 1961b; Orr, 1964a).The failure of oogenesis in blow flies that have been allatectomized beforetheir protein meal is attributed to aberrant protein and Vg biosynthesis (Mjeniand Morrison, 1975, 1976). JH therapy studies show that restoration of oo-genesis occurs if dietary protein is ingested by flies prior to JH treatment.Without the protein meal, JH alone cannot initiate Vg biosynthesis in blowflies (Huybrechts and De Loof, 1982). Similarly, Vg or Vt cannot be detectedin sugar-fed P. regina, even after three treatments of methoprene, a JH analog(Stoffolano et al., 1992). In contrast, Schwartz et al. (1985) show that applicationof 50 ng of methoprene to sugar-fed, adult females of D. melanogaster stimulateda transient increase in vitellogenesis and in ovarian ecdysteroidogenesis. Sinceat adult eclosion of D. melanogaster, Vg synthesis is already initiated (Jowettand Postlethwait, 1980), a sugar-fed D. melanogaster is not physiologicallyequivalent to a sugar-fed blowfly such as C. erythrocephala, P. regina, or L.cuprina. The physiological differences between D. melanogaster and blow fliesin their dietary requirement, as well as their lack of ovariole synchrony, mayhelp to explain the above-observed discrepancy in the JH effect on sugar-fedflies. This is a good example, showing that overt differences in JH actionmay really reflect the differences in other factors that are also necessary forVg synthesis.
At the molecular level, a comprehensive understanding is available onlyon Drosophila. The relative molecular mass of the three yolk proteins, YP1,YP2, and YP3 of D. melanogaster is 47,000, 45,700, and 44,700 Da, respectively.
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Even within the genus Drosophila, not every member possesses three yolkproteins.
To support vitellogenesis, these proteins are synthesized either in the fatbody (major site) and secreted into the hemolymph, or synthesized in the folliclecells (minor site) and secreted toward the oocyte membrane (Butterworth et al.,1992). These proteins are then selectively endocytosed into developing oocytesand stored as yolk granules (spheres). This complicated vitellogenic processcould be potentially controlled at a number of places along the process of Vgsynthesis, secretion, uptake, and storage. It is known that the initial step, i.e.,expression of these genes, are regulated by the correct expression of the sexdetermination (regulatory protein) hierarchy and tissue-specific factors, thehormones (JH and ecdysteroids), and the nutritional status of the fly.
Two of the YP genes, yp1 and yp2, are in close proximity and separatedonly by 1,225 bp. They are divergently transcribed with a shared promoterlocated within the 1,225 bp that separated them. Both genes have single in-trons (Hung and Wensink, 1983). The yp3 gene is located more than 1,000 kbaway from yp1 and has two introns (Garabedian et al., 1987). The tremen-dous increase in YP synthesis during vitellogenesis is accomplished not bygene amplification, but by production of protein in tissues that are polyteneand polyploid. Using a p-element-mediated transformation with specific frag-ments of yp genes (after deletion of a certain fragment), a 125 bp fragment isidentified as the fat body enhancer (FBE). Likewise, a 300 and a 107 bp ova-rian enhancers (OE1 and OE2) are found for yp1 and yp2, and an OE3, aswell as a 747 bp promoter, for yp3 (Logan et al., 1989; Logan and Wensink,1990; Ronaldson and Bownes, 1995).
Analyses of the DNA regions upstream for the expression of a number ofgenes in the fat body reveal that several regulatory proteins, including, AEF-1(an adult enhancer factor-1 protein), BBF-2 (box B-binding factor-2) and C/EBP(a CCAAT/enhancer binding protein, whose binding is inhibited by AEF-1),etc. (Abel et al., 1992; Falb and Maniatis, 1992), overlap in their binding siteswith the binding site of DSX (doublesex) protein on the yp1, 125 bp FBE re-gion. This makes the study of the DNA binding by these individual regula-tory proteins extremely difficult. However, deletion of FBE does not preventthe expression of a reporter gene in a sex-specific manner; and we know thatfor yp3 the binding of all the above-mentioned regulatory proteins does notshow the same overlapping conformation seen in the 125 bp FBE of yp1 andyp2. This suggests that the overlapping binding conformation may not beessential (Bownes et al., 1993).
Regulation of YP synthesis in the minor site (follicle cells) appears quitedifferent from that in the fat body. Ecdysone can increase transcript levels ofYP synthesis in the fat body but has no effect on follicle cell synthesis (Bowneset al., 1983). YP synthesis in follicle cells occurs only at specific stages ofoogenesis and is independent of the sex-determination hierarchy (Bownes etal., 1983). As can be expected, follicle cell YP synthesis relies on the OE1 andOE2 enhancers, rather than the FBE.
The fact that the expression of YP genes is regulated by ecdysone has longbeen known. It is also demonstrated that this regulation is tissue-specific (i.e.,only affects the YP gene expression in the fat body) but not sex-specific (i.e.,
Juvenile Hormone Regulation of Reproduction 527
can increase transcript levels in both male and female fat body) (Bownes etal., 1983). Ecdysone can override the sex-determination hierarchy because flymutants in dsx or tra can be induced by ecdysone to make YPs (Bownes andNöthiger, 1981).
JH also regulates the expression of YP genes. Methoprene, a JH analog, canincrease yp transcript levels up to twofold in the fat body. Methoprene alsoincreases transcript levels in the ovary. However, attempts to use JH to stimu-late the expression of reporter genes inserted in the 5' fragments of the yp1and yp2 genes have failed so far, indicating that JH might indirectly exert itseffect. These indirect mechanisms may include the stimulation of other regu-latory gene expression and the stabilization by JH of the YP gene transcripts(Bownes et al., 1993). Again, the complex nature of oogenesis may mask thehormonal regulation mechanism. For instance, the yp transcript levels arevery different between starved and nutritionally adequate flies, yet the lev-els of ecdysone and JH are similar in both groups (Bownes, 1989). This, how-ever, cannot be interpreted as there being no endocrine regulation of oogenesisin the light of other evidence.
Uptake and Internalization of Vitellogenin
Even though Koeppe et al. (1985) state that “It is difficult to demonstratethat JH acts directly upon the ovaries to stimulate Vg uptake by oocytes,”many investigators have used different experimental designs to show theeffect of JH on Vg uptake. As for the overall process of Vg uptake, the fol-lowing areas will be briefly discussed: general uptake, patency, the endocy-totic apparatus, and recognition of the yolk protein by the oocyte receptors.
The earliest suggestion that JH was involved in the uptake of Vg in thisgroup of flies was provided by Postlethwait and Weiser (1973). They showedthat the ovaries in isolated abdomens of the sterile D. melanogaster mutant,apterous-four, took up Vg when JH was topically applied. Otherwise the ova-ries failed to develop in the mutant. Later, Gavin and Williamson (1976), us-ing the same mutant, but this time intact females, showed the same effect forJH. In addition, they demonstrated that JH had no effect on either the amountof hemolymph Vg or the hemolymph profiles of the wild type versus the JH-treated mutant.
Pratt and Davey (1972), using R. prolixus, demonstrated that JH was re-quired for “patency,” or the development of extracellular spaces between thefollicle cells. They noted that patency was essential for Vg to be taken up bythe oocyte. Researchers studying cyclorrhaphous flies have not adopted thepatency index scheme proposed by Davey and his colleagues.
A comparison at the ultrastructural level between the ovaries of sugar-fedand protein-fed P. regina showed that in sugar-fed flies the follicle cells werein close apposition, thus no patency, while in protein-fed flies extracellularspaces (i.e., patency), plus the endocytotic apparatus, were present (Fig. 4)(Mazzini et al., 1987). Even though these authors did not directly show thatJH was essential for patency to occur, later work (Stoffolano et al., 1992) onthe same species showed that uptake was dependent on JH and occurredonly in protein-fed flies.
In addition to patency, the development of the receptor-mediated endocy-
528 Yin and Stoffolano
totic apparatus is essential for Vg to become internalized by the oocyte onceit reaches the interface between the oolemma and the follicle cells. Again,even though it was not direct evidence, the work of Mazzini et al. (1987) onP. regina showed that the endocytotic apparatus was present only in protein-fed flies (Fig. 5). Giorgi et al. (1993), however, provide direct evidence for theinvolvement of JH on the appearance of the endocytotic apparatus. Theytreated, in vitro, ovaries of D. melanogaster with JH and showed ultrastruc-turally that the uptake mechanism appeared. Without JH, the endocytoticapparatus was lacking and no Vg was internalized.
Recognition of vitellogenins by oocyte receptors is an important step intheir internalization by the oocyte. To date, we know of no reports on thedirect involvement of this step of internalization in the cyclorrhaphousDiptera. As discussed earlier, Martinez and Bownes (1992) addressed the im-portance of yolk protein specificity on uptake. Their conclusion was that inthe six different cyclorrhaphous species studied, uptake by D. melanogasterfemales of the yolk proteins of the other five species occurred, and the recep-tors involved in all six species appear to be highly conserved. No attempts,however, were made to show the involvement of JH on this process.
Oostatic Hormone(s)
The concept of an oostatic hormone was first proposed by Iwanoff andMestsherskaja (1939) for cockroaches. Since then, many investigators, usingdifferent insects, have worked on this hormone. In cyclorrhaphous flies, Ad-ams et al. (1968) first demonstrated the presence of an oostatic hormone inM. domestica. They proposed that this hormone, which is produced by theprimary oocytes, inhibits the development of secondary oocytes by inacti-vating the biosynthesis and release of JH by the CA. Later studies showedthat oostatic hormone may exert its effect by inhibiting the release of eggdevelopment neurosecretory hormone (EDNH) by the median neurosecre-tory cells of the pars intercerebralis (Adams, 1980). Bioassays have been de-veloped to facilitate characterization and purification of this hormone, butfinal purification has not been reported (DeMilo et al., 1991).
More recently, the amino acid sequences of two folliculostatic peptides ofthe fleshfly, N. bullata, have been determined (Bylemans et al., 1993, 1994,1995). One is named Neb-TMOF (trypsin modulating oostatic factor) and theother Neb-colloostatin. They consist of 6 and 19 amino acids, respectively.Knowledge about the chemical structure will be most valuable to elucidatethe mechanism of the hormone. One interesting observation is worth men-tioning here. Despite the recent progress in the study of oostatic hormone(s),we must remember that none of the so-called hormones has been tested (orbioassayed) according to the originally proposed function of an oostatic hor-mone. So far, all investigators have used the ability of an extract or a prepa-ration to inhibit the development of primary oocytes as the criterion to provethe oostatic property of this extract or preparation. No one has tested theirpreparation or substance against the ability to suppress the development ofthe secondary oocytes (in the presence of the primary oocytes) as the origi-nal definition of oostatic hormone would have dictated. The danger of notadhering to the originally proposed function is real. Since oogenesis is regu-
Juvenile Hormone Regulation of Reproduction 529
Fig. 5. A TEM (1 and 3) and freeze fracture replica (2) showing the endocytotic apparatus ofthe ovaries of P. regina. FC = follicle cells; D = desmosomes; MV = microvilli; L = lipid droplet;Y = yolk sphere; CV = coated vesicle; CP = coated pit; VE = vitelline envelope; OC = oocyte(taken from Mazzini et al., 1987).
530 Yin and Stoffolano
lated at so many different levels, many substances of different modes of ac-tion can cause failure anywhere along the oogenesis pathway. For example, asubstance that mimics the satiety sensation can result in a partial protein-feeding, a failure of oogenesis, and a claim of a new oostatic hormone.
Oosorption
Many of the cyclorrhaphous flies survive as adults in environments lack-ing predictable food, especially protein. If enough protein has been ac-quired to activate the neuroendocrine cascade leading to oogenesis, matureoocytes will develop, but the number depends on the amount ingested.The number of initial oocytes, however, depends on the size of the fly,which is determined by larval nutrition. One way for the fly to deal withthese uncertainties is oosorption. Presumably, oosorption removes essen-tial materials from some oocytes and makes them available to those des-tined to reach maturity.
In insects other than the cyclorrhaphous flies, JH has been shown to bethe major factor in preventing oosorption. Unfortunately, little, if anything,is known about how JH is involved in preventing resorption (Koeppe etal., 1985). An interesting report is that of Fraenkel and Hollowell (1979)on P. regina and N. bullata. They showed that 20-hydroxyecdysone, whenadded to flies whose primary follicles just started to develop, will causeprimary oocytes to degenerate, while development of the secondary oo-cytes occurs.
SUMMARY
The various effects JH has on reproduction in the cyclorrhaphous Dipteraare summarized in Table 2. As illustrated in Figure 1, JH influences the on-togeny of sexual behavior in both sexes, is involved in pupal fat body disap-pearance and adult fat body hypertrophy, development of the ARG in bothsexes, and various aspects of oogenesis
TABLE 2. Possible Involvement of JH’s on Reproduction in the Cyclorrhaphous Dipterans
1. Maturation of gonads No2. Pupal fat body disappearance Yes3. A.R.G. development Indir*4. Ontogeny of sexual behavior Yes5. Mate location and pheromone production No6. Courtship behavior and copulation No7. Ovarian competence N.R.8. Egg laying and larvipositioning N.R.9. Spermatogenesis No
10. Oogenesisa. Fat body competence Yesb. Vitellogenin biosynthesis Yesc. Vg uptake and internalization Yesd. Oostatic hormone Yes
*Indirect evidence only but implicates JH involvement.N.R. = none reported.
Juvenile Hormone Regulation of Reproduction 531
In conclusion, we believe that many interesting aspects of JH involvementin reproduction within this important group of flies remains to be investi-gated. Even though specific dipteran systems seem to dominate the litera-ture, investigations on different species may shed new light on variousevolutionary aspects of JH involvement in the reproduction of this excitinggroup of flies.
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