Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1993 Genetical and physiological mechanisms of face fly, Musca autumnalis DeGeer, diapause Yonggyun Kim Iowa State University
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Genetical and physiological mechanisms of face fly, Musca autumnalis DeGeer, diapause
Kim, Yonggyun, Ph.D.
Iowa State University, 1993
UMI 300N.ZeebRd. Ann Arbor, MI 48106
Genetical and physiological mechanisms
of face fly, Musca autumnalis DeGeer, diapause
by
Yonggyun Kim
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirement for the Degree of DOCTOR OF PHILOSOPHY
Department: Entomology Major; Entomology
Approved: Members of the Committee;
Signature was redacted for privacy.
Signature was redacted for privacy.
For the Ma]or Department Signature was redacted for privacy.
Signature was redacted for privacy.
Iowa State University Ames, Iowa 1993 ii
TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iv GENERAL INTRODUCTION AND LITERATURE REVIEW 1 Genetic mechanisms 2
Physiological mechanisms 10
Diapause studies in face flies 33
Explanation of dissertation format 35
PAPER I. MODE OF INHERITANCE OF FACE FLY DIAPAUSE AND ITS CORRELATION WITH OTHER DEVELOPMENTAL TRAITS 37 ABSTRACT 39 INTRODUCTION 41 MATERIALS AND METHODS 44 RESULTS 51 DISCUSSION 65 ACKNOWLEDGEMENTS 70 LITERATURE CITED 71
PAPER II. VITELLIN, VITELLOGENIN, AND OTHER STORAGE PROTEINS IN Musca autumnalIs 77
ABSTRACT 79 INTRODUCTION 80 MATERIALS AND METHODS 82 RESULTS 86 DISCUSSION 101 ACKNOWLEDGEMENTS 106 LITERATURE CITED 107
PAPER III. IN VIVO AND IN VITRO EFFECTS OF 20- HYDROXYECDYSONE AND METHOPRENE ON REPRODUCTIVE DEVELOPMENT IN DIAPAUSING Musca autumnalis 114
ABSTRACT 116 INTRODUCTION 117 iii
Page MATERIALS AND METHODS 120 RESULTS 125 DISCUSSION 134 ACKNOWLEDGEMENTS 138 LITERATURE CITED 139
GENERAL SUMMARY 145 LITERATURE CITED 150 iv
ACKNOWLEDGEMENTS
I would like to thank the members of my committee, Drs., Carl L. Tipton, Elliot S. Krafsur, Joel R. Coats, Kenneth J. Koehler, and Stephen H. Bishop for their guidance and assistance with this research project. I owe a particular debt of gratitude to my advisor Dr. Krafsur, who provided a great deal of support and patience with this work and always allowed me the time and opportunity to pursue a variety of other research interests. I am very grateful for the assistance of laboratory technicians, Suling Zhao and Nariboli Pramoda. Thanks also to fellow graduate student, Armando Resales for his assistance, support, and friendship. Finally, very special thanks to my entire family, particularly my parents and most of all to Youngim, my wife, for their extraordinary support, encouragement, and patience over many years. 1
GENERAL INTRODUCTION AND LITERATURE REVIEW
Predictably unfavorable conditions, such as extreme temperatures and photoperiods, drought, and a short food
supply are inevitably encountered by most insects established in temperate climates. To survive such unfavorable periods,
insects have developed numerous mechanisms ranging from a
simple reduction in metabolic rates with quick recovery to complicated, genetically-programmed diapause development. Diapause is a developmental option in a specific stage
according to species. Adult diapause occurs in some Coleoptera, Lepidoptera, Diptera, Homoptera, Hemiptera, Orthoptera, Neuroptera,
Trichoptera, and Acarina (Beck, 1980; Danilevsky, 1965; Saunders, 1982). The unique feature of adult diapause is suppression of reproduction. In females, the primary oocytes within the germarium are formed, but do not undergo vitellogenesis. In males, mature spermatozoa are already formed at adult emergence but the accessory glands are involuted and do not have secretory activity (de Wilde, 1983).
Adult diapause also has several metabolic characteristics: a low rate of oxygen consumption (Denlinger, 1985), synthesis of diapause-associated proteins (de Loof and 2
de Wilde, 1970; Dortland, 1978; Turunen and Chippendale,
1980), and high activity of JH specific esterase (Kramer and de Kort, 1976). These diapause traits are expressed in response to environmental stimuli during the pre-diapause and diapause induction period. Diapause development is mediated through specific endocrine changes (Denlinger, 1985; Tauber et al., 1986). Depending on the amount of hormone produced, the hormonal target tissues undergo reproductive or diapause development.
Genetic mechanisms
Diapause as a quantitative trait
Diapause expression and its interaction with environment are under genetic control (Danks, 1987). Diapause is a threshold trait. The threshold levels of critical photoperiod are normally distributed in a population (Gibbs, 1975; Beck, 1980). Thus, diapause is a quantitative trait. The quantitative trait is measurable and has a continuous property because of simultaneous segregation of many genes affecting the trait and interactions between genotype and environment. Quantitative traits are also called "polygenic" because they are concerned with many small, additive gene effects. 3
Genetic mechanisms in diapause focus on mode of inheritance by actions of diapause-associated genes. Estimations of heritability and number of effective genes are valuable to understand a mode of diapause inheritance.
Variance components
In the simplest case with no interaction between genotype and environment, measured phenotypic value (P) results from
the effects of genotype (G) and environment (E). P = G + E (1)
Genotypic value is composed of additive (A), dominance (D) and
interaction (I) terms. G = A + D + I (2)
In this way, phenotypic variation (ff^p) can be divided into
genetic (a^g) and environmental (a^g) components. Genetic variance is further partitioned into additive , dominance
(CT^p) , and interaction (a^,) components. a\ = + a\ (3)
= + cT^j + (4)
Estimation of heritability
The relative importance of heredity in determining phenotypic values is called heritability (review by Falconer, 1981). Estimates of the proportion of phenotypic variation 4
that is genetic are called broad-sense heritabilities and are based on estimates of genetic (a^^) and phenotypic (a^p) variances. The general formula for broad-sense heritability
(H) is H = oZg/oZp.
Broad-sense heritability is a general estimate of degree of genetic determination. It includes each average gene effect (additive effect), intra and interlocus interactions
(dominance and epistasis). The contribution of intralocus interaction, as well as some interlocus interactions, is not transmitted from parent to progeny because two alleles, or sets of alleles, are separated at meiosis. Therefore, broad- sense heritability would give the breeder an inaccurate measure as to what extent selection would be effective for a trait of interest.
Narrow-sense heritability limits measurement of genetic variation to that associated with the additive effects of individual alleles influencing a character. It determines the degree of resemblance between relatives and so gives the most important information to breeders. The additive variance (a^^) is separated from and the general formula for narrow-sense heritability (hf) is h2= a\lo\.
Difference between narrow-sense heritability and broad- 5
sense heritability estimates calculated from the same data
sets are caused primarily by nonadditive gene effects.
Several techniques to estimate heritability have been proposed (Warner, 1952; Falconer, 1981):
1) parent-offspring regressions 2) variance analysis among sibs 3) variance partitioning using generation variance
Estimation of minimum number of genes Estimating the number of the genes contributing to
quantitative genetic variance is necessary to understand physiological mechanisms as well as evolutionary mechanisms of a biological phenomenon.
The original method of Wright (in Castle, 1921) used two inbred lines to estimate the minimum effective gene number. Wright later (1968) included backcrosses. Lande (1981)
generalized Wright's formulas to crosses between genetically heterogeneous populations to minimize the inbreeding depression in each parental line and to reduce the
experimental time. For a normally distributed trial, the minimum number (Ng) of genes contributing to quantitative traits is
N, = (Pj - Pi)2 / Qa\, where P, and Pg are phenotypic values of both parents (Lande, 6
1981). Under the assumptions of no dominance, epistasis, and no environmental interaction, genetic variances (a^g) may be calculated by one of the four equations:
(1) a\= (T^F2 - (2) = + Wpz)'
(3) = - *^2' (4) = o\^ + + ho^p^)'
where a^pg, cr^pg' °'^bi' are variances of parents, F,, Fg, and backcrosses. Lande (1981) also mentioned that the effective number of genes estimated by his method cannot exceed the number of crossing-overs at meiosis
("recombination index" of Darlington, 1937), which equals the number of recombination events per gamete. Cockerham (1986) corrected the squared difference between the means of two parents in Lande's equation by subtracting the sum of their experimental variances;
(Pg - Pi)' = (Pg - Pi): - (o%i + c%2)' While Lande (1981) gave four measures of genetic variance, Cockerham (1986) estimated only one genetic variance by a least squares method using information from all crosses:
= 0.2 (4 + 0^2) - 0.4 (a%i + . Lande (1981) formulated an approximate variance of Ng on the assumption that Ng is normally distributed:
V(Ng) = NgZ {4 (a\^ + 0^2) / (Pa - Pi): + V(o\) / cj\}, 7
where / Np,. Cockerham (1986) gave one estimate of V(a^g) by an unweighted least square method: V(a\) = 0.08 (16 / Ngg) +
0.32 (a\i / Npi + a^p2 / Np2 + . From several examples (Lande, 1981) in which the parental mean differences ranged from 6 to 30 phenotypic standard
deviations, the effective minimum gene numbers were 5 to 10 with occasional values up to 20. Linkage of loci influencing a trait causes Ng to be underestimated because it increases
the genetic variance (a^^).
Modes of diapause inheritance Inheritance patterns of insect diapause vary among species. Diapause inheritance in many species has been
explained by a polygenic control because of a continuous variation among populations in quantitative characteristics such as critical photoperiod, duration of diapause, and rate of diapause development (Lees, 1968; Masaki, 1984; Tauber et al., 1986). Geographical variation in diapause response was analyzed through hybridization tests with naturally diversified populations of the knotgrass moth, Acronycta rumicis and suggested a variation of gene frequencies according to local populations (Danilevsky, 1965; Lees, 1968).
Estimations of the number of the genes involved in polygenic 8
diapause were conducted in the flesh fly, Sarcophaga bullata
Parker (Henrich and Denlinger, 1983), the fruit fly, Drosophila littoralis Meigen (Lankinen, 1986), and the bug, Oncopeltus fasciatus (Dallas) (Hayes et al., 1987). These studies showed that a small number of loci may be involved in polygenic control of diapause.
Some species, in contrast, showed a simple Mendelian inheritance of diapause such as in two sibling species of the antlion, Chrysopa (Tauber et al., 1977) and in the blow fly, Calliphora vicina Robineau-Desvoidy (Vinogradova and Tsutskova, 1978), where one or two genes control diapause with complete dominance. In D. littoralis, an autosomal unit of closely linked genes ("supergene") is responsible for diapause inheritance. This supergene is segregated in a Mendelian way, but its phenotypic variance follows a polygenic trait (Lumme and Oikarinen, 1977; Lumme, 1981).
Sex-linkage of diapause-associated genes was reported in many species. The gene carried by the heterogametic sex is normally fully expressed, but the gene in the homogametic sex is expressed according to a dominance relationship. In most sex-linked species in diapause, the inheritance is chiefly through the female parent. Paternal inheritance, however, was reported in the moths, Pionea forficalis (Linnaeus) (King, 1974), Heliothis species (Stadelbacher and Martin, 1981) and 9
some other Lepidoptera. This may be due to the fact that the heterogametic sex is the female in Lepidoptera (Danks, 1987).
Epistasis and pleiotropism are also involved in diapause inheritance (Danks, 1987). Epistasis, the interaction of two or more loci, was involved in diapause inheritance of S. bullata (Henrich and Denlinger, 1983). Pleiotropism is indicated when a single gene has more than one phenotypic effect such as both diapause response and body color in D. littoralis (Lumme and Oikorinen, 1977). Genetic analysis of diapause gave high heritability estimates (=high additive genetic variance); ca. 0.70 for the ages at first reproduction in O. fasciatus (Dingle et al.,
1977), 0.43 to 0.82 for heat requirements for the completion of diapause in the moth, Hyphantria cunea (Drury) (Morris and Fulton, 1970), 0.77 ± 0.36 for the number of days to emergence in H. zea (Holtzer et al., 1976), and 0.31 to 1.13 (sic) for diapause in the cricket, Allonemobious fasciatus DeGeer
(Mousseau and Roff, 1989). Diapause was genetically correlated with developmental rates in some species. Selection for fast development in the mosquito, Wyeomyia smithii (Coquillett), produced a correlated response of a decrease of diapause induction (Istock et al., 1976). Selection for late pupariation in S. bullata resulted in a line that showed a higher diapause incidence than the 10
parental line (Henrich and Denlinger, 1982).
Physiological mechanisms
Adult diapause (== imaginai diapause) is maintained by the suppression of reproductive development. Thus, adult diapause
mechanisms are very closely related to reproductive pathways.
Spermatogenesis
Spermatogenesis produces haploid spermatozoa from diploid spermatogonia in male reproductive systems (review by Chapman,
1982). In each follicle of the testis, a range of development is present with the earliest stages distally in the germarium and the oldest in the proximal part of the follicle adjacent to the vas deferens. Typically, three developmental zones are formed below the germarium. A zone of growth is located just below the germarium, in which spermatogonia, enclosed in cysts, divide and increase in size to form spermatocytes. In a zone of maturation, each spermatocyte undergoes meiosis to produce spermatids. Spermiogenesis, the process in which the spermatids develop into spermatozoa, then occurs in a zone of transformation.
In most male insects, meiosis is complete before adult emergence. Especially in species which do not feed and have a 11
short life span as adults, spermatogenesis may be complete before adult emergence (Engelmann, 1970). Endocrine studies on spermatogenesis are not as frequent as on vitellogenesis. Several early workers suggested that juvenile hormone (JH) inhibited differentiation of sperm, but ecdysone promoted this process (review by Dumser, 1980). This model of spermatogenesis indicates that adults are unfavorable for spermatogenesis because they lack ecdysteroids and have active corpora allata. Dumser and Davey (1975) proposed that the hormones affect only the rate of spermatogonia! mitosis and not differentiation and that there is a basal rate of mitosis even in the presence of JH. Thus in adults, spermatogenesis continues at the basal rate, despite the presence of juvenile hormone in the hemolymph. Male accessory glands are fully differentiated at adult emergence. During sexual maturation, they become active and increase in size. In many insects, there is a clear correlation between the corpora allata (CA) activity and secretory activity of accessory glands (Engelman, 1970; Gillott and Friedel, 1976). In most species, diapause in males is characterized by involution of the accessory glands and the absence of sexual behavior (Denlinger, 1985). 12
Vitelloaenesis Vitellogenesis is the process of egg maturation in the
female reproductive system and is mediated by endocrines under the control of external factors such as photoperiod,
temperature, and feeding (Adams, 1981; Hagedorn and Kunkel,
1979). JH, which is synthesized in the CA, acts as a gonadotropin and activates vitellogenin (Vg) synthesis in the fat body of most insect species. Ecdysteroids are produced by the ovaries and stimulate the fat body to synthesize Vg in some dipteran species, e.g., the mosquitoes, Aedes aegypti (Linnaeus) (Hagedorn and Kunkel, 1979), the fruit fly,
Drosophila melanogaster (Meigen) (Postlethwait et al., 1980), and the house fly, Musca domestica Linnaeus (Adams and Filipi,
1988). The median neurosecretory cells (mNSCs) produce allatotropin, which then activates JH synthesis in the CA, and may also produce egg development neurosecretory hormone (EDNH) that acts as gonadotropin in some insect species. The insect ovary produces ovarian hormones that inhibit either gonadotropin release or the activation of the receptor on the follicle cells by the gonadotropin. According to the synchrony of egg maturation in an ovariole, vitellogenesis is divided into asynchronous and synchronous types (Adams, 1981). Synchronous vitellogenesis is further subdivided into intraovariole, interovariole, and 13
uniovariole synchronous types.
Vitellogenin synthesis Vitellogenins are the egg proteins. They are synthesized by the fat body, secreted into hemolymph, and taken up by the
growing oocytes, where they are called "vitellins"
(Engelmann, 1979; Hagedorn and Kunkel, 1979). In D. melanogaster, both the ovary and the fat body synthesize
vitellogenin (Bownes, 1980; Postlethwait et al., 1980).
Ovarian synthesis of yolk proteins was also found in M.
domestica (de Bianchi et al., 1985). Few differences have been found to exist between vitellin
and vitellogenin, although a little difference was found in lipid content and protein subunit structure in some species (Hagedorn and Kunkel, 1979). Vitellogenins of many insects, vertebrates, nematodes and sea urchins are very similar in size and amino acid composition (Blumenthal and Zucker-Aprison, 1986). It appears that they are encoded by the same family of genes. Higher dipteran species, however, have a little different protein and gene structures than the other insects. In contrast to those
of other insects, their vitellogenins consist of small
peptides (45-50 Kd) and have a single copy gene (Rina and Savakis, 1991; Spieth et al., 1985). 14
In D. melanogaster, three yolk proteins were found and range 44-47 Kd in molecular weights. They are encoded by single copy genes (Ypl, Yp2, and Yp3) which are localized on the X chromosome. Genes Ypl and Yp2 are separated by 1.2 Kb and transcribed divergently (Hung and Wensink, 1982, 1983). Yp3 gene is ca. 1000 Kb from the Ypl and Yp2 genes (Barnet et al., 1980). These genes have their own promoters and are coordinately transcribed in response to ecdysone (Hung and
Wensink, 1982). Yolk protein synthesis is under hormonal control. In some insects, such as the grasshopper, Locusta migratoria (Linnaeus) (Wyatt, 1988) and the cockroach, Leucophaea maderae (Fabr) (Engelman, 1979), vitellogenin synthesis is said to be regulated only by juvenile hormone. But, in many species of Diptera, both juvenile hormone and ecdysone are involved in yolk protein synthesis in the fat body and/or the ovary
(Hagedorn, 1985).
Vitellogenin uptake
JH is known to act on the cell membrane of the follicular epithelium and activate a specific Na^,K^-ATPase (Pratt and Davey, 1972; Abu-Hakiwa and Davey, 1979). Activated Na*,K*- ATPase induces reduction of the follicle cell volume which forms intercellular spaces in the follicular epithelium. 15
These spaces allow the entry of hemolymph containing Vg. Ilenchuk and Davey (1985) suggested that JH specific receptors
are located on the cell membrane of the follicular epithelium. Protein kinase C is involved in the linking steps between JH- specific receptor and JH-sensitive Na*,K*-ATPase (Sevala and
Davey, 1989). Vg is specifically taken up and accumulated in the growing oocyte by receptor-mediated endocytosis (Raikhel and
Lea, 1986). In Rhodnius prolixus Staal, this process is facilitated by the presence of Ca** and JH. This facilitation may be explained by the increase of the number of receptors
(Wang and Davey, 1992).
Roles and structures of JH
Since Wigglesworth (1936) first demonstrated the effects of JH in morphogenies and reproductive development in R. prolixus, many other workers have established the
physiological roles of JH in different insects (Engelman,
1970). JH also plays a crucial role in various types of polymorphism (Novak, 1975): (a) caste polymorphism in ants and termites (b) phase polymorphism in locusts (c) seasonal polymorphism in aphids, and (d) polymorphism in wing development in many species in Heteroptera.
JHs have a common sesquiterpenoid structure (Fig. 1). 16
JH
JH II
JH I
JH 0
JH B3
4-Me-JH I
Fig. 1. Naturally occurring juvenile hormones (modified from Schooley and Baker, 1985). 17
The first characterized JH was JH I from the silk moth,
Hyalophora cecropia (Linnaeus) (Roller et al., 1967). It was the first known natural product with a dihomosesquiterpenoid skeleton (Schooley and Baker, 1985). JH II was identified by Meyer et al. (1968) from the same species. JH III, JH 0, and
4-methyl JH I were discovered by July et al. (1973) and Bergot et al.
(1980) from the sphinx moth, Manduca sexta (Linnaeus). JH III is the predominant hormone in most adult insects (Schooley et al., 1976) while JH I and JH II have been identified mainly in larvae of Lepidoptera and in nymphs of the cockroach, Nauphoeta cinerea (Lanzrein et al., 1975). Recently, JH III bisepoxide (JH Bj) has been found to be the major JH in higher
Diptera such as D. melanogastex:, S. bullata (Richard et al., 1989) and Calliphora vomitoria (Linnaeus) (Cusson et al., 1991).
Biosynthesis and degradation of JH The JH titer in the hemolymph is a dynamic equilibrium between the rate of JH synthesis and release by the corpora allata (CA) and the rate of uptake and degradation of the hormone by the tissues (Feyereisen, 1985). After synthesis,
JH is not stored in the gland cells but secreted almost immediately (Tobe and Pratt, 1974). Therefore, the synthetic 18
Acetate + Acetyl CoA Acetate + Acetyl CoA i i Acetoacetyl CoA Acetoacetyl CoA
4. HMG-CoA Synthase -» I 3-Hydroxy-3-inethylglutaryl CoA 3-Hydroxy-3-methylglutaryl CoA I *- HMG-CoA Reductase I
Mevalonate Mevalonate i 1 1 Isopentenyl pyrophosphate Isopentenyl pyrophosphate
Farnesyl pyrophosphate Farnesyl pyrophosphate
Farnesoic acid Squalene i Methyl transferase I i Methyl farnesoate Cholesterol i Epoxidase
Juvenile hormone III
Fig. 2. Biosynthetic pathways of JH III and cholesterol
(modified from Khan, 1988). 19
activity gives a direct effect on JH titer in hemolymph. The synthetic activity is dependent on the precursor amount and on the activity of the key enzymes in the synthetic pathway. Regulation of the JH synthetic activity by the CA has been considered as the primary cause of the low JH titer during adult diapause (Denlinger, 1985).
The biosynthetic pathway (Fig. 2) of JH III is similar to cholesterol biosynthesis in vertebrate liver (Schooley and Baker, 1985). Up to the formation of farnesyl pyrophosphate ('early steps'), the two biosynthetic pathways are almost identical. Thereafter, several final enzymatic steps ('late steps') catalyze the formation of JH.
JH II, JH I, JH 0, and 4-methyl JH I do not follow the synthetic pathway of JH III because of their ethyl branches. Two main explanations for the generation of ethyl branches were postulated; methylation after the formation of JH III by a methyl donor such as S-adenosyl-methionine or condensation of a homoisoprenoid synthesized from propionate via homomevalonate. Most studies so far support the homomevalonate hypothesis from the degradative analysis of radiolabelled products after in vitro culturing in radiolabelled precursors such as acetate, propionate, and mevalonate (Schooley et al, 1973; Peter and Dham, 1975).
The major pathways for JH degradation in insects are 20
hydrolysis of the ester moiety by JH esterase (JHE) and hydration of the epoxide ring by epoxide hydrolase. The
primary products of these reactions are JH acid and JH diol (Slade and Zibbit, 1972). Besides JHE and epoxide hydrolase, mixed function
oxidases (MFO) are involved in JH catabolism in the house fly, M. domestica and some other Diptera (de Kort and Granger, 1981). Piperonyl butoxide and carbon monoxide, which are
inhibitors of MFO, inhibit JH catabolism in vitro (Yu and Terriere, 1978). Phénobarbital, a potent inducer of MFO activity, blocks metamorphosis and vitellogenesis (Yu and
Terriere, 1974). In the hemolymph, JH is degraded only by JHE, which is
synthesized in the fat body and released into hemolymph (Whitmore et al., 1974). Unlike general esterases, JHE are capable of rapidly hydrolyzing both free JH and JH bound to JH
binding protein (Hammock, 1985). JHE is insensitive to 10"' M diisopropylfluorophosphate, while general carboxy-esterases are totally inhibited. The importance of high JHE activity in adult diapause has been shown in L. decemlineata (Kramer and de Kort, 1976). The activity of JHE increases about 10-fold in prediapausing beetles. High JHE activity is not the primary cause for the low JH titer during early diapause. Rather, high JHE activity 21
may make it possible to attain low JH levels rapidly early in diapause induction (de Kort, 1990).
Pathways of stimuli affecting CA activity It has been known from classical endocrinology that insect CA are under the control of the brain (Engelmann,
1970). Several factors affect CA activity. These factors can be classified as external and internal stimuli (Khan, 1988). External stimuli include phoroperiod, food availability, and mating. Internal stimuli are further subdivided into neural and humoral factors from the brain and the target organ (e.g. ovaries). The feedback control of JH titer to CA activity is also one of internal stimuli. Regulation of CA activity in response to external and internal stimuli can be separated into rapid and slow physiological and biochemical responses (Feyereisen, 1985). The effects of both stimuli are expressed via controlling the actions of rate-determining enzymes in JH biosynthesis in the CA. Changes in non-rate-determining enzymes, cell volume or number, and cell ultrastructure also affect the total biosynthetic capacity of the CA.
Corpora allata are innervated from both the brain and the suboesophageal ganglion (Tobe and Stay, 1985). The connection between the gland and the suboesophageal ganglion does not 22
play a role in controlling CA activity because severance of
this nerve did not influence the gland activity in the cockroaches, Diploptera punctata (Stay and Tobe, 1977) and Periplaneta americana (Linnaeus) (Pipa, 1982) , and the
grasshopper, L. migratoria (Couillaud et al, 1984). Neural transport of allatostatins from the brain has been
reported in many species. Williams (1976) proposed the existence of both an allatotropin and an allatostatin for activating and inactivating JH biosynthesis, respectively. Recently, four related peptides with allatostatic activity in vitro have been isolated and characterized from the brain of D. punctata (Woodhead et al., 1989). An allatotropin has been also isolated from M. sexta (Kataoka et al., 1989).
Adult diapause of L. migratoria is under dual control by allatotropic and allatostatic factors (Baehr et al., 1986). The allatostatic factor is located in the lateral NSCs whose
axons reach the CA through NCC II and NCA I (Poras et al., 1983). NCC II or NCA I section inhibited diapause development by removal of CA inhibition. The denervated CA which were
implanted into diapausing hosts continued to be active until 3
days after the operation. When the denervated CA were cultured in vitro, they became active. But, their activity declined rapidly. This difference between in vivo and in vitro results of the denervated CA can be explained by the 23
presence of a blood-borne allatotropic factor. Neural and humoral pathways between the brain and the CA are also found in L. decemlineata (Khan et al., 1983; Khan, 1988). The results consistently indicated that any influence by way of the humoral pathway on the CA activity was subordinate to neural inhibition. The biochemical targets of allatostatin in the CA are as yet unidentified, although they usually appear to be located early in the JH biosynthetic pathway (Feyereisen and
Farnsworth, 1987a). It is likely that cAMP and protein kinase (e.g., protein kinase C) play some role in the regulation of gland activity (Meller et al., 1985; Feyereisen and
Farnsworth, 1987b). The control of JH biosynthesis occurs at the early steps as well as the late steps, and varies among species. The control at the early steps is characterized by stimulating the synthetic activity to many-fold its normal value after addition of farnesoic acid to the incubation medium. This control type has been found in S. gregaria, D. punctata, and L. decemlineata (Feyereisen et al., 1981; Khan et al., 1982). 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase is a key enzyme in the control of JH biosynthesis. This enzyme catalyzes the reduction of HMG-CoA to mevalonate which is an irreversible and an important regulatory step in cholesterol 24
biosynthesis (Brown and Goldstein, 1980). HMG-CoA reductase is a glycoprotein and located in the endoplasmic reticulum (Daughtery-Varsat and Lodish, 1983). Several mechanisms are
involved in the control of HMG-CoA reductase activity during feedback control of cholesterol biosynthesis; by phosphorylation/dephosphorylation (Brown et al., 1979) or by
the control at the level of transcription of the enzyme-coding
gene (Luskey et al., 1983). A good correlation between the activity of HMG-CoA
reductase and the rate of JH biosynthesis has been reported in M. sexta (Kramer and Law, 1980). Its activity seemed to be
controlled by phosphorylation/dephosphorylation (Feyereisen et
al., 1981). Compactin, a substance which inhibits HMG-CoA reductase specifically, inhibited the rate of JH biosynthesis (Edwards and Price, 1983).
Diploptera punctata, however, did not show any correlation between the activity of HMG-CoA reductase and the
rate of JH biosynthesis (Feyereisen and Farnsworth, 1987a).
When the rate of JH biosynthesis was inhibited in vitro by exposure to high K*-concentration, the activity of the enzyme was not significantly lowered (Feyereisen and Farnsworth,
1987b). The control of JH biosynthesis at the late steps was reported in the inactivation of CA at the end of the last 25
larval instar in M. sexta (Bhaskaran et al., 1986). The
inactivation was due to the loss of JH-methyl transferase activity but not the HMG-CoA reductase.
Ecdvsteroids in adult diapause
Ecdysteroids, the molting hormone in insects, are produced in a prothoracic gland during immature stages. This
gland is under brain control, via a neuropeptide hormone known as prothoracicotropic hormone. Prothoracic glands degenerate with adult development.
The production of ecdysteroids depends on dietary cholesterol because insects cannot synthesize cholesterol, a- Ecdysone is the first characterized ecdysteroid in Calliphora
(Karson et al., 1965). Later, 20-hydroxyecdysone (8-ecdysone) was found in Bombyx mori Linnaeus and other insects. 2 0- Hydroxyecdysone is a more effective form than a-ecdysone in many insects. In higher diptera, ecdysteroids as well as JH have a role
in inducing vitellogenin gene expression in the fat body
(Bownes, 1980; de Bianchi et al., 1985). The major source of ecdysone in adults is the ovaries and the testes even though oenocytes or epidermal cells synthesize a little ecdysone in some species (Hagedorn, 1985). In A, aegypti, egg developing neurohormone is released from the corpora cardiaca after a 26
blood meal and stimulates the synthesis of ecdysone in the ovaries (Shapiro and Hagedorn, 1982). Adams and Filipi (1988) have shown that both JH and 20-hydroxyecdysone are necessary for the production of normal levels of vitellogenin in M. domestica. In L. decemlineata, ecdysteroid level slowly increased throughout diapause (Briers et al., 1982). Lefevere (1989) suggested that ecdysone plays a key role in diapause termination by modifying the regulation of the JH metabolism.
Metabolic rate depression
Diapausing insects are usually biochemically distinct from nondiapausing counterparts (Denlinger, 1985). Two major stresses in overwintering species are limited food sources and subzero temperatures. Lowering metabolic rates is of adaptive significance in many species of animals in the face of unfavorable conditions such as anoxia, aestivation, and diapause (Storey and Storey,
1990). Diapause can lower aerobic metabolic rates up to 20- fold (Storey, 1988). Three biochemical mechanisms have been known to control metabolic rates: covalent modifications such as phosphorylation or dephosphorylation of glycolytic enzymes, pathway regulation by association or dissociation of enzymes 27
into active complexes, and control of carbohydrate usage by an allosteric modulator. Fructose-2,6-bisphosphate is a strong activator of phosphofructokinase which is a key regulating enzyme of glycolysis. Reduced level of the activator limits the use of carbohydrate and leads to metabolic rate depression.
Subzero temperatures pose a severe threat to insects. Injuries caused by subzero temperatures are divided into cold injury and freezing injury. Cold injury occurs above freezing temperatures and is caused by malfunction of lipid bilayers and disruption of metabolic regulation (Quinn, 1985). Freezing injury includes physical and metabolic damages by ice crystals (Lee and Denlinger, 1991). Storey and Storey (1990) explained both devastating effects caused by subzero temperatures in disruption of cell membrane potential; responsiveness of a cell depends upon the presence of a membrane potential. Generally, animal cells actively extrude [Na*], but maintain higher [K*] and [Cl'] inside than outside. This ionic concentration gradient establishes a membrane potential. This ionic concentration gradient is maintained by an ATP-driven ion pump to compensate for the tendency of ions to flow down their concentration gradients. Under hypoxia or hypothermia, ATP production is affected. ATP-driven ion pumps rapidly fail and the backward flow of ions through ion 28
channels leads to rapid membrane depolarization. When
membrane depolarization reaches a critical level, unregulated
Ca'*"^ influx is initiated and triggers a variety of Ca**- stimulated disruptions.
Cold tolerance Many species survive in subzero temperatures by physiological and biochemical adaptations conferring cold
hardiness (Storey, 1990). Freeze-avoidance and freeze- tolerance are two major kinds of overwintering adaptation.
Freeze-avoiding insects avoid harmful ice formation by deep depression of the supercooling point (SCP) of body fluids. They synthesize polyols such as glycerol and sorbitol in
response to low temperatures. The low temperatures activate
glycogen phosphorylase and allow an unidirectional change of
glycogen to polyols (Lee et al., 1987). Antifreeze proteins or thermal hysteresis proteins also play a role in
cryoprotection, lowering the freezing and SCPs (Duman, 1977;
Duman and Horwath, 1983). Freeze-tolerant insects tolerate ice formation in the extracellular fluid. Survival of freezing comes from limiting the ice formation only to the extracellular compartment and preventing cell volume reduction and dehydration. Ice- nucleating proteins induce and control extracellular ice 29
formation. Thermal hysteresis proteins may attach to the surface of ice crystals and prevent recrystallization of ice
crystals. The accumulation of polyols are common to both survival strategies. Besides glycerol and sorbitol, other polyols are
present in cold-hardy insects; - trehalose and sucrose,
- glucose and fructose, Cg - ribitol, - erythritol and threitol. They are nontoxic and freely penetrate cell membranes to achieve concentration equilibria across the
membrane. These polyols act as antifreeze agents by their colligative properties and stabilize membrane proteins by hydrogen bonds to prevent denaturation as a consequence of low temperature or freezing (Storey, 1988). Polyols are synthesized from glycogen reserves in the fat
body (Fig. 3). Sorbitol is synthesized from glycogen by the sequential actions of glycogen phosphorylase, phosphoglucomutase, glucose-6-phosphatase, and polyol dehydrogenase. Glycerol is formed from glyceraldehyde-3- phosphate by triose phosphate isomerase, «-glycerophosphate dehydrogenase, and a-glycerophosphatase. Both syntheses differ in their ATP requirements; glycerol synthesis depends on the presence of ATP, but sorbitol synthesis does not. Gall fly larvae, Eurosta solidaginis, produce cryoprotectants under a Ng atmosphere (Baust and Lee, 30
Glycogen phosphorylase (inactive) Glycogen I *- Low temperature (0-5°c) I < Glycogen phosphorylase (active)
Glucose-l-phosphate
Glucose-6-phosphate —• Glucose » Sorbitol Polyol I dehydrogenase
Fructose-6-phosphate
Fructose-1,6-bisphosphate
Glyceraldehyde-3 Dihydroxyacetone -phosphate phosphate
4 I 1 Glycerol-3-phosphate " Glycerol Pyruvate Glycerol-3 -phosphatase 4 TCA cycle
Figure 3. Synthesis of major cryoprotectants in response to low temperature: glycerol and sorbitol (modified from Storey and Storey, 1988). 31
1982). Production of glycerol was reduced to 57%, but
sorbitol biosynthesis was activated. Total cryoprotectants were 93% of those produced in aerobic conditions. Synthesis of polyols requires reducing power in the form
of NADH or NADPH. Several experiments (Kageyame, 1973; Tsumuki et al., 1987; Wood and Nordin, 1976) support the hypothesis that the pentose phosphate cycle is the source of
these reducing agents rather than the tricarboxylic acid cycle. Polyol synthesis is under enzymatic regulation by
glycogen phosphorylase and phosphofructokinase (Storey, 1988). Low temperatures initiate polyol production by activating glycogen phosphorylase. This enzyme can change between an
active and an inactive form. Phosphorylase kinase phosphorylates glycogen phosphorylase to the active form and phosphorylase phosphatase dephosphorylates the enzyme to the
inactive form. Low temperatures do not change the activity of the kinase, but inactivate the phosphatase. The result is a net activation of glycogen phosphorylase. Once polyol biosynthesis occurs, phosphofructokinase (PFK) determines the kind of a cryoprotectant to be synthesized. Differential regulation of PFK controls the synthesis of glycerol and sorbitol in E. solidaginis (Storey et al., 1981). At high temperatures, activated PFK leads to 32
the production of glycerol, but at low temperatures, inactivated PFK results in the production of sorbitol.
Diapause-associated proteins Some proteins may be unique to diapausing individuals.
These proteins are usually called diapause-associated proteins
(DAP). DAP have been detected in the hemolymph and fat body of some Lepidoptera and Coleoptera such as the southwestern
corn borer, Diatraea grandiosella Dyar (Brown and Chippendale, 1978), stem borer Busseola fusca (Fuller) (Osir et al., 1989), pink bollworm, Pectinophora gossypiella (Saunders) (Salama and Miller, 1992), codling moth, Cydia pomonella (Linnaeus) (Brown, 1980), Colorado potato beetle, L. decemlineata (de Loof and de Wilde, 1970), and leaf beetle, Gastrophysa atrocyanea Motch (Ichimori et al., 1990). Molecular weights of native proteins vary from 41 kDa in L. decemlineata to 500 kDa in S. fusca. DAP have been suggested to play a role in diapause induction and maintenance, but the exact physiological function has not been determined. The suggested functions include JH storage and transporting (Yin and Chippendale, 1976), a storage protein (Brown and Chippendale, 1978), and a lipid carrier (Turunen and Chippendale, 1980). 33
Diapause studies in face flies
Literature review Face flies undergo a facultative reproductive diapause characterized by hypertrophied fat body and arrested
vitellogenesis (Stoffolano and Matthysse, 1967). Other diapause traits include an increase of total lipid content (Valder et al., 1969; Read and Moon, 1986; Kim and Krafsur, unpublished data), cold hardiness (Resales et al., 1993), depressed feeding (Stoffolano, 1968), no mating behavior (Ode and Matthysse, 1967; Krafsur et al., 1985), and a negative phototaxis (Stoffolano and Matthysse, 1967).
Photoperiod, temperature, and light intensity have significant effects on diapause induction in laboratory simulations (Stoffolano and Matthysse, 1967; Caldwell and
Wright, 1978; Read and Moon, 1986; Evans and Krafsur, 1990; Burks et al., 1992). In natural Iowa face fly populations, diapause is induced in the second week of September even though substantial temperature variation among years occurs during this week (Krafsur et al., 1986). Temperature was not predictive of the time when diapause was induced.
Valder et al. (1969) stated that only adult flies were sensitive to diapause-inducing conditions in the first two days after adult eclosion. Read and Moon (1986) observed a 34
progressive change in diapause commitment according to the duration adults were kept in a diapause-inducing environment. Moreover, a recent study found an additive effect of the environment experienced during immature stages on adult development (Kim and Krafsur, unpublished manuscript). There seems to be genetic variation among different face fly strains in diapause incidence. Caldwell and Wright (1978) showed that a recently established strain could be induced to diapause more readily than an older strain. The change in diapause response may be caused by inadvertent selection in laboratory culture (Danks, 1987). Application of the mean Iowa outdoor diapause-inducing environment to face fly adults in the laboratory did not induce diapause development in a strain derived from Iowa flies (Evans and Krafsur, 1990; Kim and Krafsur, unpublished data). Krafsur and Black (1992) explained that significant genetic differentiation at 12 allozyme loci among six laboratory strains was probably caused by random genetic drift rather than geographical variation in the populations from which the flies originally were sampled.
Female face flies have polytrophic ovaries and the ovarioles undergo vitellogenesis synchronously (Miller and Treece, 1968). About 70 degree days over a 12°C threshold
(DD^^g.g) are needed for females to become gravid and oviposit (Krafsur et al., 1985). Diapausing females had smaller 35
corpora allata than reproductive flies and topical application
of methoprene induced vitellogenesis in diapausing face flies
maintained in a diapause environment (Burks et al., 1992). Thus, low JH titer can be used as a major signal for diapause development in face flies.
Research objectives
This study was to determine the mode of diapause
inheritance, including the estimation of the number of
diapause-associated genes and diapause heritability. The other purpose of this study was to identify endocrine signals during diapause induction in face flies. For this purpose, face fly vitellogenin was isolated and characterized for use as a molecular indicator of reproductive development for in vitro hormonal experiments.
Explanation of dissertation format
This dissertation consists of three papers that will be submitted for publication. These papers are preceded by a general introduction and literature review and are followed by a general summary. References that are cited in the general introduction, literature review, and general summary follow the general summary. Paper I analyzes the inheritance pattern 36
of face fly diapause and determines the factors influencing
the inheritance. Paper II examines the specific hemolymph proteins in reproductive and diapausing face flies. Paper III determines the roles of juvenile hormone and ecdysone in diapause development. Paper I will be submitted to The Journal of Heredity, paper II to Insect Biochemistry and
Molecular Biology, and paper III to Journal of Insect Physiology. 37
PAPER I. MODE OF INHERITANCE OF FACE FLY DIAPAUSE AND ITS CORRELATION WITH OTHER DEVELOPMENTAL TRAITS 38
Mode of Inheritance of Face Fly Diapause
and Its Correlation with Other Developmental Traits
Y. Kim\ E. S. Krafsur\ and T. B. Bailey^
^Department of Entomology and ^Department of Statistics Iowa State University Ames, Iowa 50011 39
ABSTRACT Face flies, Musca autumnalis DeGeer, respond to short photoperiods by entering a facultative diapause. Diapause responses at 17°C to different photoperiods varied among seven laboratory face fly strains. Old laboratory strains lost much of their diapause responses to short days. To analyze the nature of inheritance of diapause, hybrids and backcrosses were made between two strains that showed the greatest difference in diapause induction at 10:14 h (L:D), and a mean 17°C. Hybrids had responses to the short photoperiod that were intermediate to those of their parents. Joint scaling tests showed that diapause did not fit an additive model. The lack of fit to the additive model was attributed to incomplete dominance of diapause-associated genes. No cytoplasmic effect was detected. Another characteristic of diapause inheritance in face flies was sex-linkage of some diapause-associated genes. Estimates of the minimum number of genes involved in diapause suggested that a small number were involved. Heritabilities varied among replications with mean estimations of ca. 50% and 30% for broad and narrow sense heritabilities, respectively. Developmental rates of egg to adult, follicle growth, and pterin accumulation correlated significantly with diapause propensities among lines. High diapause lines had slower developmental rates than low diapause lines, suggesting 40
that diapause may be a pleiotropic effect of genes controlling developmental rates. Alternatively, diapause-associated genes may be closely linked to genes controlling developmental rates. 41
INTRODUCTION Diapause, in most insects, plays an important role in accommodating seasonal change (Dingle et al., 1977; Tauber et al., 1986; Danks, 1987). During diapause, there is suppression of both developmental and metabolic rates (Denlinger, 1985; Storey and Storey, 1990).
Face flies, Musca autumnalis DeGeer (Diptera; Muscidae), undergo a facultative reproductive diapause characterized by hypertrophied fat body and arrested vitellogenesis (Stoffolano and Matthysse, 1967; Valder et al., 1969). Photoperiod, temperature, and light intensity demonstrated significant effects on diapause induction (Stoffolano and Matthysse, 1967;
Caldwell and Wright, 1978; Read and Moon, 1986; Evans and Krafsur, 1990; Burks et al., 1992). The environmental threshold for diapause induction differs between laboratory and field populations (Krafsur et al., 1986). Caldwell and Wright (1978) reported a difference in diapause incidence between two laboratory strains of face flies, a difference inversely correlated with the colonization period. Various laboratory strains of face flies showed significant genetic differentiation at 12 polymorphic loci, probably the result of random genetic drift (Krafsur and Black, 1992). Diapause is genetically programmed and influenced by environment (Tauber and Tauber 1979; Beck 1980; Saunders 42
1982). Diapause inheritance in most insects has been explained by polygenic control because quantitative values related to diapause show a continuous variation among different populations (Lees, 1968; Masaki, 1984; Tauber et
al., 1986). Geographical variation in diapause response was analyzed (Danilevsky, 1965) and interpreted as local
differences of polygene frequencies (Lees, 1968).
Some insect species, including the sibling species of the antlion, Chrysopa (Tauber et al., 1977) and the blow fly, Calliphora vicina Robineau-Desvoidy (Vinogradova and Tsutskova, 1978), showed simple Mendelian inheritance of diapause, where one or two loci with complete dominance
control diapause characteristics.
In some species, sex-linkage, maternal or paternal effects, and epistasis have been reported to be involved in diapause inheritance (Danks, 1987). Genetic analysis of
diapause indicated a high proportion of additive genetic variance in the bug, Oncopeltus fasciatus (Dallas) (Dingle et al., 1977) and the cricket, Allonemobious fasciatus DeGeer
(Mousseu and Roff, 1989). Diapause, in some species, is genetically correlated with other quantitative traits related to developmental rates.
Selection for fast development in the mosquito, Wyeomyia smithii produced a correlated decrease in diapause induction 43
(Istock et al., 1976). Selection for late pupariation in the flesh fly, Sarcophaga bullata Parker resulted in a line that showed a higher diapause incidence than the parental line (Henrich and Denlinger, 1982). The purposes of the research reported in this paper were to investigate genetic mechanism of diapause in face flies and to explain the variation of diapause incidence among their laboratory populations. We conducted a genetic analysis to determine the mode of diapause inheritance and to estimate the minimum number of diapause-associated genes and the heritability. We also estimated correlations of diapause with several other developmental traits. 44
MATERIALS AND METHODS Establishment of laboratory strains Seven laboratory strains were used. IA83 strain was colonized from flies near Ames, Iowa in 1983 (Evans and Krafsur, 1990). IA91 strain was the second generation progeny from flies collected near Ames, Iowa in 1991. The MN strains
were established by R. D. Moon from flies collected in Stevens Co., Minnesota, in 1990 and 1991. KS was a laboratory strain from Kansas. MDl was a laboratory stain from Maryland, established in 1972. MD2 was a subcolony of MDl maintained in the laboratory at Kansas State University. KY was a laboratory strain from Kentucky.
Flies were fed water, pollen candy and a mixed dry diet of sugar, powdered egg and powdered milk (Arends and Wright,
1981) ad libitum at 16:8 h (L:D) at 26° ± 4°C. Ovipositions by the gravid adults were allowed for 4 h on bovine dung. Rearing conditions were 16:8 h (L:D) at 30:25°C (12:12 h).
Puparia were harvested daily. Emerged adults were collected daily between 1000 h and 1200 h (lights-on at 0800 h) and were used in testing for diapause or reproductive development. More than 99% of the flies emerged between 2200 h and 1200 h in an emergence day (Kim and Krafsur, 1993). 45
Diapause responses for seven laboratory strains to photoperiods The newly emerged flies from the old laboratory strains
{KS, MD2, and KY) were treated with photoperiods of 0:24 h,
4:20 h, 10:14 h, 16:8 h, and 24:0 h (L:D) at 23°:11°C (12:12 h). An experimental unit consisted of ten female flies in a cage of a strain and was replicated three times. After 80
degree-days above a 12°C threshold, flies were collected and
kept at -70° until dissection. Evaluation of diapause and reproductive development followed the criteria of Evans and
Krafsur (1990), where females having both previtellogenic
ovaries and hypertrophied fat body were defined as being in diapause and males were judged to be in diapause if they had
hypertrophied fat body. Flies from the other strains (IA83, MDl, IA91, and MN) were treated with photoperiods of 4:20 h, 8:16 h, and 10:14 h (L:D) at 23°:11°C (12:12 h).
Crossing design Crosses between the IA83 and MN strains were performed to obtain F^. F^ were selfed to obtain an Fg. F, were backcrossed to the lA and MN parents to obtain lABC and MNBC, respectively. Fourteen crosses were made; the crosses were classified into the following generation types: 46
Generation types Cross lines (cf * 9)
lA (lA * lA) MN (MN * MN) (lA * MN) , {MN * lA) F2 (lA * MN)*(IA * MN) , (MN * lA)* (MN * lA) lABC (lA * MN)*IA, IA*(JA * MN) (MN * IA)*IA, IA*(MN * lA) MNBC (JA * MN)*MN, MN*(lA * MN) (MN * IA)*MN, MN*(MN * lA)
The experiment was replicated five times. Some replications did not have all 14 lines because of unsuccessful crossings.
The total number of cross lines among five replications was 42. In each cross, » 50 adult flies of each sex were tested for diapause induction in 10:14 h (L;D) at 24°:11°C (12:12 h).
Analysis of diapause inheritance Effects of replication, among-generation types, and reciprocal crosses within generation types were analyzed by PROC GLM in SAS (SAS Institute, 1988). The analyzed variable was mean follicle length. The estimated correlations (r) between follicle length and diapause incidence were similar 47
among different generation types with a range of -0.74 to - 0.87. The joint scaling test of Cavalli (1952) was used for testing the goodness of fit of the observed data to an additive model. To analyze further the same hypothesis on the basis of incomplete dominance, the model of Henrich and
Denlinger (1983) was used.
Estimation of minimum number of genes Lande's (1981) and Cockerham's (1986) methods were used for the estimation of the minimum number of genes involved in diapause. Lande's method in BASIC (Angus, 1983) was used to make the estimations for each replication. Cockerham's method was applied by hand.
Estimation of heritability Heritabilities of diapause in the broad (Mahmud and Kramer, 1951) and narrow (Cockerham, 1986) senses were estimated by using the variance of follicle length: Heritability in the broad sense:
H = - (V„ X V„)«) / V„. Heritability in the narrow sense:
- (0.2 (4V„ + + V,,,,) - 0.4 (V„ + V,, + V,,)) / V,^, where Vj^ and are the variances of lA and MN parents, Vp, and Vp2 are the variances of F., and Fg, and and are the 48
variances of backcrosses to lA and MN parents. Standard deviations of heritabilities were calculated by Dickerson's approximation (1969):
S.D. (n/d) = 1/d X V(n)%, where n and d are numerator and denominator used for estimating heritability, respectively and V(n) is the variance
of the numerator.
Estimation of egg to adult developmental times A cohort of « 100 puparia was replicated three to four times. Daily emergence frequencies of a cohort were the
fractions of the daily numbers of the emerged flies over the
total numbers eclosed. Mean egg to adult developmental time was estimated by summing the products of the daily emergence
frequencies and the corresponding days after oviposition.
Measurements of follicle length and pterin lA, MN, F,, and Fg crosses were used. In each cross, ca.
10 female flies were collected at each of 0, 40, 50, 60, 70, 80, 90, 100, 120, and 140 DD 16:8 h (L:D) at 30°:25°C (12:12 h). Before dissecting flies, heads were removed with a
forceps and transferred into centrifuge tubes. These tubes were kept in the dark in a desiccator at room temperature. Dissections were performed under a stereoscope. Follicle 49
lengths were measured under stereoscopic optics with an ocular micrometer at 25 X. Sample preparation and spectrofluorimetry to measure pterin amounts followed the procedure described by Krafsur et al. (1992). A fly head was ground in 200 jul of grinding buffer (sterile 50 mM Tris-HCl buffer, pH 8.6) and diluted with 800 lil of the same buffer. , After centrifugation, 450 jul of supernatant was delivered to a plastic 1.5 ml semimicro cuvette (Fisher Scientific Company) and diluted with 450 /nl of the grinding buffer. Five known concentrations of pterin (2- amino-4-pteridinol, Aldrich Chemical Company) were prepared as
0, 0.5, 1, 3, and 5 x 10'^ M in the grinding buffer to get a standard curve: CONC (M) = (- 0.03 + 5.04 SPF) X 10'^, = 0.9997, where CONC is the pterin concentration in molar units and SPF is the fluorescence reading from an SLM6000 spectrofluorimeter. From the estimated pterin concentration in a sample, the total amount of pterin in a fly head was calculated: pterin (^g) = CONC x DF x VGB x weight of 1 mole pterin
= CONC X 2 X 10"^ X 163140
= 326.28 X CONC (1) where DF is the dilution factor and VGB is the volume of the grinding buffer. Where a fly head is regarded as a half 50
ellipsoid with the same diameters in two out of three axes, the volume can be calculated from the longitudinal width (frontal to occipital: FO) and the lateral width (compound eye to compound eye: CC) of the head from a dorsal view by the following equation: Volume of a fly head (mm') = n/6 x FO x CC^. (2) From equations (1) and (2), the amount of pterin per unit volume of a fly head (ng/mm^) was calculated. 51
RESULTS Diapause response to photoperiod
Photoperiodic induction of diapause in seven laboratory face fly strains was investigated. To determine the photoperiods necessary to induce diapause at 23°:11°C (12:12 h), KS, KY, and MD2 were exposed to each of six photoperiods.
Responses were linear to photoperiods of 4-16 h in KS and KY, but the MD2 strain did not show any diapause response (Table
1). The extreme photoperiods (0:24 h and 24:0 h) deviated from the linear response and caused high mortalities. Four additional strains were exposed to three short photoperiods between 4-10 h. Two old laboratory strains (IA83 and MDl) showed no diapause response (Table 1). Two recently established laboratory strains (IA91 and MN), however, readily entered diapause. MW seemed to have a longer critical daylength than IA91.
Mode of inheritance of diapause Crosses between IA83 and MN flies showed different diapause inductions (Table 2). IA83 parents showed less variation among replications in diapause response than did MN flies. Males had a higher diapause incidence than females in most lines; lA and lABC flies which had low diapause incidence, however, showed no sex differences. Table 1. Diapause frequencies (± SE) in response to photoperiods among seven laboratory strains® of female face flies at 23°:11°C (12:12 h).
Photoperiods (L:D)
Strains 0:24 4:20 8:16 10:14 16:8 24:0 df P
KS 0.00 0.19 - 0.06 0.02 0.12 9.07 4 0.06 ± 0.00 ± 0.13 + 0.06 + 0.03 ± 0.11
KY 0.00 0.37 - 0.24 0.03 0.11 15.70 4 <0.01 ± 0.00 + 0.08 + 0.16 + 0.06 + 0.19
MD2 0.00 0.02 - 0.03 0.00 0.03 1.72 4 0.79 ± 0.00 + 0.03 + 0.04 + 0.00 + 0.06
MDl - 0.00 0.00 0.00 - - 0.20 2 0.90 + 0.00 + 0.00 + 0.00
IA83 - 0.00 0.00 0.00 - - 0.00 2 1.00 + 0.00 + 0.00 + 0.00
IA91 - 0.89 0.86 0.56 - - 8.63 2 0.01 + 0.19 + 0.13 + 0.05
Ml^ - 0.89 0.64 0.83 - - 4.17 2 0.12 + 0.19 + 0.15 + 0.15
® Iowa (JA83 and JA91), Kansas {KS), Kentucky {KY), Maryland (MDl), subcolony of MDl {MD2), and Minnesota (MN) laboratory strains. ^ Flies collected in 1991. 53
Table 2. Diapause incidence and sexual differences in different face fly generation types at 10:14 h (L:D), 23°:11°C (12:12h).
Generation Diapause
types Sex n Incidence lA a 410 0. 066 9 365 0.041 2.309 0.129 MN a 438 0.820 9 675 0.636 43.538 < 0.001 a 716 0.500 9 832 0.276 51.647 < 0.001 cf 382 0.497 9 378 0.370 12.476 < 0.001 JABC cf 710 0.239 9 684 0.222 0.581 0.446 MNBC a 459 0.776 9 415 0.518 63.817 < 0.001 54 Diapause responses of the foregoing lines were analyzed by ANOVA on their mean follicle lengths (Table 3). There were significant replication effects. Approximately 44% of the variance was explained by different lines. Of the variance caused by different cross lines, 73% was attributed to the different generation types. There was no significant difference in diapause induction among reciprocal crosses within any generation type. The inheritance pattern of diapause was not additive (Table 4). Cavalli's joint scaling test showed that the data did not adequately fit the additive model. The responses of flies were significantly different from expectations in both diapause rate and follicle length. This suggested a hypothesis of incomplete dominance of diapause-associated gene(s). We therefore retested the goodness of fit of diapause responses to an additive model with a consideration of an incomplete dominance of diapause. Both the Fg and lABC adequately fitted the modified additive model, but MNBC deviated significantly (Table 4). Estimation of the heritabilities and the minimum number of diapause-associated genes showed much variation among replications (Table 5). The mean estimates of heritabilities in the broad and narrow senses were 50% and 30%, respectively. The estimated minimum number of genes indicated that at 55 Table 3. ANOVA in face fly follicle lengths in response to diapause inducing conditions among lines, types, and crosses. Means are set forth in Table 4. Source df SS MS F P Replication 4 4.18 1.05 3.28 0.028 Line 13 9.24 0.71 2.22 0.044 Generation type 5 6.71 1.34 4.19 0.007 Reciprocal cross 8 2.53 0.32 1.00 0.461 Lines within 1 0.16 0.16 0.50 0.486 Lines within Fg 1 0.02 0.02 0.06 0.808 Lines within lABC 3 0.56 0.19 0.59 0.628 Lines within MNBC 3 1.79 0.60 1.88 0.160 Error 24 7.60 0.32 Total 41 21.02 56 Table 4. Fitting of face fly diapause frequency data to an additive model. Diapause (%) Follicle length (mm) n observed expected^ observed expected® expected^ lA cf 410 6.6 9 365 4.1 2.39 ± 0.05 1.96 MN a 438 82.0 9 675 63.6 0.86 ±0.04 0.99 Fl cf 716 50.0 44.3** 9 832 27.6 33.9** 1.76 ± 0.04 1.63 1.60** Fz cf 382 49.7 47.2 9 378 37.0 30.7** 1.65 ± 0.07 1.55 1.68 lABC cf 721 27.9 28.3 9 673 18.0 15.9 2.18 ±0.04 1.80 2.06** MWBC cf 459 77.6 66.0** 9 415 51.8 45.6* 1.18 ± 0.06 1.31 1.31* Joint scaling test (Cavalli, 1952) on follicle length X^df=3 = 182.730, P < 0.01 Test model of Henrich and Denlinger (1982) * P < 0.05. ** P < 0.01. ® Expected values based on an additive model. Expected values based on incomplete dominance. 57 Table 5. Estimations of the minimum number of loci in diapause and heritability of face fly diapause. Replication Estimates 1st 2nd 3rd Mean* Heritabilities Broad (H) 0.68 0. 40 0.34 (0.47)b Narrow (hf) 0,.43 ± 0.25 0.38 ± 0.17 0.19 ± 0.11 0.30 Minimum number of genes Lande First -1.82 ± 1.78 0.70 ± 0.50 88.82 ± 0.15 6032.3 Second 10.55 ±38.88 0.54 ± 0.29 0.93 ± 0.63 0.71 Third -1.56 ± 1.59 0.96 + 2.12 0.39 ± 0.21 0.23 Fourth 1. 20 ± 0.91 0.37 ± 0.21 -2.38 ± 5.52 0.44 Cockerham 1.98 ± 1.18 0.44 ± 0.24 0.84 ± 0.54 0.74 ® Weighted mean = (S Xi/SD^.) / (S l/SD^j), where Xi is the estimate of number of genes or heritability in the ith replication. ^ Unweighted mean. 58 least one major factor was involved in the inheritance of face fly diapause. Correlation between diapause and other developmental traits Egg to adult developmental times differed among generation types (Table 6). Phenotypic correlation (r) between diapause incidence and egg to adult developmental time was 0.90 in males and 0.84 in females. lA flies developed faster than MN flies. F,, Fj, and backcrosses had developmental rates intermediate to those of their parents. Females developed faster than males in all lines. Both generation type and sex effects were highly significant (Table 7). Temporal patterns of follicle growth and pterin accumulation in females were measured (Fig. 1). These two traits were significantly dependent on age (Table 7). Both follicle growth and pterin accumulation varied by generation types. Developmental rates of these traits also differed according to the diapause propensities of different generation types. 59 Table 6. Egg to adult developmental times (± SE) among face fly lines in a 16:8 h (L:D) photoperiod and 30°:25°C (12:12 h). Generation types Sex n Egg to adult (days) lA a 165 9.14 ± 0.04 9 123 9.01 ± 0.02 WW cf 113 11.44 ± 0.02 9 140 10.99 ± 0.02 F, cf 195 9.99 ± 0.05 9 99 9.85 ± 0.15 Fg cf 120 9.77 ± 0.15 9 165 9.30 ± 0.08 JABC cf 138 9.82 ± 0.01 9 98 9.44 ± 0.00 MWBC cf 157 10.59 ± 0.03 9 137 10.32 ± 0.05 60 Table 7. ANOVA on developmental rates of face fly lines and crosses. Source df SS F P Egg to adult (days) Sex 1 0.61 223.90 0.0001 Gen* 5 17.18 1265.58 0.0001 Sex * Gen 5 0.19 14.17 0.0001 Error 28 0.08 Total 39 18.06 Follicle length (mm) DD 9 184.26 31.59 0.0001 Gen 3 55.27 28.43 0.0001 DD * Gen 13 35.63 4.23 0.0001 Error 382 247.57 Total 407 522.73 [Pterin] in head (fig/mm^) DD 9 0.33 91.71 0.0001 Gen 3 0.04 31.80 0.0001 DD * Gen 13 0.03 4.99 0.0001 Error 510 0.20 Total 535 0.60 ® 'Gen' represents generation types. Fig. 1. Temporal patterns of follicle growth and pterin accumulation in female heads in 16:8 h (L:D) at 30°:25°C (12:12 h) according to degree days over 12°C threshold. lA Iowa strain, MN is Minnesota strain, and and Fg are the first and second progeny of the cross between Iowa and Minnesota parents. 62 • 4.0 3.5 3.0 2.5 2.0 0.5 0.0 CP 4.0 3.5 3.0 2.5 2.0 0.5 0.0 60 80 100 120 80 100 120 140 Degree Days > i2°c Fig. 1. (continued) 64 160 140 120 100 80 ) 60 E 2 40 60 80 100 120 0 40 60 80 100 120 O 160 C 120 S 100 CL 80 60 40 20 60 80 100 1200 40 60 80 100 120 140 Degree Days > i2°c 65 DISCUSSION Diapause response to photoperiod Laboratory face flies varied in diapause responses to photoperiods. Krafsur and Black (1992) explained that significant genetic differentiation at 12 allozyme loci among these laboratory strains was probably caused by random genetic drift rather than geographical variation. There seems, however, to have been a directional change in diapause responses of face flies in laboratory culture. Old laboratory strains (MDl, MD2, IA83, and KS) seemed to have lost their ability to diapause in response to short photoperiods. Recently colonized strains {IA91 and MN) were easily induced to diapause. Caldwell and Wright (1978) also showed that a recently established face fly strain could be induced to diapause more readily than an older strain. The change of diapause response may be caused by inadvertent selection in laboratory culture (Banks, 1987). Diapause inheritance We showed that face fly diapause is under genetic control. Different crosses had significant effects on diapause responses and their variances were about half of the total diapause variation. Hybrids showed intermediate diapause responses to those of their corresponding parents. This is a characteristic of polygenic systems (Danilevsky, 66 1965; Lees, 1968; Beck, 1980). The progeny of reciprocal crosses in any generation showed no differences in diapause. This indicated that there was no cytoplasmic effect. Face fly diapause did not follow an additive model. This suggests that nonadditive genetic factors had significant effects on diapause inheritance. One such factor could be incomplete dominance of diapause genes. Based on incomplete dominance, the diapause responses in Fg and JABC were explained by the additive model, but still not those in MWBC. Another source of nonadditive factors could be sex- linkage of diapause-associated gene(s). Males were induced to diapause more readily than females, confirming earlier works (Stoffolano and Matthysse, 1967, Valder et al., 1969, Caldwell and Wright, 1978, Evans and Krafsur, 1992). Also, significant differences between sexes correlated with the presence of diapause-associated genes; sexual differences were found in high diapause lines (WW, F^, Fg, and MNBC), but not in the low diapause lines (lA and JABC). The foregoing suggests that some diapause-associated genes are sex-linked. Diapause may be a sex-linked recessive phenotype because males show higher diapause incidence than females. The nonadditive proportion of diapause variance was obtained from the heritability estimates. The small heritabilities (H = 50% and h^ = 35%) support much 67 nonadditivity in diapause. In contrast, high heritabilities of diapause were reported in other species; 0.70 for the ages at first reproduction in O. fasciatus (Dingle et al., 1977), 0.43 to 0.82 for degree-day requirements for the completion of diapause in Hyphantria cunea (Drury) (Morris and Fulton, 1970), 0.77 ± 0.36 for the number of days to emergence in Heliothis zea (Boddie) (Holtzer et al., 1976), and 0.31 to 1.13 for diapause in A. fasciatus (Mousseau and Roff, 1989). The relatively low diapause heritability in laboratory face flies suggests that this trait had been under strong selection for an optimal phenotype (Falconer, 1981). The selection on diapause in laboratory culture might give a reduction of additive genetic variance which led to low estimations. Different populations gave different diapause heritability estimates in A. fasciatus (Mousseau and Roff, 1989). Face fly diapause also may have a specific norm of reaction with respect to temperature. Different temperatures give different phenotypic variations. Another genetic experiment using multiple strains at different temperatures may give a more useful indicator of heritability in face fly diapause. Estimation of the minimum number of diapause-associated genes suggested that a small number were involved. There was much replicate variation in the estimations. The estimation assumed that there were no linkage, dominance, or epistasis. 68 Inheritance of face fly diapause was affected by the nonadditive genetic factors of incomplete dominance and sex- linkage. These deviations from assumptions would lead to an increase in Fg variance, which is the denominator for estimating the minimum number of genes. An increase in Fg variance would underestimate the minimum number of genes (Dempster and Snyder, 1950). Diapause in S. bullata (Henrich and Denlinger, 1983), O. fasciatus (Hayes et al., 1987), and Drosophila littoralis Meigen (Lumme, 1981) was explained by a small number of genes. Correlation with other developmental traits Three other developmental traits were correlated with diapause. The high diapause lines had slower developmental rates: longer egg to adult developmental times and slower follicle growth and pterin accumulation rates than the low diapause lines. Diapause incidence was positively correlated with pupal period in S. bullata (Henrich and Denlinger, 1982). Fast developing flies seem to have higher viabilities in laboratory culture. Thus, diapause, as a correlated response, is under selection. Diapause incidence decreased according to duration of laboratory colonization. The correlation between diapause and other developmental traits may come from the pleiotropic effects of genes 69 associated with developmental rates (Falconer, 1981). Another possibility may be that diapause-associated genes could be closely linked to genes for developmental rates. 70 ACKNOWLEDGEMENTS We would like to thank Dr. Roger Moon, University of Minnesota for supplying face fly puparia, to Dr. Sheldon Shen, Iowa State University for sharing his spectrofluorimeter, and to Suling Zhao for helping this research in many ways. 71 LITERATURE CITED Angus, R.A. 1983. A program in BASIC for estimating the number of genes contributing to quantitative character variation. J. Hered. 74; 386. Arends, J.J. and Wright, R.E. 1981. Mass rearing of face flies. J. Econ. Entomol. 74: 355-358. Beck, S.D. 1980. Insect Photoperiodism. 2nd ed. Academic Press, New York. Burks, C.D., Evans, L.D., Kim, Y. and Krafsur, E.S. 1992. Effect of precocene and methoprene application in young adult Musca autumnalis. Physiol. Entomol. 17: 115-120. Caldwell, E.T.N, and Wright, R.E. 1978. 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Kerkut, G.A. and Gilbert, L.I.), pp. 353-412. Pergamon Press, Oxford. Dickerson, G.E. 1969. Techniques for research in quantitative animal genetics. In Techniques and Procedures in Animal Science Research, pp. 36-79. American Society Animal Science, New York. Dingle, H., Brown, C.R. and Hegmann, J.P. 1977. The nature of genetic variance influencing photoperiodic diapause on a migrant insect, Oncopeltus fasciatus. Am. Nat. Ill: 1047- 1058. Evans, L.D. and Krafsur, E.S. 1990. Light intensity, photoperiod, and diapause induction in face flies (Diptera: Muscidae). Environ. Entomol. 19: 1217-1222. Falconer, D.S. 1981. Introduction to Quantitative Genetics. 2nd ed. Longman, New York. Hayes, J.L., Bandy, D.J. and Dingle, H. 1987. Genetic analysis of diapause differences between populations of the large 73 milkweed bug. J. Hered. 78: 266-270. Henrich, V.C. and Denlinger, D.L. 1982. Selection for late pupariation affects diapause incidence and duration in the flesh fly, Sarcophaga bullata. 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Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation. Quart. Rev. Biol. 65; 145-174. Tauber, M.J. and Tauber, C.A. 1979. Inheritance of photoperiodic responses controlling diapause. Bull. Entomol. Soc. Amer. 25: 125-128. Tauber, M.J., Tauber, C.A. and Masaki, S. 1986. Seasonal Adaptations of Insects. Oxford University Press, New York. Tauber, M.J., Tauber, C.A. and Nechols, J.R. 1977. Two genes control seasonal isolation in sibling species. Science 76 197: 592-593. Valder, S.M., Hopkins, T.L. and Valder, S.E. 1969. Diapause induction and changes in lipid composition in diapausing and reproducing faceflies, Musca autumnalis. J. Insect Physiol. 15: 1199-1214. Vinogradova, E.B. and Tsutskova, I.P. 1978. The inheritance of larval diapause in the crossing of intraspecific forms of Calliphora vicina (Diptera, Calliphoridae). Entomol. Rev. 57: 168-176. , 77 PAPER II. VITELLIN, VITELLOGENIN, AND OTHER STORAGE PROTEINS IN Musca autumnalis 78 Vitellin, Vitellogenin, and other storage Proteins in Musca autumnalis Yonggyun Kim and E. S. Krafsur Department of Entomology Iowa State University Ames, Iowa 50011 79 ABSTRACT Vitellin and vitellogenin in face flies were analyzed by SDS-polyacrylamide gel electrophoresis. These proteins are composed of four subunits with molecular weights of approximately 46, 49, 53, 55 kD and are similar to those in house flies. Western blotting showed that anti-house fly vitellin reacted with face fly, horn fly, and stable fly vitellin as well as house fly vitellin. We developed an indirect ELISA with anti-house fly vitellin for a quantitative analysis of face fly vitellogenin. Temporal changes of vitellogenin and total proteins in the haemolymph of vitellogenic and diapausing females were compared. Reproductive females began to synthesize vitellogenin soon after adult eclosion, between 0 and 15 degree-days above a 12°C threshold. Vitellogenin synthesis was sigmoid with respect to time. Diapausing flies did not synthesize vitellogenin except pre-diapause, teneral adults which synthesized small amounts of vitellogenin. We also report the presence of other storage proteins in face flies. 80 INTRODUCTION Vitellogenins are the yolk proteins synthesized by the fat body in female insects (Engelmann, 1979; Hagedorn and Kunkel, 1979). Vitellogenins are secreted to the haemolymph and taken up selectively by the growing oocytes through receptor-mediated endocytosis (Ilenchuk and Davey, 1987; Raikhel and Lea, 1986). Yolk proteins in the ovary are termed "vitelline". In Drosophila melanogaster (Meigen) and Musca domestica Linnaeus, both the ovary and the fat body synthesize vitellogenin (de Bianchi et al., 1985; Bownes, 1980; Brennan and Mahowald 1983; Postlethwait et al., 1980). There are some differences between vitellin and vitellogenin in lipid content and protein subunit structure in some species (Hagedorn and Kunkel, 1979). Yolk protein synthesis is under hormonal control. In Locusta migratoria (Linnaeus) (Wyatt, 1988) and Leucophaea maderae (Fabr.) (Engelman, 1979), vitellogenin synthesis is said to be regulated only by juvenile hormone. In many species of Diptera, both juvenile hormone and ecdysone are needed for vitellogenin synthesis (Hagedorn, 1985). Yolk proteins of higher Diptera have unique characteristics in their molecular weights, gene structure, and amino acid sequences in comparison with those of other egg laying invertebrates (Rina and Savakis, 1991; Spieth et al., 81 1985). In D. melanogaster, there are three yolk proteins that range from 44 to 47 kD. They are encoded by single copy genes (Ypl, Yp2, and Yp3) on the X chromosome. Genes Ypl and Yp2 are separated by 1.2 kb and transcribed divergently (Hung and Wensink, 1982). The Yp3 locus is separated from Ypl and Yp2 by « 1000 Kb (Barnet et al., 1980). These loci have their own promoters and are transcribed in concert in response to ecdysone (Hung and Wensink, 1982). Female face flies have polytrophic ovaries and the ovarioles undergo vitellogenesis synchronously (Miller and Treece, 1968). About 70 degree-days above a 12°C threshold (D]0i,^2°c) needed for teneral females to become gravid and oviposit (Moon and Kaya, 1981; Krafsur et al., 1985). Our earlier studies suggested that juvenile hormone is responsible for ovarian development of face flies because corpora allata increased in size during vitellogenesis, precocene inhibited vitellogenesis, and topically applied methoprene induced vitellogenesis in diapausing face flies (Burks et al., 1992). To investigate differences in vitellogenin and other protein synthesis between reproductive and diapause development of face flies, we identified the yolk proteins and compared vitellogenin titers in reproductive and diapausing females' haemolymph during the first gonotrophic cycle. We also examined other development-specific proteins. 82 MATERIALS AND METHODS Insects We used a face fly strain established by R.D. Moon, U. Minnesota, from collections made in Stevens Co., MN in 1992. Flies were provided with powdered milk, sugar, and egg in a ratio of 6:6:1. A piece of raw liver was suspended in each fly cage to provide additional protein and encourage ovarian development. Flies were maintained at 27.5°C, 16h light-8h dark for reproductive development or at 17.5°C, lOh light-14h dark for diapause induction. House flies (M. domestica), stable flies (Stomoxys calcitrans (Linnaeus)), and horn flies {Haematobia irritans (Linnaeus)) were sampled from field populations in Story Co., lA in 1992. Ovary and haemolymph samples Female flies were dissected in sterile house fly saline (Fye and LaBrecque, 1966). To extract vitellin and prepare it for SDS-PAGE, 10 pairs of ovaries (an experimental unit) were ground in 100 ml grinding buffer (40% sucrose, 0.1% bromophenol, 0.04% basic fuchsin, 1.54% dithiothreitol, 0.372% EDTA in 0.125 mM Tris-HCl, pH 8.3). We then added 100 ml of sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol in 0.125 M tris HCl, pH 6.8). Samples were boiled for 90 seconds 83 and centrifuged at 14,000 rpm for 3 minutes. To get haemolymph samples, flies were anesthetized with COg and punctured on their dorsal vessels with fine forceps. The emerging haemolymph was collected in microcapillary tubes. For electrophoresis, the haemolymph was diluted with distilled water and then combined with an equal amount of sample buffer. For measuring titers of vitellogenin and total proteins, haemolymph of ten flies of uniform age was combined and represented an experimental unit. Three measurements were made on each age of flies and analyzed by PROC GLM in SAS (SAS Institute, 1988). Regression of temporal change in vitellogenin titers was accomplished by using PROC REG in SAS. Gel electrophoresis and Western blotting Egg proteins were separated in vertical slab SDS- polyacrylamide electrophoresis (Laemmli, 1970) at 30 mA/gel until the tracking dye migrated to the end of the gel (120 mm). Molecular weight markers (Sigma Chemical Co.) included carbonic anhydrase (29 kD), egg albumin (45 kD), bovine serum albumin (66 kD), phosphorylase B (97.4 kD), B-galactosidase (116 kD), and myosin (205 kD). Gels were stained in 0.125% Coomassie Blue R-250 in 50% methanol and 10% acetic acid. Stained gels were destained in 50% methanol and 10% acetic acid for 2 h, then immersed in 5% methanol and 7% acetic acid 84 for « 48 h. The separated proteins were transferred electrophoretically from gels to nitrocellulose by using a semi-dry transfer apparatus (Kyhse-Andersen, 1984). The electroblotting was run for 40 min at constant 40 V. After hybridization with antibody raised against house fly vitellin, the membrane was incubated with -protein A for 1 h at room temperature. Kodak X-ray film was exposed to the labelled membrane for 7 h at -70°C. Indirect ELISA We developed a modification of an indirect, competitive ELISA assay (Ishiguro et al., 1986; Voiler et al., 1979) for quantitative analysis of face fly vitellogenin by using the primary antibody raised against house fly vitellin. House fly vitellin and its antibody were gifts from Dr. T.S. Adams who prepared them by the procedure of Adams and Filipi (1983). The antigen was dissolved in 10 mM, pH 7.4 phosphate buffer containing 140 mM NaCl and 0.05% Triton-X (PBS-TX) and diluted (5 /xg/ml) with 50 mM, pH 9.6 carbonate-bicarbonate coating buffer. Diluted primary antibodies (1:1000) with antibody dilution buffer (PBS-TX containing 1 % bovine serum albumin) were preincubated with known concentrations of antigen (0-40 )Ltg/ml) as standards or with samples overnight at 4°C in 85 microcentrifuge tubes. The preincubated mixtures (100 nl) were added to wells and incubate for 2 h at 24°C. Diluted (1:2500) goat antirabbit IgG-alkaline phosphatase conjugate (100 /il) with antibody dilution buffer was added and incubated for 2 h at 24°C. p-Nitrophenyl phosphate (1 mg/ml, 100 /il) in 10%, pH 9.6 diethanolamine buffer was added to the wells. The reaction was stopped after 3 0 min incubation by adding 50 /il of 3 M NaOH. Absorbance at 405 nm was read on a Molecular Devices Kinetic Microplate Reader. Protein determination Protein was determined by the method of Bradford (1976). Bovine serum albumin was used as the standard. 86 RESULTS Vitellin and vitellogenin The proteins in the ovary and the haemolymph of face flies were separated in 7% SDS-PAGE (Fig. 1). Mature ovary extracts (lane 1) showed four clear bands, which were also found at the same positions in reproductive female haemolymph (lane 3). Estimated molecular weights of the bands were 46, 49, 53, and 55 kD, respectively. These four bands were also seen in diapausing female haemolymph (lane 5), but were much fainter. Haemolymph from reproductive and diapausing males (lanes 2 and 4) showed only a weakly staining band at 53 kD. To examine the immunological relationship among the vitellins of face fly and other Muscidae, we conducted Western blotting with the primary antibody raised against house fly vitellin (Fig. 2). The vitellins of house flies, stable flies, and horn flies had similar molecular weights with a range of 45-55 kD, and all reacted to anti-house fly vitellin, showing a high degree of homology. Indirect ELISA We developed an indirect ELISA to measure vitellogenin in face flies with the primary antibody raised against house fly vitellin. Observed absorbances at 405 nm were negatively related to the amounts of vitellin (Fig. 3). Unknown Fig. 1. Vitellin (Vn) and vitellogenin (Vg) of face flies on 7% SDS-PAGE. From left to right are shown the extract of a pair of mature ovaries (lane 1), 45 DD reproductive male (lane 2) and female (lane 3) haemolymph, 80 DD diapausing male (lane 4) and female (lane 5) haemolymph. Each molecular marker is shown by arrow following the weight size. 88 1 2 3 4 5 f-i m rr 116k 'i ^ 66k 45k- K •I 29k Fig. 2. Western blotting of purified house fly vitellin (Vn) and the extracts of ovaries of house fly (HF), face fly (FF), horn fly (HnF), and stable fly (SF) with antibody raised against house fly vitellin in rabbit after they were separated on 10% SDS-PAGE. 90 Vn hf FF HnF SF 97.4k w. 66k -> 45k •Iff 29k -y. Fig. 3. Standard curve of indirect ELISA for titration of face fly vitellogenin generated by known concentrations of house fly vitellin against their absorbances at 405 nm. Absorbance at 405 nm 93 amounts of vitellogenin in haemolymph samples of face flies were estimated by using the equation, Log^Q([Vg] + 1) = 1.94 - 4.61 X Absorbance, = 0.94. Vitellogenin and total protein titers in haemolymph Vitellogenin and total proteins in haemolymph were measured in reproductive and diapausing females. Both reproductive and diapausing females began to accumulate vitellogenin in haemolymph just after adult emergence (Fig. 4). After 30 DD, however, reproductive and diapausing flies showed different patterns of vitellogenin synthesis. Diapausing females showed no further increases in vitellogenin after 30 DD. In contrast, vitellogenin titers in reproductive females increased as ovarian development proceeded, reaching 110-100 jug/ml at 45-60 DD. Thereafter, titers decreased until 120 DD and increased again at 150 DD. The vitellogenin levels at 45-60 DD and at 75-120 DD were significantly different (F = 13.51, df = 1,16, P = 0.002). The vitellogenin levels at 120 DD and at 150 DD were significantly different (F = 5.77, df = 1,15, P = 0.029). This pattern of haemolymph vitellogenin could be visualized in whole body extracts on 10% SDS-PAGE (Fig. 5). Vitellin and vitellogenin bands occurred only in adults. The bands were clearly shown by 30 DD flies, even though 15 DD females had slightly stained vitellogenic bands. Fig. 4. Temporal changes in haemolymph titers in total proteins (upper) and vitellogenin (lower) in vitellogenic (solid line) and diapausing (dot line) female face flies. 95 25 r 20 tlfl Ë 15 M i=l • 1—1 CD 4-) 10 O a 5 15 o H 0 Vitellogenic Diapausing 40 r 100 ^ 60 0 10 20 30 40 50 60 70 80 90 1 00110120130140150 O Degree days > 12 C Fig. 5. Larval-pupal storage proteins (LPSP) on 10% SDS-PAGE. 'L', 'P', and 'A' stand for larval, pupal, and adult. The age, in days, of each stage at 27.5°C, 16h light-8h dark is shown. LARVAL FDPAL ADULT LO LI L2 L3 PO PI P2 P3 P4 P5 AO A1 A2 A4 116k -* ; r 66k LPSP -••» r>-H vo 45k - Vn/Vg 3 29k 98 Total haemolymph proteins of all females were greatest in newly emerged flies (Fig. 4). Reproductive flies and diapausing flies began to accumulate proteins in their haemolymph at 30 and 45 DD, respectively. Diapausing flies had greater protein levels than reproductive flies after 60 DD. Young adults, whether in reproductive or in diapause development, showed a unique, dense band in both sexes, compared to older adults ('LPSP' in Fig. 5). The molecular weight of LPSP was estimated to be « 90 kD. Third instar larvae and pupae also showed the protein. The staining density of the protein band was strongest in 1 day old pupae. In young adults, both fat body and haemolymph expressed the protein band, but lost it as adults aged (Fig. 6). The protein seemed to disappear earlier in fat body than in haemolymph. Fig. 6. Mobilization of larval-pupal storage proteins (LPSP) from fat body to haemolymph during early adult development. 100 Hemolymph - — 4- LPSP Oh 3h 6h 12h 24h 48h 101 DISCUSSION Ovarian extracts and female haemolymph showed four clear bands in common. These bands must be subunits of vitellin and vitellogenin. Molecular weights of the four subunits were 46, 49, 53, and 55 kD, similar to subunit structure of M. domestica (de Bianchi et al., 1985). Generally, in higher Diptera, yolk proteins are composed of relatively small polypeptides (44-50 kD) (Rina and Savakis, 1991) compared to those of other insects, nematodes, and sea urchins (Blumenthal and Zucker-Aprison, 1987). Our results also indicated the similarity among vitelline of house flies, stable flies, horn flies, and face flies in molecular weights of their subunits and immunoreactivity to anti-house fly vitellin. This shows the highly conserved nature of vitellogenin in Muscidae. Face fly vitellogenin is specific by sex and developmental stage. Only females showed vitellogenin in their haemolymph. Larvae and pupae did not have any vitellogenin. This specificity is the common property of vitellogenins of most insects except in some Lepidoptera, including Bombyx mori Linnaeus and Pieris brassicae Linnaeus, where males have vitellogenin in the haemolymph (Hagedorn and Kunkel, 1979; Lamy, 1984). By using the immunological reactivity of face fly vitellogenin to antibodies against house fly vitellin, we 102 developed an indirect ELISA to measure face fly vitellogenin titer in haemolymph. The standard curve using the purified house fly vitellin was typical of an indirect ELISA, where the measured absorbances are negatively related to the amounts of antigen in samples (Ishiguro et al., 1986; Rosell and Coons 1991; Voiler et al., 1979). Vitellogenin titers in the haemolymph of reproductive females varied over the gonotrophic cycle; vitellogenin titers were low during pre- and post-vitellogenesis and high at mid- vitellogenesis. This temporal pattern of vitellogenin titers is a well known phenomenon in insects with an intraovariole synchrony of vitellogenesis including L. migratoria (Ferenz and Lubzens, 1981), Diploptera punctata (Mundall et al., 1981), Aedes aegypti (Linnaeus) (Hagedorn and Kunkel, 1979), Calliphora vicina Robineau-Desvoidy (Jensen et al., 1981), and M. domestica (Adams and Filipi, 1983). The dynamics of vitellogenin concentrations in haemolymph may be controlled by the relative rates of vitellogenin synthesis and uptake by the fat body and the ovary (Hagedorn and Kunkel, 1979). Juvenile hormone stimulates both synthesis and uptake of vitellogenin in most insects (Tobe and Stay, 1985; Wyatt, 1988). Juvenile hormone titer is controlled mainly by the synthetic activity of the corpora allata (Tobe et al., 1985). In face flies, increase of juvenile hormone 103 titer was suggested to be necessary for vitellogenesis by a measured correlation between corpora allata growth and ovarian development (Burks et al., 1992). One of the characteristics of face fly diapause is an arrested vitellogenesis (Stoffolano and Matthysse, 1967). Haemolymph from diapausing females, however, showed relatively large amounts of vitellogenin which seemed to have been formed in adults younger than 30 DD. Read and Moon (1986) observed that diapause commitment increased continuously with duration of adult face flies in a diapause environment. Therefore, adults less than 30 DD of age must be too young to be fully committed to diapause. Vitellogenin synthesis in teneral adults may be initiated by the initial juvenile hormone level accruing from a basal activity of the corpora allata, as in L. decemlineata (Khan, 1988). After diapause induction, diapausing females do not have enough juvenile hormone to initiate egg development. Vitellogenin synthesis before diapause induction would remain in the haemolymph without being taken up by the ovary. Haemolymph from diapausing flies 45 DD of age or older showed an increasing amount of total proteins, but no further change in vitellogenin titers. The occurrence of large amounts of total proteins in diapausing fly haemolymph may be explained by one of two hypotheses: production of a diapause- 104 associated protein or excess production of some haemolymph protein(s). We did not find, on SDS-PAGE, a diapause- associated protein from diapausing flies older than 3 0 DD (unpublished data). This contradicts the hypothesis that there is a diapause-associated protein in face flies, but there remains the hypothesis of an excess production of typical haemolymph protein(s). Diapausing face flies have large lipid reserves (Valder et al., 1969; Read and Moon, 1986). We observed recently that diapausing flies begin to accumulate lipids at 30 DD and reach a maximum level (« 43 ng/mg of fresh weight) at 125 DD (unpublished data). During this lipid accumulation period, the haemolymph proteins also increased. This suggests that the protein(s) accumulated in diapausing haemolymph may involve lipophorin which is one of the typical haemolymph proteins. In L. decemlineata, the concentration of lipophorin in a short-day regime is two to three times greater than in a long-day regime (de Kort and Koopmanschap, 1987). A protein band of » 90 kD was found among third instar larvae, pupae, and teneral adults. This protein disappeared early in young adults, before reproductive or diapause commitment. The foregoing characteristics are similar to those of arylphorin, a general storage protein in insects characterized by its high aromatic amino acid content (Munn 105 and Greville, 1969; Telfer et al., 1983). Arylphorin forms hexamers in the 500 Kd range and dissociates into peptides of 70-90 Kd. The expression of the arylphorin gene is developmentally regulated and restricted to the fat body. Large amounts of a lipo-glycoprotein are synthesized by the fat body and secreted into the haemolymph at the final larval instar of many insects (Scheller et al., 1990). Arylphorin supplies amino acids necessary for the remodeling of tissues and sclerotization of cuticle in adult development (Peter and Scheller, 1991; Levenbook and Bauer, 1984). Also, arylphorin may be a carrier of ecdysteroid hormones (Enderle et al., 1983). The storage protein of face flies needs further study to be identified as an arylphorin. 106 ACKNOWLEDGEMENTS We are grateful to Dr. Roger Moon, University of Minnesota for supplying face fly pupae, to Dr. Theodore Kramer, College of Veterinary Medicine, Iowa State University for sharing his ELISA reader, to Juwhan Eom for advice on Western blotting, and to Dr. Terry Adams, ARS, USDA, Fargo, ND for gifts of house fly vitellin and its antibody. 107 LITERATURE CITED Adams, T.S. and Filipi, P.A. 1983. Vitellin and vitellogenin concentrations during oogenesis in the first gonotrophic cycle of the house fly, Musca domestica. J. Insect Physiol. 29; 723-733. Barnet, T., Pachl, C., Gergen, P.J. and Wensink, P.C. 1980. The isolation and characterization of Drosophila yolk protein genes. Cell 21: 729-738. de Bianchi, A.G., Coutinho, M., Pereira, S.D., Marinotti, O. and Targa H. J. 1985. Vitellogenin and vitellin of Musca domestica. Quantification and synthesis by fat bodies and ovaries. 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Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. 110 Biochem. Biophys. Methods 3: 97-128. Laenunli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680—685. Lamy, M. 1984. Vitellogenesis, vitellogenin and vitellin in the males of insects: a review. Int. J. Invert. Reprod. 7: 311-321. Levenbook, L. and Bauer, A. 1984. The fate of the larval storage protein calliphorin during adult development of Calliphora vicina. Insect Biochem. 14: 77-86. Miller, T.A. and Treece, R.E. 1968. Gonadotrophic cycles in the face fly, Musca autumnalis. Ann. Entomol. Soc. Amer. 61: 690-696. Moon, R.D. and Kaya, H.K. 1981. A comparison of methods for assessing age structure and abundance in populations of nondiapausing female, Musca autumnalis (Diptera: Muscidae). J. Med. Entomol. 18: 289-297. Mundall, E.G., Tobe, S.S. and Stay, B. 1981. Vitellogenin fluctuations in haemolymph and fat body and dynamics of uptake into oocytes during the reproductive cycle of Diploptera punctata. J. Insect Physiol. 27: 821-827. Munn, E.A. and Greville, G.D. 1969. The soluble proteins of developing Calliphora erythrocephala, particularly calliphorin, and similar proteins in other insects. J. Ill Insect Physiol. 15: 1935-1950. Peter, M.G. and Scheller, K. 1991. Arylphorin and the integument. In The Physiology of Insect Epidermis (Eds. Retnakaran, A. and Binnington, K.), pp. 115-124. CSIRO, Australia. Postlethwait, J.H., Bownes, M. and Jowett, T. 1980. Sexual phenotype and vitellogenin synthesis in Drosophila melanogaster. Dev. Biol. 79; 379-389. Raikhel, A.S. and Lea, A.O. 1986. Internalized proteins directed into accumulative compartments of mosquito oocytes by the specific ligand, vitellogenin. Tisse Cell 18: 559-574. Read, N.R. and Moon, R.D. 1986. Diapause induction and commitment in face fly, Musca autumnalis (Diptera;Muscidae). Environ. Entomol. 15; 669-677. Rina, M. and Savakis, C. 1991. A cluster of vitellogenin genes in the mediterranean fruit fly Ceratitis capitata: Sequence and structural conservation in dipteran yolk proteins and their genes. Genetics 127: 769-780. Rosell, R. and Coons, L.B. 1991. Determination of vitellogenin titer in the hemolymph of Dermacentor variabilis (Acarina: Ixodidae) using an indirect enzyme- linked immunosorbent assay. J. Med. Entomol. 28: 41-44. SAS Institute, Inc. 1988. SAS/STAT User's Guide, Release 6.03 112 ed. Cary, NC. 1028 pp. Scheller, K., Fischer, B. and Schenkel, H. 1990. Molecular properties, functions and developmentally regulated biosynthesis of arylphorin in Calliphora vicina. In Molecular Insect Science (Eds. Hagedorn, H. H., Hildebrand, J.G., Kidwell M.G. and Law J.H.), pp. 155- 162. Plenum Press, New York. Spieth, J., Denison, K., Hirtland, S., Cane, J. and Blumenthal, T. 1985. The C. elegans vitellogenin genes; short sequence repeats in the promoter regions and homology to the vertebrate genes. Nucl. Acids Res. 13: 5283-5295. Stoffolano, J.G.,Jr. and Matthysse, J.G. 1967. Influence of photoperiod and temperature on diapause in the face fly, Musca autumnalis (Diptera: Muscidae). Ann. Entomol. Soc. Am. 60; 1242-1246. Telfer, W.H., Keim, P.S. and Law, J.H. 1983. Arylphorin, a new protein from Hyalophora cecropia: Comparisons with calliphorin and manducin. Insect Biochem. 13; 601-613. Tobe, S.S., Ruegg, R.P., Stay, B., Baker, F.C., Miller, C. A., and Schooley, D.A. 1985. Juvenile hormone titer and regulation in the cockroach Diploptera punctata. Experientia 41; 1028-1034. Tobe, S.S. and Stay, B. 1985. Structure and regulation of the 113 corpus allatum. Adv. Insect Physiol. 18: 305-432. Valder, S.M., Hopkins, T.L. and Valder, S.E. 1969. Diapause induction and changes in lipid composition in diapausing and reproducing faceflies, Musca autumnalis. J. Insect Physiol. 15; 1199-1214. Voiler, A., Bidwell, D.C. and Bartlett, A. 1979. The Enzyme Linked Immunosorbent Assay (ELISA). A Guide with Abstracts of Microplate Applications. Dynatech Laboratories, Alexandria, Va. Wyatt, G.R. 1988. Vitellogenin synthesis and the analysis of juvenile hormone action in locust fat body. Can. J. Zool. 66: 2600-2610. 114 IN VIVO AND IN VITRO EFFECTS OF 20- HYDROXYECDYSONE AND METHOPRENE ON REPRODUCTIVE DEVELOPMENT OF DIAPAUSING Musca autumnalis 115 In vivo and In vitro Effects of 20- Hydroxyecdysone and Methoprene on reproductive development in diapausing Musca autumnalis Yonggyun Kim and E. S. Krafsur Department of Entomology Iowa State University Ames, Iowa 50011 116 ABSTRACT Face flies overwinter as adults in reproductive diapause. Administration of 20-hydroxyecdysone and/or methoprene to diapausing flies induced reproductive development even though flies were maintained in diapause conditions. The effects of the hormones were additive and females were more sensitive than males. Fat body from diapausing flies synthesized vitellogenin in vitro only in the presence of 20- hydroxyecdysone or methoprene. Transfer of diapausing flies from diapause to reproductive conditions induced them to enter the reproductive mode, but this effect decreased with the age of transferred flies. In contrast, topical application of methoprene on diapausing face flies induced reproductive development irrespective of their ages. Therefore, both hormones are needed for reproductive development and should be maintained at low levels during diapause induction. The effects of methoprene on diapausing flies varied according to diapause propensities of different genetic lines. Lines showing high frequencies of diapause required greater amounts of methoprene for reproductive development in diapause conditions than did lines showing low frequencies of diapause. 117 INTRODUCTION Face flies (Musca autumnalis DeGeer) overwinter in an adult diapause characterized by arrested vitellogenesis and hypertrophied fat body (Stoffolano and Matthysse, 1967; Valder et al., 1969). Photoperiod, temperature, and light intensity have significant effects on diapause induction in laboratory simulations (Stoffolano and Matthysse, 1967; Caldwell and Wright, 1978; Read and Moon, 1986; Evans and Krafsur, 1990). Juvenile hormone (JH) is important in reproductive development by inducing vitellogenin (Vg) synthesis in the fat body (Engelmann, 1979; Hagedorn and Kunkel, 1979). JH is also necessary for the uptake of Vg from the haemolymph by the growing oocytes (Ilenchuk and Davey, 1986; Raikhel and Lea, 1986). Thus, lowering JH titer can be used as a common tool for diapause induction of insects undergoing adult diapause (Denlinger, 1986). Our earlier study (Burks et al., 1992) showed that in face flies diapausing females had smaller corpora allata than reproductive flies and that topical application of methoprene, a JH analog, induced vitellogenesis in diapausing face flies that were maintained in a diapause environment. This suggested that low JH titer can be used as a signal for diapause induction in face flies. In higher Diptera, however, ecdysteroids as well as JH have a role in inducing 118 vitellogenin gene expression in the fat body (Bownes, 1980; de Bianchi et al., 1985). In the Colorado potato beetle, Leptinotarsa decemlineata (Say), ecdysteroid level slowly increased throughout diapause (Briers et al., 1982). Lefevere (1989) suggested that ecdysteroids play a key role in diapause termination by modifying the regulation of JH metabolism. Another characteristic of face fly diapause is variation in diapause incidence among different strains (Caldwell and Wright, 1978; Krafsur and Black, 1992; Kim and Krafsur, 1993). Diapause propensities of different laboratory strains decreased with duration of laboratory colonization. Saunders (1982) suggested a photoperiodic counter mechanism in diapause induction, where insects are induced to diapause only after being exposed to a required number of short days. Four main components in the diapause induction mechanism include the photoperiodic receptor, clock, counter, and hormonal target tissues. The photoperiod receptor and counting center are located in the brain and act neurally on controlling CA activity in adult diapause of L. decemlineata (Khan et al., 1986). Depending on the amount of hormone produced, the hormone target tissues, e.g., fat body and ovaries, undergo reproductive development or diapause induction (Chippendale, 1983; Denlinger, 1985). Thus, sources of variation in diapause incidence may lie in the neuroendocrinological and 119 hormonal target tissue levels. We tested the effects of 20-hydroxyecdysone and methoprene on diapausing face flies to examine the role of JH and ecdysteroid levels in reproductive development. We also analyzed methoprene effects on diapausing flies of different genetic backgrounds to examine the physiological sources of genetic variation in diapause induction. 120 MATERIALS AND METHODS Insects Flies were provided with dried non-fat milk, powdered sugar, and powdered egg at a ratio of 6:6:1 (Arends and Wright, 1981). Bovine dung was used for oviposition and larval development. Egg to pupal stages were kept at 27.5°C, 16h light-8h dark. Newly emerged flies were exposed to 27.5°C, 16h light-8h dark or to 17.5°C, lOh light-14h dark for reproductive or diapause development, respectively. Light intensities were « 1000 lux at lights-on and 0 lux at lights- off. An Iowa laboratory face fly strain (JA), which had been established in 1983, was used for a "low diapause" strain (i.e., low frequency of diapause). The Minnesota strain (MN) was obtained from flies established by R. D. Moon from flies collected in Stevens Co., Minnesota in August, 1991, and used for the "high diapause" strain (i.e., a high frequency of diapause). Morphological criteria of diapause development Flies were dissected to evaluate diapause or reproductive development at 80 degree-days above a 12°C threshold (DD>,2°c) (Moon and Kaya, 1981; Krafsur et al., 1985). The evaluation followed the criteria of Evans and Krafsur (1990), where females having both previtellogenic ovaries and hypertrophied 121 fat body were defined as being in diapause and males were judged as being in diapause if they had hypertrophied fat body. Enzyme-linked immunosorbent assay (ELISA) Vg was measured quantitatively by an indirect, competitive ELISA by using the primary antibody raised against house fly vitellin. House fly vitellin and its antibody were a gift from Dr. T. S. Adams, USDA-ARS, Fargo, North Dakota, who prepared them by the procedure of Adams and Filipi (1983). The antigen was dissolved in 10 mM, pH 7.4 phosphate buffer containing 140 mM NaCl and 0.05 % Triton-X (PBS-TX) and diluted (5 jug/ml) with 50 mM, pH 9.6 carbonate-bicarbonate coating buffer. Primary antibodies (1:1000) diluted with antibody dilution buffer (PBS-TX containing 1 % bovine serum albumin) were preincubated with known concentrations of antigen (0-40 jug/ml) or with samples overnight at 4°C. The preincubated mixtures (100 jul) were added to wells and incubated for 2 h at 24°C. Diluted (1:2500) goat antirabbit IgG-alkaline phosphatase conjugate (100 fil) with antibody dilution buffer was added and incubated for 2 h at 24°C. p- Nitrophenyl phosphate (1 mg/ml, 100 fj,l) in 10%, pH 9.6 diethanolamine buffer was added to the wells. The reaction was stopped after 30 min with 50 /itl of 3 M NaOH. Absorbance 122 at 405 nm was read on a Molecular Devices Kinetic Microplate Reader. Fat body culture Fat body from 30 DD females was prepared in a sterile and cold (5®C) house fly saline (Fye and LaBrecque, 1966) to test in vitro hormonal effect on producing vitellogenin. Although the isolated tissue contained muscle, epidermis, and cuticle, it consisted mainly of fat body. After washing the fat body three times with fresh Grace's medium, fat body from three flies was cultured in 15 jul of Grace's medium supplemented with streptomycin (50 /Ltg/ml) and a certain amount of 20- hydroxyecdysone or methoprene in a microcentrifuge tube. After 24 h incubation at room temperature in a moist chamber, Vg amounts in the culture media were measured by ELISA. In vivo and in vitro hormonal treatments Face flies were treated with 2 0-hydroxyecdysone dissolved in methanol to make concentrations of 0-4 /xg/jul. The hormone was injected with a 10 /il Hamilton syringe through the intersegmental membrane of the abdominal pleuron. Methoprene was dissolved in acetone to make concentrations of 0-3 nq/fil, and topically applied to flies on the ventral side of the abdomen. To study the combined effect of both hormones, flies 123 were treated with 1 jug of methoprene and then injected with different concentrations of 20-hydroxyecdysone. The experimental unit for a hormone treatment consisted of ten diapausing flies and these were replicated three times. Hormone effects on diapausing flies were expressed as the frequencies of reproductive flies "rescued" from diapause. For in vitro experiments, both hormones were dissolved in 0.55% Triton-X and added to Grace's medium to make final concentrations of lO'^-lO"' M. Each culture medium contained three fat bodies and represented an experimental unit, and each unit was replicated five times. Reproductive environment vs methoprene To examine the effect of a reproductive-inducing environment at different ages of early diapausing flies, flies were transferred at 1000 - 1200 h every day from a diapause to a reproductive environment. At the same time, some remaining flies in the diapause environment were treated individually with 3.5 fig of methoprene. After 80 DD had elapsed in each environment, flies were collected and dissected, and the frequencies of diapause were estimated. Variation in methoprene effects on diapausing flies Six lines of face flies were constructed by crosses 124 between the lA (low diapause) and MN (high diapause) strains: two parental lines ('lA' and 'WW), an 'F^' (MN cf x lA Ç) , an •Fg* (F^ X F^) , a backcross of F^ to lA (F, cf x lA 9: 'lABC'), and a backcross of F^ to MN (MN cf x F^ 9; 'WWBC'). Because the original diapausing conditions induced only a low frequency of diapause in lA flies, a new diapause regime was established. The new diapause regime was 15"C, 8h light-16h dark. Three day old adults from each of the six lines maintained in this diapause environment were treated with a series of methoprene doses (0-7 jug/jul) and returned to the same environment. Statistical analysis All experiments were analyzed by using a factorial design. Reproductive frequency data indicating hormonal effects were transformed by arcsin and analyzed by PROC GLM in SAS (SAS Institute, 1988). 125 RESULTS Hormonal effects on diapausing flies Injection of 20-hydroxyecciysone or topical methoprene application to 30 DD-old diapausing flies could induce the flies to undergo reproductive development in a diapause environment (Table 1). The three hormonal treatments each had significant effects on inducing reproductive development, but were significantly different in the magnitude of their effects (F = 24.85; df = 2, 58; P < 0.01). Methoprene was more effective than 20-hydroxyecdysone in males. The two hormones were additive in their effects. Males required greater amounts of hormones to become reproductive than did females. Female fat body synthesized significant amounts of Vg in Grace's medium supplemented with 20-hydroxyecdysone or methoprene (Table 2). 20-hydroxyecdysone had a significant effect only at high concentrations, while methoprene was effective at low concentrations. Both hormone treatments induced fat body from reproductive or diapausing flies to synthesize significant amounts of Vg. The two hormones were not additive in their effects in vitro. Reproductive environment vs methoprene The effects of reproductive environment or methoprene on diapausing flies of different ages were compared to examine 126 Table 1. In vivo effects of 20-hydroxyecdysone and methoprene applied to presumptively diapausing face flies 30 DD old at 17.5°C in a lOh light-14h dark photoperiod. Treatment Reproductive Proportion ± SD 9 cf COg 0.35 ± 0.14®* 0.00 ± 0.00* Methoprene O.OOt/xg 0.33 ± 0.25* 0.00 ± 0.00* 0.50 0.38 ± 0.16* 0.20 + 0.08*b 1.00 0.67 ± 0.00^ 0.50 ± 0.15® 1.50 0.82 ± 0.20^ 0.72 ± 0.24^ 3.00 1.00 ± 0.00^ 0.80 ± 0.22=d 20-HE O.OOt^g 0.22 ± 0.25* 0.00 ± 0.00* 0.50 0.36 ± 0.19* 0.18 ± 0.17*" 1.00 0.70 ± 0.30^ 0.24 ± 0.12^ 2.00 0.64 ± 0.27b 0.36 + 0.13^ 4.00 0.80 ± 0.00^ 0.37 ± 0.11^ 2 0-HE + Methoprene 0.00t+ 0.00* 0.46 ± 0.24* 0.05 ± 0.08* 0.50 + 1.00 0.93 ± 0.12^ 0.89 ± 0. i9cd 1.00 + 1.00 0.92 ± 0.14^ 0.67 ± 0.29^ 2.00 + 1.00 0.89 ± 0.19^ 0.89 + 0. i9cd 4.00 + 1.00 1.00 ± 0.00^ 0.82 ± 0.17^ * Means followed by different letters are significantly different (Least-Squares Means test, P < 0.05). t Acetone only. $ Methanol only 127 Table 2. Effects of 20-hydroxyecdysone and methoprene on vitellogenin synthesis in vitro from 30 DD^^g-old female fat body after 24 h incubation. Treatment Vitellogenin in Culture Medium (Mg/ml) From reproductive From diapausing flies* flies* o Control 0.10 ± 0.17*f 0.63 ± vo 20-hydroxyecdysone lO'S M 2.66 ± 2.81^ 1.03 + 0.54®'' 10"^ M 1.40 ± 0.76*b 3.26 ± 1.20= 10"' M 3.50 ± 1.22^ 2.46 ± 1.75'"= Methoprene 10'5 M 3.92 ± 1.30*' 2.25 ± 1.64'"= 10"'^ M 2.07 ± 1.60®'' 2.09 ± 0. 80*"= 10"' M 1.80 ± 3.06»^ 2.05 ± 1. 09abc Both 10"^ M 3.68 ± 1.58'' 2.46 ± 0. 53'"= Flies developed in a reproduction mode at 27.5°C, 16h light- 8h dark or in a diapause mode at 17.5°C, lOh light-14h dark, t Means followed by different letters are significantly different in each column (Least-Squares Means test, P < 0.05). 128 the importance of a low level of juvenile hormone for maintaining diapause. The reproductive environment effectively induced young, male or female diapausing flies to undergo reproductive development (Fig. 1). The effects, however, decreased with the age of diapausing flies (F = 12.64; df = 5, 24; P < 0.01). In contrast, methoprene was effective in reversing diapause at all ages tested (F = 0.89; df = 5, 24; P = 0.51). Genetic variation in response to methoprene Untreated (00% only) lA and MN flies showed much different responses to the diapause environment (Fig. 2). lA parents showed low diapause frequencies while MN flies did high diapause frequencies in both sexes. Hybrids (F^, lABC, and MNBC) showed intermediate responses to those of their parents. The effects of methoprene on diapausing flies varied among lines and sexes (Fig.2). As shown by a significant interaction between lines and doses (Table 3), low diapause lines (e.g., lA and lABC) were highly sensitive to smaller amounts of methoprene than were the high diapause lines (e.g., MN and MNBC). Females were more sensitive to methoprene than males. The variances attributed to lines, sex, and their interactions explained 34% of the total experimental variance. Fig. 1. Comparison of the effects of transferring flies (• 9} from a diapause (17.5*0, lOh light-14h dark) to a reproductive environment (27.5°C, 16h light-8h dark) and applying methoprene (3.5 fig: o O) on diapausing face flies at 17.5°C, lOh light-14h dark. 130 "T" 1.0 C) 0.8 - 1 0 0.6 - 0 0 0.4 u 0 3 0.2 TD 0 Transferred O Females 0.0 O Methoprene Q_ CD O 0.8 iVlales 0 12 3 4 5 6 /\ge c)f trfsoted flies (cdcjyis 131 Table 3. ANOVA on the effects of methoprene on face fly lines of different genetic backgrounds, where the analyzed variable is the arcsin transformed frequencies of reproductive flies. Source df SS F P Dose 11 32.38 198.67 0.0001 Line 5 11.30 152.49 0.0001 Sex 1 11.27 761.65 0.0001 Dose * Linet 34 2.79 5.55 0.0001 Dose * Sex 11 1.35 8.28 0.0001 Line * Sex 5 0.32 4.38 0.0008 Dose * Line * Sex 34 4.29 8.52 0.0001 Error 197 2.92 Total 298 66.65 t Test of homogeneity of slopes Fig. 2. Effects of methoprene on six lines of face flies at 15°C, 8h light-16h dark. 133 Reproductive proportions Females 0.8 0.6 MN 0.4 -G- MNBC lABC -©- lA COj 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Methoprene (ug) Reproductive proportions Males 0.8 0.6 0.4 -S- MNBC 0.2 lABC lA 00' ig CO; 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Methoprene (ug) 134 DISCUSSION A common characteristic of adult diapause is suppression of reproductive development (Denlinger, 1985). We have shown in face flies that 20-hydroxyecdysone and methoprene effectively induced diapausing flies to undergo reproductive development. The fat body of reproductive and diapausing flies synthesized Vg in vitro in the presence of 20- hydroxyecdysone or methoprene. The foregoing observations suggest that vitellogenesis in face flies requires both hormones. This hypothesis is suggested by the additivity of hormonal effects. Methoprene was effective at relatively high concentrations both in vivo and in vitro, compared to a normal physiological level of JH. In vivo results clearly indicated that application of 20-hydroxyecdysone decreased the concentration of methoprene needed. Ecdysteroids and JH participate in vitellogenin synthesis in other Diptera including Aedes aegypti (Linnaeus) (Hagedorn, 1985), Drosophila melanogaster (Meigen) (Jowett and Postlethwait, 1980), and Musca domestica Linnaeus (Adams, 1988). Both hormones are additive in their effects on inducing Vg synthesis and are required to maintain a normal Vg hemolymph titer during vitellogenesis in M. domestica (Adams, 1988). In vivo and in vitro results in face flies suggest that both hormones are maintained in the haemolymph at low levels during 135 diapause of face flies. Males also responded to 20-hydroxyecdysone and methoprene, but needed greater amounts of each than did the females. This suggests that males enter diapause more readily than females at a specific hormonal concentration in their haemolymph. Diapause studies on face flies in the laboratory have consistently shown a greater diapause incidence in males than in females (Stoffolano and Matthysse, 1967; Valder et al., 1969; Evans and Krafsur, 1990). Different hormonal thresholds between sexes to express Vg synthesis were found in M. domestica (Adams et al., 1989). Males synthesized Vg when they were treated with 20-hydroxyecdysone. But, the absence of Vg in normal males was explained by a high threshold of male fat body to the hormone. The importance of a low JH level in diapause maintenance was further supported by comparing results of transferring flies from diapause to reproductive conditions and methoprene effects on diapausing flies. Our data clearly showed that responses of diapausing flies to a reproductive environment decreased with duration of the flies in diapause conditions, but that responses to methoprene did not change with the ages of treated flies. In diapausing L. decemlineata, there is little JH synthesis in the CA (Khan et al., 1982). Khan et al. (1986) showed that the source of CA inhibition lies in the 136 lateral NSCs of the brain. In diapausing Locusta migratoria (Linnaeus), cutting the nerve between the lateral NSCs and CA activated the JH-synthesis in the CA (Baehr et. al., 1986). de Kort (1990) stressed the importance of the connection between lateral NSC and CA for the photoperiodic counter mechanism. In face flies, the decreasing responsiveness to reproductive environment according to ages of diapausing flies may have been the result of a gradual accumulation of putative inhibitory factor(s) from the brain, which then inhibits JH and ecdysone synthesis. The constant effect of methoprene on different ages of diapausing flies and the demonstration of Vg synthesis in diapausing fat body in response to exogenous JH all suggest that the hormone receptors in fat body cells are prepared to respond to these hormones even in diapause. Diapause incidence varied among lines with different genetic backgrounds. The parental lines showed extremely different diapause incidences. Hybrids showed intermediate responses to a diapause-inducing environment. Our data suggested that face fly diapause is a polygenic trait controlled by a small number of genes (unpublished data). Variation among lines in diapause incidence can be explained by different JH or ecdysteroid synthesis in the same environment or a different responsiveness of hormonal target tissues to the same amount of JH or ecdysteroids. 137 The effects of methoprene on diapausing flies in six lines were closely related to their diapause propensities. High diapause lines seemed to be resistant to methoprene and so required greater amounts for reproductive development than did the low diapause lines. Methoprene resistance was explained by different binding affinities of cytosolic JH receptors between susceptible and resistant strains of D. melanogaster (Shemshedini and Wilson, 1990). The variation in methoprene effects on face fly lines suggests that some variation in diapause comes from hormonal target tissues. Thus, phenotypic variation in face fly diapause may be attributed to the combined actions of the brain, endocrine, and target systems. 138 ACKNOWLEDGEMENT We are grateful to Dr. Roger Moon, University of Minnesota for supplying face fly puparia, to Dr. Theodore Kramer, College of Veterinary Medicine, Iowa State University for sharing his ELISA reader, and to Dr. Terry Adams, ARS, USDA, Fargo, ND, for gifts of house fly vitellin and its antibody. 139 LITERATURE CITED Adams, T.S. and Filipi, P.A. 1983. Vitellin and vitellogenin concentrations during oogenesis in the first gonotrophic cycle of the house fly, Musca domestica. J. Insect Physiol. 29: 723-733. Adams, T.S. and Filipi, P.A. 1988. Interaction between juvenile hormone, 20-hydroxyecdysone, the corpus cardiacum-allatum complex, and the ovaries in regulating vitellogenin levels in the housefly, Musca domestica. J. Insect Physiol. 34; 11-19. Adams, T.S., Filipi, P.A. and Kelly, T.J. 1989. Effect of 20-hydroxyecdysone and a juvenile hormone analogue on vitellogenin production in male houseflies, Musca domestica. J. Insect Physiol. 35: 765-773. Arends, J.J. and Wright, R.E. 1981. Mass rearing of face flies. J. Econ. Entomol. 74: 355-358. Baehr, J.C., Caruelle, J.P. and Poras, M. 1986. The activity of denervated corpora allata in a diapausing strain of Locusta migratoria: in vivo and in vitro studies. Int. J. Invertebr. Rep. 10: 143-150. Beck, S.D. 1980. Insect Photoperiodism. 2nd ed. Academic, New York. de Bianchi, A.G., Coutinho, M., Pereira, S.D., Marinotti, 0. and Targa, H.J. 1985. Vitellogenin and vitellin of Musca 140 domestica. Quantification and synthesis by fat bodies and ovaries. Insect Biochem. 15: 77-84. Bownes, M. 1980. Ovarian synthesis of yolk proteins in Drosophila melanogaster. Genetika 12: 13-2 0. Briers, T., Peferoen, M. and de Loof, A. 1982. Ecdysteroids and adult diapause in the Colorado potato beetle, Leptinotarsa decemlineata. Physiol. Entomol. 7: 379-386. Burks, C.S., Evans, L.D., Kim, Y. and Krafsur, E.S. 1992. Effects of precocene and methoprene application in young adult Musca autumnalis. Physiol. Entomol. 17: 115-120. Caldwell, E.T.N, and Wright, R.E. 1978. Induction and termination of diapause in the face fly, Musca autumnalis (Diptera: Muscidae), in the laboratory. Can. Entomol. 110: 617-622. Chippendale, G.M. 1983. Larval and pupal diapause. In Endocrinology of Insects (Eds. Downer, R.G.H. and Laufer, H.), pp. 343-356. Alan R. Liss, New York. Denlinger, D.L. 1985. Hormonal control of diapause. In Comprehensive Insect Physiology Biochemistry and Pharmacology (Eds. Kerkut, G.A. and Gilbert, L.I.) Vol. 8, pp. 353-412. Pergamon Press, Oxford. Engelmann, F. 1979. Insect vitellogenin: identification, biosynthesis and role in vitellogenesis. Adv. Insect Physiol. 14: 49-108. 141 Evans, L.D. and Krafsur, E.S. 1990. Light intensity, photoperiod, and diapause induction in face flies (Diptera: Muscidae). Environ. Entomol. 19: 1217-1222. Fye, R.C. and LaBrecque, G.C. 1966. Sexual acceptability of laboratory strains of male house flies in competition with wild strains. J. Econ. Entomol. 59: 538-540. Hagedorn, H.H. 1985. The role of ecdysteroids in reproduction. In Comprehensive Insect Physiology Biochemistry and Pharmacology (Eds. Kerkut, G.A. and Gilbert, L.I.) Vol. 8, pp. 205-262. Pergamon Press, Oxford. Hagedorn, H.H. and Kunkel, J. 1979. Vitellogenin and vitellin in insects. Ann. Rev. Entomol. 24: 475-505. Ilenchuk, T.T. and Davey, K.G. 1987. The development of responsiveness to juvenile hormone in the follicle cells of Rhodnius prolixus. Insect Biochem. 17: 525-529. Jowett, T. and Postlethwait, J.H. 1980. The regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body of 20-hydroxyecdysone and a juvenile hormone analog. Devel. Biol. 80: 225-234. Khan, M.A., Doderer, A.B., Koopmanschap, A.B. and de Kort, C.A.D. 1982. Improved assay conditions for measurement of corpus allatum in vitro in the adult Colorado potato beetle, Leptinotarsa decemlineata. J. Insect Physiol. 28: 142 279-284. Khan, M.A., Romberg-Privee, H.M. and Koopmanschap, A.B. 1986. Location of allatostatic centers in the pars lateralis regions of the brain of the Colorado beetle. Experientia 42; 836-838. Kim, Y. and Krafsur, E.S. 1993. Adult emergence patterns in the face fly, Musca autumnalis DeGeer (Diptera: Muscidae). J. Med. Entomol. 30: 816-819. de Kort, C.A.D. 1990. Thirty-five years of diapause research with the Colorado potato beetle. Entomol. Exp. Appl. 56: 1-13. Krafsur, E.S. and Black, W.C. 1992. Analysis of isozyme loci in the face fly, Musca autumnalis DeGeer. Biochem. Genet. 30: 625-634. Krafsur, E.S., Moon, R.D. and Kaya, H.K. 1985. Age structure of breeding face fly populations (Diptera: Muscidae). Environ. Entomol. 14: 590-596. Lefevere, K.S. 1989. Endocrine control of diapause termination in the adult female Colorado potato beetle, Leptinotarsa decemllneata. J. Insect Physiol. 35: 197- 203. Moon, R.D. and Kaya, H.K. 1981. A comparison of methods for assessing age structure and abundance in populations of nondiapausing female Musca autumnalis (Diptera: 143 Muscidae). J. Med. Entomol. 18; 289-297. Raikhel, A.S. and Lea, A.O. 1986. Internalized proteins directed into accumulative compartments of mosquito oocytes by the specific ligand, vitellogenin. Tissue Cell 18: 559-574. Read, N.R. and Moon, R.D. 1986. Diapause induction and commitment in face fly, Musca autumnalis (Diptera:Muscidae). Environ. Entomol. 15: 669-677. SAS Institute, Inc. 1988. SAS/STAT User's Guide, Release 6.03 ed. Gary, NO. 1028 pp. Saunders, D.S. 1982. Insect Clocks, 2nd ed. Pergamon Press, Oxford. Shapiro, J.P. and Hagedorn, H.H. 1982. Juvenile hormone and the development of ovarian responsiveness to a brain hormone in the mosquito Aedes aegypti. Gen. Comp. Endocr. 46: 176-183. 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. Stoffolano, J.G.,Jr. and Matthysse, J.G. 1967. Influence of photoperiod and temperature on diapause in the face fly, Musca autumnalis (Diptera; Muscidae). Ann. Entomol. Soc. 144 Am. 60; 1242-1246. Valder, S.M., Hopkins, T.L. and Valder, S.E. 1969. Diapause induction and changes in lipid composition in diapausing and reproducing faceflies, Musca autumnalis. J. Insect Physiol. 15: 1199-1214. 145 GENERAL SUMMARY The primary objectives of this research were to analyze the mode of inheritance of face fly diapause and to determine the roles of JH and ecdysteroids in face fly diapause. These studies advanced our knowledge and understanding of reproductive and diapause physiology in face flies. The first significant result was the estimation of genes involved in face fly diapause. Even though there is a significant deviation from the general assumptions in estimating the number of genes because of much nonadditive genetic variance, the estimation suggests that face fly diapause is controlled by a small number of genes. Genetic correlation between diapause propensities of different genetic lines and their responsiveness to methoprene, a JH analog, indicates that these diapause-associated genes act at the hormonal target tissues as well as on the brain center that controls hormonal synthesis. The second significant result is a genetic and physiological explanation of sexual dimorphism in diapause induction. Males showed higher diapause induction in most lines of different genetic backgrounds. The differences between male and female diapause induction rates were greater in high diapause lines than in low diapause lines. These indicate a sex-linkage of some diapause-associated gene(s). 146 This sexual difference is explained by different responsive thresholds to both JH and ecdysteroids: males needed larger amount of methoprene or 20-hydroxyecdysone for reproductive development than did females. The third significant result is the correlation of diapause with other developmental traits. High diapause lines showed slower developmental rates than did low diapause lines. This explains why old laboratory strains showed low diapause induction. As duration of laboratory colonization increases, the decreasing diapause incidence is a correlated response to selection because laboratory rearing favors fast developing individuals. The fourth significant result is the characterization of face fly egg proteins. Face fly vitellogenin was detected mainly in female reproductive flies. Males do not have any vitellogenin in their hemolymph. Vitellogenin has a similar subunit structure with vitellin. They are composed of four subunits with molecular weights of 46, 49, 53, and 55 kD. Antibody raised against house fly (M. domestica) vitellin reacts with vitellins of face flies, stable flies (Stomoxys calcitrans), and horn flies (Haematobia irritans). This suggests the highly conserved nature of vitellogenin within the Muscidae. Reproductive females began to synthesize vitellogenin 147 soon after adult emergence. Their hemolymph vitellogenin titers followed a sigmoid pattern. Total protein amounts in the hemolymph of reproductive females changed directly with vitellogenin titers, except for high protein levels in teneral adults. Initial high level of total proteins were explained by the presence of a storage protein which had been synthesized and accumulated since the third larval instar, but disappeared in young adults. Diapausing females also had vitellogenin in their hemolymph. Vitellogenin synthesis in diapausing females occurred only in young adults. Later, vitellogenin titers decreased while total protein concentrations in the hemolymph increased. Initial vitellogenin synthesis of diapausing females seems to be a response to the endogenous, initial JH synthetic activity of the corpora allata before the flies are induced fully to diapause. Increase of total protein amounts suggests an excess production of some "typical" hemolymph protein rather than the presence of a diapause-specific protein. The last significant result of my research concerns the role of JH and ecdysteroids in diapause development. Methoprene and 20-hydroxyecdysone induced diapausing face flies maintained in a diapause environment to undergo reproductive development. The effects of the hormones were 148 additive. Fat body from diapausing flies synthesized vitellogenin in vitro in the presence of methoprene or 20- hydroxyecdysone. Therefore, both hormones must be maintained at low levels during diapause development. In conclusion, face fly diapause is under the genetic control of a small number of genes, and induced and maintained by low levels of JH and ecdysteroids. Future studies of face fly diapause should investigate how hemolymph titers of JH and ecdysteroids are regulated early in diapause induction. One of my studies suggests that there may be a cumulative effect of an inhibitory factor on JH production. Transfer of diapausing flies from diapause to reproductive conditions induced them to enter the reproductive mode, but this effect decreased with age of transferred flies, confirming earlier work by Read and Moon (1986). In contrast, topical application of methoprene on diapausing face flies induced reproductive development irrespective of their ages. Khan and Buma (1985) observed an accumulation of a putative, unidentified inhibitory material in the corpora allata (CA) via neural transport from the brain in the diapausing Colorado potato beetle, L. decemlineata. Khan et al. (1986) showed that the inhibitory signal for JH synthetic activity of the CA comes from lateral NSCs in the brain. This inhibitory material has been regarded as an allatostatin (de Kort, 1990). 149 An experiment of cutting the nerves between the brain and CA in early diapausing flies would be helpful to verify the presence of allatostatic material in diapausing face flies. Application of cyclic AMP analogs or inhibitors of phosphodiesterase, or controlling Ca*^ ion level by using an ionophore in the CA growing medium would be helpful to study the presumptive allatostatic effect and determine its signal transduction pathway. A study of the mechanisms controlling ecdysteroid levels could be performed in at least two steps. One is to find the source of ecdysteroid production in adult face flies. Detection of ecdysteroids in the culture medium of the ovary would be helpful because the ovary is the major ecdysteroid- producing organ in most adult insects (Hagedorn, 1985). The other is to examine the ecdysiotropic factor because the release of this factor is probably blocked in diapause induction in face flies. A study of JH-specific esterase would be helpful to understand how JH in teneral adults (which may be caused by the initial, endogenous JH production) is cleared off early in diapause induction. It would be desirable also to examine the relationship between onset of diapause and the activity of JH- specific esterase. 150 LITERATURE CITED Adu-Hakima, R. and Davey, K.G. 1979. A possible relationship between ouabain-sensitive (Na*-lC)-dependent ATPase and the effect of juvenile hormone on the follicle cells of Rhodnius prolixus. Insect Biochem. 9: 195-198. Adams, T.S. 1981. The role of ovarian hormones in maintaining cyclical egg production in insects. In Advances in Invertebrate Reproduction (Eds. Clark, W.H. and Adams, T.S.), pp. 109-125. Elsevier Press, North Holland. Adams, T.S. and Filipi, P.A. 1988. 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