- Email: [email protected]
Biosynthesis and release of juvenile hormone during the reproductive cycle of the ring-legged earwig
Camp. Biochem. Physiol. Vol. I IOC, No. 3, pp. 241-251, 1995 Elsevier Science Ltd Printed in Great Britain 0742+X413/95 $9.50 + 0.00
0742-8413(95)00016-X
Biosynthesis and release of juvenile hormone during the reproductive cycle of the ring-legged earwig Susan M. Rankin,* James 0. Palmer,* Grant L. Scott* and Stephen S. Tobet *Department of Zoology,
of Biology, Allegheny University of Toronto,
College, Toronto,
Koichiro
J. Yagi,“f
Meadville, PA 16335, U.S.A.; Ontario, Canada M5S IA1
and TDepartment
Corpora allata of adult female Euborellia annulipes, incubated in medium containing 3H-metbionine, synthesized and released juvenile hormone III. Labelled material co-migrating with methyl farnesoate was also found, suggesting this as an intermediate in the pathway of juvenile hormone III production. Juvenile hormone was not appreciably stored in the glands, but was released into the medium. In normal medium, 93.6 f 1.6% of the total juvenile hormone III synthesized was released and 96.5% f 0.3 in medium supplemented with 60 PM farnesoic acid. The rate of juvenile hormone III biosynthesis/release in vitro remained constant for at least 8 hr for glands of different activities. The rate of juvenile hormone production was closely correlated with the gonadotrophic cycle. In females with previtellogenic ovarian follicles (0.26 f 0.004 mm), hormone production was only 0.59 + 0.13 fmol hr-‘/corpus allatum; production increased to 1.52 + 0.25 fmol hr-‘/corpus allatum when basal follicles were growing rapidly, and remained high during the period of oviposition. By 3 days following oviposition when females were brooding clutches, hormone production had declined to 0.46 + 0.13 fmol hr-‘/corpus allatum. The addition of 60 gM farnesoic acid to the medium enhanced juvenile hormone biosynthesis at each stage examined. Lastly, elevating the level of L-methionine in Maximal hormone production was the medium also enhanced hormone biosynthesis. 32.8 + 10.9 fmol hr-‘/corpus allatum, at an L-methionine concentration of 51 FM. Key words: Reproductive cycle; Egg development; Corpus allatum; In vitro incubations. Comp. Biochem.
Physiol.
IlOC, 241-251,
Earwig;
Juvenile
hormone;
Farnesoic
acid;
1995.
Introduction Corpora allata are endocrine glands that synthesize and release juvenile hormone (JH). In most insects, JH regulates larval development and adult reproduction [see Tobe and Stay (1985) for a review]. The use of direct radiochemical in vitro techniques for monitoring JH production has demonstrated a close relationship between corpus allatum activity and
ovarian
development
in cockroaches
such
as
Diploptera Periplaneta
punctata (Tobe and Stay, 1977), americana (Pratt et al., 1975; Weaver and Pratt, 1977; Weaver et al., 1975; Edwards et al., 1990) and Nauphoeta cinerea (Lanzrein et al., 1978, 198 1). Additionally, such a correlation has been suggested in Schistocerca gregaria (Tobe and Pratt, 1975a,b), Melanoplus sanguinipes (McCaffery and McCaffery, 1983), Leptinotarsa decemlineata (Kramer, 1978) and Tenebrio molitor (Weaver et al., 1980).
Correspondence fo: S. M. Rankin, Department of Biology, Allegheny College, Meadville, PA 16335, U.S.A. Tel. (+ 1) 8 14-332-5375; Fax (+ 1) 8 14-332-2789; e-mail [email protected]. Abbreviations: JH, juvenile hormone; FEAR, fractional endocrine activity ratio. Received 29 September 1994; revised 23 December 1994; accepted 23 December 1994.
To our knowledge, neither corpus allatum activity of earwigs nor its potential correlation with ovarian development has been reported. Studies in these animals are particularly interesting for two reasons. (1) Morphologically, 241
242
S. M. Rankin
earwigs possess highly unusual primitive polytrophic ovaries, in which a single nurse cell (trophocyte) contributes to the development of its primary oocyte partner (Bonhag, 1956; Nath et al., 1959). Studies to date using radiochemical assays for JH production in adult female insects have been performed on orthopteroid insects with panoistic ovaries (cockroaches and locusts) or on beetles (see above) with complex polytrophic ovaries. (2) Behaviorally, these animals are subsocial, providing extensive maternal care for clutches during embryogenesis and for newly hatched larvae. No similar studies to date have been performed on subsocial insects: most are restricted to solitary insects, although JH production has been addressed in some social insects, such as honey bees (Huang et al., 1991) and primitive termites (Greenberg and Tobe, 1985). Preliminary studies of JH production must include identification of the type(s) of JH produced as well as some delineation of the pathways of JH synthesis. The identities of several synthesized juvenile hormones have been established (Bergot et al., 1981a,b; Tobe and Stay, 1985; Cusson et al., 1991). For example, in the lepidopteran, Munducu sextu, JH 0, JH I, JH II, JH III and 4-methyl JH I have been reported (Bergot et al., 1981a,b; Peter et al., 1981) as well as acids of three of the JH homologues (Bhaskaran et al., 1986). In Pseuduletiu unipunctu, corpora allata from males also release the acids of the homologues, and perhaps homo- and/or dihomo farnesoic acid, whereas those of females release the three common homologues and an additional esterified product (Cusson et al., 1991). In the lower dipterans, only JH III has been reported (Baker et al., 1983; Guilivard et al., 1984; Readio et al., 1988) whereas in Drosophila melunoguster, the ring glands release both bis-epoxide JH III and methyl farnesoate (Richard et al., 1989) and in Culliphoru vomitoriu, the corpora allata may also release 6,7-epoxide as well (Cusson et al., 1991). In the cockroach Diplopteru punctutu, fourth instar larvae release farnesoic acid as well as JH III (Yagi et al., 1991). Although no information is available for in vitro synthesis of JH by corpora allata of any dermapteran, in the earwig Labidura ripuria, JH III is the primary gonadotropic hormone (Baehr et al., 1982; Vantassel, 1984; Vancassel et al., 1984). We report here our identification of the JH produced by corpora allata in vitro of post-tenera1 adult female earwigs, Euborellia unnulipes using a direct, radiochemical assay. We further establish the degree to which biosynthesized hormone is stored or released from corpora allata in vitro, and the suitability of incubation conditions for incubations of up to 8 hr. We
et
al
assess the correlation between adult female age, basal follicle length, and JH production. Finally, we determine the effects of farnesoic acid on JH production in vitro in glands of varying physiological stages. We thus address the degree to which JH production is tightly coupled to this gonadotrophic cycle in a primitively polytrophic ovary and provide the basis for investigating key regulatory events at the endocrine level that may affect the initiation, maintenance, or termination of subsocial behavior.
Materials and Methods Earwigs Our colony is descended from a founder population collected in College Station, Texas in 1989-1990. Individuals were field-collected as second instar-to-adult animals, and reared subsequently in our laboratories. Generations descended from the founder population were used for our experimental observations. A stock population of animals was groupreared in polypropylene containers (30 x 16 x 8 cm’) with moist paper towels, cotton-stoppered vials and dry catfood ad libitum in incubators at 28°C 12: 12 hr light :dark cycle. For most experiments, O-day adult females were removed from stock cultures and each maintained with one or two post-teneral males in 8 oz polypropylene containers with dry catfood and cotton-stoppered water vials. The moist cotton in each container maintained high humidity and provided oviposition sites. Thus, for these studies, animals were reared as families; however, the adult males were routinely removed on the day of oviposition, since preliminary experiments in which the male remained with the family frequently resulted in the death of the male (presumably by the mother defending her clutch against predation by the male). Similar anecdotal observations of dead males near nests of brooding females or of female aggression have been made for the earwigs E. annulipes 1942; Bharadwaj, 1966) (Klostermeyer, E. cincticollis (Knabke and Grigarick, 1971) Forficulu uuriculuriu (Lamb, 1976; Crumb et al., 1941) and L. ripuriu (Caussanel, 1970). Dissections Surgery was performed on chilled animals. The corpora cardiacacorpus allatum complexes were removed through a dorso-posterior window in the head cuticle. No attempt was made to isolate the corpus allatum from the corpora cardiaca. Earwigs have two corpora cardiaca and a single median corpus allatum in close association with the dorsal aorta (DeLerma, 1942; Cazal, 1948; Ozeki, 1958,
Juvenile
hormone
biosynthesis
1961; Lhoste, 1957; Awasthi, 1975; Juberthie and Caussanel, 1980). Ovaries were dissected from chilled animals following removal of abdominal sternites. Basal follicle lengths were determined using a dissecting microscope with an ocular micrometer, and the number of ovarioles (typically five) was recorded. In vitro incubations
of corpora allata
For identification of the JH produced in this insect, corpora cardiacacorpus allatum complexes were excised from preovulatory females whose ovaries were nearing completion of vitellogenesis (basal follicle length > 0.9 mm), then incubated at 28°C in medium 199 (GIBCO) lacking methionine and supplemented with [methyl-3H]methionine (NEN; 1 PM 3H-met; 80 mCi/pmol), 2% Ficoll and 5 mM CaCl,. The effect of farnesoic acid on JH synthesis by corpora allata from pre-ovulatory females with ovaries in late vitellogenesis was determined in two ways: (1) by incubating glands in medium containing 60 PM farnesoic acid and comparing production with that of glands in normal medium. There was no significant difference in basal follicle length between females of the farnesoic acid-treated groups and those of untreated controls; (2) by incubating glands in normal medium for 3 hr, then transferring the glands to medium supplemented with 60 PM farnesoic acid for an additional 3-hr incubation. Another (control) group was incubated in normal medium for the second 3-hr incubation. The dose-relationship for JH synthesis as a function of L-methionine concentration was determined in medium supplemented with the appropriate concentrations of unlabelled Lmethionine (Sigma Chemical Co., St Louis, MO). Extraction
of biosynthesized
products
High-performance liquid chromatography (HPLC) analysis was performed as described by Cusson et a/. (1991) to identify radiolabelled compounds synthesized by the corpora allata. In this test, six glands with media were extracted as a single sample; following extraction, only one-third of the resulting extract comprised a single application. No difference was seen between the three runs of each sample. Unlabelled standards were applied with our samples to confirm elution times of potential products. Glands were not removed from media prior to extraction; hence, we monitored the total amount of juvenile hormone biosynthesized, and had the potential to determine whether methyl farnesoate which would not ordinarily be released into the medium, was an intermediate in the production of JH III, as has been
243
in earwigs
shown in some other insects [e.g. D. punctata; Feyereisen et al. (198 l)]. Thin-layer chromatography (TLC) was performed (a) to provide independent verification of radiolabelled compounds; (b) to quantify the relationship between the amount of JH released into the medium and the total amount of hormone produced; and (c) to quantify hormone release during the first gonadotrophic cycle. For these analyses, glands and media were extracted and purified separately as described elsewhere (Tobe and Pratt, 1974; Pratt and Tobe, 1974) except that three glands (rather than isolated pairs) were analyzed as single samples to ensure adequate amounts of radiolabelled products. Unlabelled standards were run with our unknown samples to monitor migration of compounds.
Results I. IdentiJication of radiolabelled products and of the efSects of farnesoic production
acid on juvenile
hormone
HPLC analysis (Fig. I), performed on extracts of incubation medium together with corpora allata, demonstrated that JH III was the only JH produced by the corpora allata of adult female earwigs in which ovaries are nearing completion of the gonadotrophic cycle. The major radioactive compound detected after incubation of glands in normal medium (i.e. not supplemented with farnesoic acid) was JH III (73.9%). Not surprisingly, labelled methyl farnesoate was also present (8.1%). Another component (retention time 8.6 min) was also detected that did not correspond to known juvenoids, and comprised 24.3% of the radioactive material. The addition of 60 p M farnesoic acid to the medium resulted in a 4-fold increase in JH production (Fig. 1). In this case, JH III accounted for 92.2 + 0.4% and methyl farnesoate for 7.8 + 0.4%. The unknown component with retention time of 8.6 min was not observed in samples incubated in medium supplemented with farnesoic acid. A second experiment was performed to confirm the results of HPLC analysis and to distinguish between JH production and hormone release into the medium. Samples of three pairs of glands each from 1 l-day females (basal follicle length 0.55 + 0.08 mm) were incubated for 3 hr in normal medium, then transferred to medium supplemented with 60 PM farnesoic acid for a second 3-hr incubation. After incubation, glands were removed from the medium and extracted and processed separately. A typical chromatogram of these results from a single
244
S. M. Rankin
0
2
4
Time
6
er al.
8
after
injection
10
12
14
(min)
Fig. I. High performance liquid chromatography (HPLC) analysis of the radiolabelled products biosynthesized by corpora allata of adult female E. annulipes. Corpora allata from six females per sample were incubated in modified TC 199 medium and ‘H[met], either with (dashed line) or without (solid line) 60 PM farnesoic acid, then assayed according to the methods of Cusson et al. (1991). Retention times of standards indicated by arrows are methyl farnesoate (MF), juvenile hormone I (JH I), juvenile hormone II (JH II) and juvenile hormone III (JH III). Basal follicle lengths from corpus allatum donors were I 17 k 0.18 and 0.96 + 0.18 mm, respectively.
sample is shown in Fig. 2. The major product released into the medium was JH III (Fig. 2B), and farneosic acid again dramatically enhanced JH production. Extraction of glands (Fig. 2A) demonstrated that JH III again was the predominant product, and that methyl farnesoate was also present. In the example shown, the amount of JH III extracted from the glands was only 4.2% of that in the medium, suggesting that the product is released from the glands into the medium. Radiolabelled methyl farnesoate was present in proportionally much greater quantities in the glands than in the medium (for example, the ratio of JH-to-methyl farnesoate in the example shown is 3.3: 1 and 43.6: 1, for glands and medium, respectively). 2. Correlation between juvenile hormone biosynthesis and release There is a linear relationship between JH biosynthesis and release for glands either incubated in normal medium for 6 hr (n = 7) or in medium supplemented with farnesoic acid for the final 3 hr (n = 35) (Fig. 3). For corpora allata in normal medium, radiolabelled JH accounted for 93.6 f 1.6% of the total biosynthesized hormone (r* = 0.994), whereas in supplemented medium, the released radiolabelled hormone accounted for 96.5 ? 0.3% (r* = 1.000). These results are consistent with the TLC analysis presented above (Fig. 2). 3. Time course of earwig juvenile synthesis
hormone
bio-
After establishing that hormone release was highly correlated with JH production in vitro
(Fig. 3) we next determined whether our incubation conditions could sustain JH production in vitro for at least 8 hr. To that end, glands were incubated in medium for 8 hr, but corpora allata were transferred to new medium at 2 hr intervals. Rates of JH production over an 8-hr incubation period were constant for the four samples with varying degrees of activity (r2 = 0.995, 0.925, 0.989, 0.986 for the samples A., 0, a, and 0, respectively) (Fig. 4). These results show that the incubation medium is sufficient to support JH synthesis for at least 8 hr. 4. Correlation between juvenile hormone thesis and the gonadotrophic cycle
syn-
To begin our investigation of the endocrine control of reproduction in this subsocial animal, we documented the relationship between age, basal follicle length, and JH biosynthesis (Fig. 5). For this laboratory population, basal follicle length was small in 3- and 5-day-old females (0.26 + 0.004 and 0.27 + 0.02 mm, respectively), then increased dramatically between days 5 and 13 to 0.84 + 0.05 mm. Oviposition typically occurred on days 15-17 after adult eclosion, over a I-2-day period. By 3 days after oviposition, the lengths of new basal follicle were small (0.40 f 0.06 mm, n = IS), and did not increase (perhaps regressed slightly) for at least 8 days after oviposition (basal follicles were 0.31 + 0.03 mm; n = 15; Fig. 5A). During that post-ovipositional period, females displayed pronounced maternal care.
Juvenile
600
“0
hormone
biosynthesis
245
in earwigs
-
2
4
6
Migration
i
Migration
6
10
distance
(cm)
12
i
distance
(cm)
Fig. 2. Thin-layer chromatographic (TLC) analysis of the biosynthetic products of corpora allata from three 1 l-day-old adult female E. annulipes. Medium (B) was assayed separately from glands (A, assayed after 6 hr) according to the methods of Tobe and Pratt (1974). Glands were incubated in the absence of farnesoic acid (solid line) for 3 hr, then transferred to medium containing 60pM farnesoic acid for an additional 3-hr incubation (dashed line). Migration distances of standards are indicated by arrows for juvenile hormone III (JH III), methyl farnesoate (MF), and JH III bis epoxide (BIS). The addition of farnesoic acid significantly enhanced JH III biosynthesis. The basal follicle length of donor females was 0.55 + 0.08 mm (mean + standard error of the mean, SEM).
JH production by corpora allata from females during the first gonadotrophic cycle was determined by TLC analysis following a 3-hr incuJH production was low bation period. (0.59 + 0.13 fmol hr-‘/corpus allatum) in previtellogenic females, then increased between days 5 and 13 to 1.52 f 0.25 fmol hrr’/corpus allatum as follicles grew rapidly (Fig. 5B,C). JH production remained high on the day of oviposition, likely related to the fact that oviposition spanned a I-2-day interval. Following oviposition, JH production declined to low levels (0.46 + 0.13 fmol hr-‘/corpus allaturn). These experiments also demonstrated (data not shown) that JH III is the major product synthesized by glands of different activities throughout the first reproductive cycle.
We investigated whether the addition of farnesoic acid to incubation medium stimulated JH III production throughout the first gonad0 trophic cycle by incubating glands first for 3 hr in unsupplemented medium, then for 3 hr in medium supplemented with 60 PM farnesoic acid. For glands from females of each physiological stage, farnesoic acid had a dramatic effect on JH III production (Fig. 5B). The cyclic nature of JH production was preserved in glands stimulated with farnesoic acid; however, glands from females on the day of oviposition (near-maximally active glands in unsupplemented medium) were stimulated to a much lesser degree than were corpora allata from females of other physiological states. The ratio between the rates of JH synthesis in
246
S. M. Rankin
et ~1.
80000
60000
0
20000 JH
40000 released
60000
80000
(DPM)
Fig. 3. Relationship between earwig juvenile hormone biosynthesis and release. For this experiment only, biosynthesis is defined as the total amount of juvenile hormone released into the medium and that present in the corpora allata. Glands were either incubated in normal medium for 668 hr (solid circles), or for 3 hr in normal medium followed by 3 hr in medium supplemented with 60 PM farnesoic acid (open circles). Each point represents the measurement of three glands incubated in a single sample.
normal medium and then in the medium enhanced with farnesoic acid, termed the fractional endocrine activity ratio [FEAR; see Tobe and Pratt (1976)], was 0.0440.06 for each physiological state, except ovipositing females, in which the FEAR was 0.13. Thus, the addition of farnesoic acid stimulated JH production by corpora allata in vitro by 7-17-fold. 5. EfSect of L-methionine juvenile hormone biosynthesis on
concentration
from females in late vitellogenesis were used, since they exhibit high rates of hormone biosynthesis in vitro (Fig. 5). At 1 PM, glands released labelled hormone at 1.35 + 0.47 fmol hr ‘/ corpus allatum (confirming relatively high activity of glands). The addition of unlabelled methionine significantly enhanced hormone production (Fig. 6). Rates of JH biosynthesis increased with increasing concentration up to 51 PM, at which concentration corpus allatum activity was 32.8 f 10.9 fmol hr-‘/corpus allaturn. Above this level, no further increase was realized.
on
To assess the dependence of JH biosynthesis methionine concentration, corpora allata
8000
6000
0
2 Incubation
4
6 time
8
10
(hours)
Fig. 4. Time course of earwig juvenile hormone biosynthesis. Each point represents the rate of cumulative biosynthesis from four samples (0, 0, A., A) each containing three glands from vitellogenic stage adult females. Note the variation in rates of synthesis. Total incubation time was 8 hr, but corpora allata were transferred to new medium after 2, 4 and 6 hr of incubation.
0.6
1.6
0.6
1.6
0.6
0.4 ’ u.2 Basal
I
I
0.4
0.6
follicle
length
I 0.6 (mm)
Fig. 5. Relationship between basal follicle length and rate of juvenile hormone synthesis in earwigs, (A) Mean basal follicle lengths (& SEM; n = 12-l 5 females, see below). (B) Juvenile hormone biosynthesis (solid circles) and the effect of farnesoic acid on juvenile hormone biosynthesis (open circles). Assays were determined on corpora allata from the same earwigs for which ovarian basal follicle lengths were determined (n = 5 samples each with corpora allata from three females, except for that of the day of oviposition, wherein four samples of three corpora allata each were assayed). Glands were incubated for 3 hr without farnesoic acid, then transferred to medium containing 60 PM farnesoic acid for an additional 3-hr incubation. (C) Relationship between basal follicle length and juvenile hormone production for females in the first gonadotrophic cycle. Data shown are from glands incubated in medium not supplemented with farnesoic acid. Vertical and horizontal bars represent SEM.
S. M. Rankin
248
1
11
L-methionine
et al.
101
51
concentration
(FM)
Fig. 6. Dose-dependence of juvenile hormone biosynthesis by corpora allata from females with ovaries in late vitellogenesis as a function of L-methionine concentration. Corpora allata (highly active glands) were incubated for 6 hr in medium supplemented with unlabelled L-methionine to the appropriate concentrations. Each sample contained three corpora allata, and each point represents six samples. Vertical bars represent SEM.
Discussion Sufficient radiolabelled product was synthesized and released from short-term incubations of corpora allata from adult female E. annulipes, in medium containing 1 PM [methyl-‘Hlmethionine, to permit identification of the hormone. A combination of HPLC (Fig. 1) and TLC (Fig. 2) has identified the major product to be JH III. We found no incorporation of radiolabel into either JH I or JH II, nor any other known JH-related compound. This finding is in agreement with studies of the hemolymph of post-teneral adult females of the earwig Labidura riparia, using HPLC analysis after derivitization of the extracted JH to the diol form which identified JH III as the principal JH (Vancassel et al., 1984). Presence
of methyl farnesoate
We detected radiolabelled material which comigrated with methyl farnesoate in two instances: (1) using HPLC analysis of samples in which glands were extracted with medium (Fig. I); (2) by TLC analysis of glands alone after incubation (Fig. 2A). These results are consistent with the hypothesis that the production of JH III in earwig corpora allata proceeds along the pathway now attributed to most insects (Cusson et al., 1991): farnesoic acid is likely methylated to form methyl farnesoate, and this product is subsequently epoxidized to form JH III.
Storage/release
of juvenile
hormone
III
JH was not stored to any appreciable extent in corpora allata in vitro (Fig. 3). JH is similarly
released into the medium when produced in vitro by corpora allata from S. gregaria (Tobe and Pratt, 1974), P. americana (Pratt et al., 1975), D. punctata (Feyereisen et al., 1981) and Apis melf@ra (Huang et al., 1991). Thus, in earwigs, as in other insects, JH III titers are likely regulated by synthesis and/or degradation of hormone, rather than by sequestration in or release from the endocrine source. Suitability
of incubation
conditions
JH biosynthesis and release occurred at a constant rate in vitro for at least 8 hr (Fig. 4). Since in our sequential 2-hr incubations, we observed no detectable lag-time in the production of labelled JH, it appears that corpora allata from these earwigs had very low methionine that levels of endogenous were rapidly diluted by the exogenous radiolabelled methionine, as has been suggested for the locust, S. gregaria (Tobe and Pratt, 1974) and the beetle, T. molitor (Weaver et al., 1980). To our knowledge, there are no reports of amino acid concentrations in earwig hemolymph. The linear rate of incorporation of label into newly biosynthesized JH III for 8 hr (Fig. 4) demonstrated that the medium is sufficient to support corpus allatum function for at least that interval. This further suggests that incubations of this duration (or shorter) yield estimates of glandular activity that reflect rates of hormone production by the corpora allata immediately prior to their removal from these adult females.
Juvenile
Correlation of juvenile hormone with age and the gonadotrophic
hormone
biosynthesis
III biosynthesis cycle
This is the first report of rates of JH production in any dermapteran (Fig. 5). Our finding that the rate of JH III production is correlated with the gonadotrophic cycle, and that synthesis remains high during the ovipositional period in this earwig is in agreement with the change in JH III hemolymph titer in L. riparia associated with the gonadotrophic cycle (Vancassel et al., 1984). These findings are consistent with the notion that maturing ovaries might stimulate JH production, as has been shown for the cockroach, D. punctata (Stay et al., 1983; Rankin and Stay, 1984). Variability in egg lengths and corresponding rates of synthesis (Fig. SC) is likely attributable, in part, to incubation of glands from three females (with potentially different basal follicle lengths) as single samples (see above). Our discovery that, in the earwig, JH III production remained high during the ovipositional period suggests that, unlike regulation in D. punctata, JH production in this earwig does not include inhibition by mature ovarian follicles (Rankin and Stay, 1985). Following oviposition (during the brooding period), JH production declined. This period of maternal care may thus be analogous to the situation in pregnant D. punctata (Rankin and Stay, 1985). These results are consistent with the finding in L. riparia that hemolymph titers of JH III are relatively low during the brooding phase (Vancassel et al., 1984). Effects of exogenous farnesoic hormone III biosynthesis
acid on juvenile
Farnesoic acid has been shown to stimulate JH production in vitro in corpora allata from a number of orthopteroids, including adult female D. punctata (Feyereisen et al., 1981; Feyereisen et al., 1984), larval D. punctata (Yagi et al., 1991), P. americana (Pratt et al., 1975) and locusts (Tobe and Pratt, 1974). It is also effective in stimulating corpus allatum activity from the adult female beetles, T. molitor (Weaver et al., 1980) and L. decemlineata (Khan et al., 1982). These findings indicate that in these species, and in the earwig, rate-limitation most likely occurs prior to the conversion of farnesoic acid to juvenile hormone. The degree of stimulation of JH III production by farnesoic acid has been expressed as the ratio between the rates of JH synthesis in normal medium and then in medium supplemented with farnesoic acid [FEAR, see Tobe and Pratt (1976); Feyereisen et al. (1981); Yagi et al. (1991)]. In D. punctata (Feyereisen et al., 1981; Yagi et al., 1991) and in locusts (Tobe and
249
in earwigs
Pratt, 1975a, 1976; Gadot and Applebaum, 1986; Couillaud et al., 1988), glands of low activity have low FEAR values, whereas glands of high spontaneous activity have high FEAR values. If farnesoic acid substantially stimulates JH III biosynthesis (a very low FEAR) as occurs, for example, at the end of last larval stadium in D. punctata males (Yagi et al., 1991), and in adult female earwigs (Fig. 5), the ratelimiting steps very likely occur prior to the O-methyl transferase. Eflects of L-methionine production
concentration
on hormone
The relationship between L-methionine concentration and JH production has been examined in a number of insects; interestingly, the optimal level of methionine is quite variable. For example, for the primitive termite, Z. angusticollis, optimal levels are only 5-10 p M (Greenberg and Tobe, 1985); in the cockroach, D. punctata, optimal levels are 20-40 PM (Tobe and Clarke, 1985); in S. gregaria, the optimum is 100-400 p M (Tobe and Pratt, 1974), similar to that observed for the beetle, T. molitor (300 PM, Weaver et al., 1980). These differences suggest potiential differences in the ability to scavenge L-methionine and in the size of the endogenous pool of L-methionine in glands from different species (Tobe and Clarke, 1985). Our result for this earwig species demonstrating 51 PM as an adequate concentration for methionine clearly places these animals well within the previously observed optimal spectrum of methionine concentrations for insects examined thus far. Conclusions The characterization of the hormone of this earwig as JH III, coupled with the demonstration of constant rates of incorporation for at least 8 hr in vitro, and the strong correlation of JH III production in uitro with the reproductive cycle indicates that the radiochemical assay for JH biosynthesis can be used for the study of the interactions between JH production, egg development and the associated behavioral phenomenon of maternal care. The ability to measure rates of JH biosynthesis in these subsocial insects will also help in our efforts to discover potential physiological linkages of JH and behavior in the evolution of insect sociality. Acknowledgements-This
work was supported
by National
Science Foundation grant no. DCB 9118275 to SMR; support for this research was additionally provided by a grant from the Howard Hughes Medical Institute through the Undergraduate Biological Sciences Education Program awarded by Allegheny College and by a grant from the National Science and Engineering Research Council of Canada. We are also indebted to undergraduate student researchers Heather Dossat and John Lunz for excellent technical assistance.
250
S. M. Rankin
References Awasthi
V. B. (1975) Neurosecretory system of the earwig riparia, and the role of the aorta as a neurohema1 organ. J. Insect Physiol. 21, 1713-1719. Baehr J.-Cl., Cassier P., Caussanel Cl. and Porcheron P. (1982) Activity of corpora allata, endocrine balance and reproduction in female Labidura riparia (Dermaptera). Cell Tiss. Res. 225, 267-282. Baker F. C., Hagedorn H. H., Schooley D. A. and Wheelock G. (1983) Mosquito juvenile hormone: identification and bioassay activity. J. Insect Physiol. 29, 465-470. Bergot B. J., Ratcliff M. and Schooley D. A. (198la) Method for quantitative determination of the four known juvenile hormones in insect tissue using gas chromatography-mass spectroscopy. J. Chromatogr. 204, 231-244. Bergot B. J., Baker F. C., Cerf D. C., Jamieson G. and Schooley D. A. (1981 b) Qualitative and quantitative aspects of juvenile hormone titers in developing embryos of several insect species: Discovery of a new JH-like substance extracted from eggs of Manduca sex&. In Jurenile Hormone Biochemistrv (Edited bv Pratt G. E. and Brooks G. T.), pp. 33-45. Eisevier, Amsterdam. Bharadwaj R. K. (1966) Observations on the bionomics of Euborellia annulipes (Dermaptera: Labiduridae). Ann. Labedura
Entomol.
Sot.
Am. 59, 44-450.
Bhaskaran G., Sparagana S. P., Barrera P. and Dahm K. H. (1986) Change in corpus allatum function during metamorphosis of the tobacco hornworm Manduca sexta: Regulation at the terminal step in juvenile hormone biosynthesis. Arch. Insect Biochem. Physiol. 3, 321-338. Bonhag P. F. (1956) The origin and distribution of periodic acid-Schiff-positive substances in the oocyte of the earwig, Anisolabis maritima (G&n&). J. Morphol. 99, 433- 463. Cazal P. (1948) Les glandes endocrines r&troctrtbrales des lnsectes (ttudes morphologiques). Bull. biol. France et Belgiaue,
spp.
32.
Couilia;d F., Mauchamp B., Girardie A. and DeKort S. (1988) Enhancement bv farnesol and farnesoic acid of juvenile-hormone biosinthesis in induced low activity locust corpora allata. Arch. Insect Biochem. Phy.siol. 7, 133-143.
Cusson M., Yagi K. J.. Ding Q., Duve H., Thorpe A., McNeil J. N. and Tobe S. S. (1991) Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: the search for a unifying arthropod endocrinology. Insect Biochem. 21, l-6. DeLerma B. (1942) Ricerche sul sistema endocrine cefalico (corpora allata e corpi faringei) e sullo stomatogastrico hei Dermatteri. Boll:Sec. Nat. Napoli 52, 25- 42. Edwards J. P.. Chambers J.. Short J. E., Price N. R., Weaver R. J., Abraham L. and Walker C. M. (1990) Endogenous juvenile hormone III levels and in vitro rates of hormone biosynthesis by corpora allata during the reproductive cycle of adult female Periplaneta americana. In Chromatography and Isolation of Insect Hormones and Pheromones (Edited by McCaffery A. R and Wilson 1. D.),
pp. l-8. Plenum Press. Feyereisen R., Friedel T. acid stimulation of C,, corpora allata of adult Biochem.
New York. and Tobe S. S. (1981) Farnesoic juvenile hormone biosynthesis by female Diploptera punctata. Insect
11, 401-409.
Feyereisen R., Ruegg R. P. and Tobe S. S. (1984) Juvenile hormone III biosynthesis: stoichiometric incorporation of 12-‘%I acetate and effects of exogenous farnesol and farneioic acid. Insect Biochem. 14, 657- 661. Gadot M. and Applebaum S. W. (1986) Farnesoic acid and allatotropin stimulation in relation to locust allatal maturation. Molec. Cell. Endocr. 48, 69-76. Greenberg S. and Tobe S. S. (1985) Adaptation of a radiochemical assay for juvenile hormone biosynthesis to study caste differentiation in a primitive termite. J. Insect Phvsiol. 31. 347-352.
et al.
Guilivard D., DeReggi M. and Rioux J. A. (1984) Changes in ecdvsteroid and juvenile hormone titers correlated . tn ._ the iiitiation of Gitellogenesis in two Aedes species (Dintera. Culicidae). Gen. coma. Endocr. 53. 218-223 ,. Hia& Z.-‘Y., Robinson G. E.,*Tobe S. S., ‘Yagi K. J., Strambi C., Strambi A. and Stay B. (1991) Hormonal regulation of behavioural development in the honey he? -__ is-based on changes in the rate of juvenile hormone biosvnthesis. J. Insect Phvsiol. 37. 733-741. Juberthie C. and Caussanel Cl. (1980) Release of brain neurosecretory products from the neurohaemal part of the aorta during egg-laying and egg-care in Labidura riparia (Insecta, Dermaptera) J. Insect Physiol. 26, 427-429.
Khan M. A., Koopmanschap A. B., Privee H. and DeKort C. A. D. (1982) The mode of regulation of the corpus allatum activity during starvation in adult females of the Colorado potato beetle, Leptinotarsa decemlineata (Say). J. Insect Physiol.
28, 791-796.
Klostermeyer E. C. (1942) The life history and habits of the _._. ring-legged earwig Euborellia annulipes (Lucas) (Dermaptera). J. Kansas Entomol. Sot. 15, 13-18. Kramer s. J. (1978) Age-dependent changes in corpus allatum activity in vitro in the adult Colorado potato beetle, Leptinotarsa decemlineata. J. Insect Physiol. 24, 461-464.
Lanzrein B., Gentinetta V. and Liischer M. (1978) Correlation between haemolymph juvenile hormone titre, corpus allatum volume, and corpus allatum in viuo and in vitro activity during oocyte maturation in a cockroach (Naupheota
cinerea).
Gen. Comp. Endocrinol.
36,339%345.
Lanzrein B.. Whilhelm R. and Gentinetta V. (1981) On relations between corpus allatum activity and oocyte maturation in the cockroach Nauphoeta cinerea. In Regulation of Insect Development and Behavior (Edited by Sehnal F., Zabza A., Menn J. J. and Cymborowski B.), pp. 523-534. Wroclaw Technical University Press, Wroclaw. Lhoste J. (1957) Donntes anatomiques et histophysiologiques sur ForJicula auricularia L. (Dermapttre). Arch. 2001.
Erp.
G&n. 95, 75-252.
McCaffery A. R. and McCaffery V. A. (1983) Corpus allatum activity during overlapping cycles of oocyte growth in adult female Melanoplus sanguinipes. J. Insect Physiol.
29, 259-266.
Nath V., Gupta B. L. and Aggarwal S. K. (1959) Histochemistry of vitellogenesis in the earwigs Labidura riparia Pall. and L. bengalensis Dohrn. Res. Bull. Punjab Univ. 10, 315-341. Ozeki K. (1958) Neurosecretion of the earwig, Anisolohi~ -I .._ maritima.
Sci. Pap. Coil. Gen. Ed. 8, 85-92.
Ozeki K. (1961) Secretion of the juvenile hormone in the ...imago 0; the earwig, Anisolabis karitima. Sci. Pap. Coil. Gen. Ed. 11, 101-107. Peter M. G.. Shirk P. D., Dahm K. H. and R(iller H. (19811 On the specificity of juvenile hormone biosynthesis in the male cecropia moth. Z. Naturf 36~. 579-585. Pratt G. E. ‘and Tobe S. S. i1974) Juvenile hormones radiobiosynthesized by farnesoic acid and its analogues. Lancer i, 1270-1273. Pratt G. E., Tobe S. S., Weaver R. J. and Finney J. R. (1975) Spontaneous synthesis and release of C,, juvenile hormone by isolated corpora allata of female locust Schistocerca gregaria and female cockroach Periplaw~~~ --I
america’Aa.VGen.
camp.
Endocr.
26, 478-484.
._‘-
Rankin S. M. and Stay B. (1984) The changing effect of the ovary on rates ofjuvenile hormone synthesis in Diploptera nunctata.
Gen. camp.
Endocr.
54. 382-388.
R&kin S. M. and ‘Stay B. (1985) Ovarian inhibition of juvenile hormone synthesis in the viviparous cockroach, Diploprera punctata. Gen. camp. Endocr. 59, 230-237.
Readio
J., Peck
K., Meola
R. and
Dahm
K. H. (1988)
Juvenile
hormone
biosynthesis
Corpus allatum activity (in vitro) in female C&x pipiens during adult life cvcle. J. Insect Phvsiol. 34, 131-135. Richard-D., Applebaum S. W., Slit& T. J., Baker F. C., Schooley D. A., Reuter C. C., Henrich V. C. and Gilbert L. I. (1989) Juvenile hormone bisepoxide biosynthesis in vitro by the ring gland of Drosophila melanogaster: a putative juvenile hormone in the higher Diptera. Proc. natn. Acad. Sci. U.S.A. 86, 1421-1425. Stay B., Tobe S. S., Mundall E. C. and Rankin S. (1983) Ovarian stimulation of juvenile hormone synthesis in the viviparous cockroach, Diploptera punctata. Gen. camp. Endocr. 52, 34 I-349. Tobe S. S. and Clarke N. (1985) The effect of L-methionine concentration on juvenile hormone biosynthesis by corpora allata of the cockroach, Diploptera punctata. Insect Biochem. 15, 175- 179. Tobe S. S. and Pratt G. E. (1974) The influence of substrate concentrations on the rate of insect juvenile hormone biosynthesis by corpora allata of the desert locust in vitro. Biochem. J. 144, 107-I 13. Tobe S. S. and Pratt G. E. (1975a) Corpus ailatum activity in vitro during ovarian maturation in the desert locust Schistocerca gregaria. J. exp. Biol. 62, 61 l-627. Tobe S. S. and Pratt G. E. (1975b) The synthetic activity and glandular volume of the corpus allatum during ovarian maturation in the desert locust Schistocerca gregaria. Life Sci. 17, 417-422. Tobe S. S. and Pratt G. E. (1976) Farnesenic acid stimulation of iuvenile hormone biosynthesis as an experimental probe-in corpus allatum phisiology. In The’Juvenile Hormones (Edited by Gilbert L. I.), pp. 147-163. Plenum Press, New York.
in earwigs
251
Tobe S. S. and Stay B. (1977) Corpus allatum activity in vitro during the reproductive cycle of the viviparous cockroach, Diploptera puncrata (Eschscholtz). Gen. romp. Endocr. 31, 138-147. Tobe S. S. and Stay B. (1985) Structure and regulation of the corpus allatum. Adv. Insect Physiol. 18, 306-432. Vancassel M. (1984) Plasticity and adaptive radiation of dermapteran parental behavior: results and perspectives. Ad. S/udy Behav. 14, 51-80. Vancassel M., Foraste M., Strambi A. and Strambi C. (1984) Normal and experimentally induced changes in hormonal hemolymph titers during parental behavior of the earwig Labidura ripario. Gen. cornp. Endocr. 56, 444-456. Weaver R. J. and Pratt G. E. (1977) The effect of enforced virginity and subsequent mating on the activity of the corpus allatum of Periplaneta americana measured in vi/ro, as related to changes in the rate of ovarian maturation. Physiol. Entomol. 2, 59-76. Weaver R. J., Pratt G. E. and Finney J. R. (1975) Cyclic activity of the corpus allatum related to gonotrophic cycles in adult female Periplaneta americana. Experientia 31, 597-598. Weaver R. J.. Pratt G. E., Hamnett A. F. and Jennings R. C. (1980) The influence of incubation conditions on the rates of juvenile hormone biosynthesis by corpora allata isolated from adult females of the beetle Tenebrio molitor. Insect Biochem. 10, 245-254. Yagi K. J., Konz K. G., Stay B. and Tobe S. S. (1991) Production and utilization of farnesoic acid in the juvenile hormone biosynthetic pathway by corpora allata of larval Diploptera punctata. Gen. cornp. Endocr. 81, 284-294.
0742-8413(95)00016-X
Biosynthesis and release of juvenile hormone during the reproductive cycle of the ring-legged earwig Susan M. Rankin,* James 0. Palmer,* Grant L. Scott* and Stephen S. Tobet *Department of Zoology,
of Biology, Allegheny University of Toronto,
College, Toronto,
Koichiro
J. Yagi,“f
Meadville, PA 16335, U.S.A.; Ontario, Canada M5S IA1
and TDepartment
Corpora allata of adult female Euborellia annulipes, incubated in medium containing 3H-metbionine, synthesized and released juvenile hormone III. Labelled material co-migrating with methyl farnesoate was also found, suggesting this as an intermediate in the pathway of juvenile hormone III production. Juvenile hormone was not appreciably stored in the glands, but was released into the medium. In normal medium, 93.6 f 1.6% of the total juvenile hormone III synthesized was released and 96.5% f 0.3 in medium supplemented with 60 PM farnesoic acid. The rate of juvenile hormone III biosynthesis/release in vitro remained constant for at least 8 hr for glands of different activities. The rate of juvenile hormone production was closely correlated with the gonadotrophic cycle. In females with previtellogenic ovarian follicles (0.26 f 0.004 mm), hormone production was only 0.59 + 0.13 fmol hr-‘/corpus allatum; production increased to 1.52 + 0.25 fmol hr-‘/corpus allatum when basal follicles were growing rapidly, and remained high during the period of oviposition. By 3 days following oviposition when females were brooding clutches, hormone production had declined to 0.46 + 0.13 fmol hr-‘/corpus allatum. The addition of 60 gM farnesoic acid to the medium enhanced juvenile hormone biosynthesis at each stage examined. Lastly, elevating the level of L-methionine in Maximal hormone production was the medium also enhanced hormone biosynthesis. 32.8 + 10.9 fmol hr-‘/corpus allatum, at an L-methionine concentration of 51 FM. Key words: Reproductive cycle; Egg development; Corpus allatum; In vitro incubations. Comp. Biochem.
Physiol.
IlOC, 241-251,
Earwig;
Juvenile
hormone;
Farnesoic
acid;
1995.
Introduction Corpora allata are endocrine glands that synthesize and release juvenile hormone (JH). In most insects, JH regulates larval development and adult reproduction [see Tobe and Stay (1985) for a review]. The use of direct radiochemical in vitro techniques for monitoring JH production has demonstrated a close relationship between corpus allatum activity and
ovarian
development
in cockroaches
such
as
Diploptera Periplaneta
punctata (Tobe and Stay, 1977), americana (Pratt et al., 1975; Weaver and Pratt, 1977; Weaver et al., 1975; Edwards et al., 1990) and Nauphoeta cinerea (Lanzrein et al., 1978, 198 1). Additionally, such a correlation has been suggested in Schistocerca gregaria (Tobe and Pratt, 1975a,b), Melanoplus sanguinipes (McCaffery and McCaffery, 1983), Leptinotarsa decemlineata (Kramer, 1978) and Tenebrio molitor (Weaver et al., 1980).
Correspondence fo: S. M. Rankin, Department of Biology, Allegheny College, Meadville, PA 16335, U.S.A. Tel. (+ 1) 8 14-332-5375; Fax (+ 1) 8 14-332-2789; e-mail [email protected]. Abbreviations: JH, juvenile hormone; FEAR, fractional endocrine activity ratio. Received 29 September 1994; revised 23 December 1994; accepted 23 December 1994.
To our knowledge, neither corpus allatum activity of earwigs nor its potential correlation with ovarian development has been reported. Studies in these animals are particularly interesting for two reasons. (1) Morphologically, 241
242
S. M. Rankin
earwigs possess highly unusual primitive polytrophic ovaries, in which a single nurse cell (trophocyte) contributes to the development of its primary oocyte partner (Bonhag, 1956; Nath et al., 1959). Studies to date using radiochemical assays for JH production in adult female insects have been performed on orthopteroid insects with panoistic ovaries (cockroaches and locusts) or on beetles (see above) with complex polytrophic ovaries. (2) Behaviorally, these animals are subsocial, providing extensive maternal care for clutches during embryogenesis and for newly hatched larvae. No similar studies to date have been performed on subsocial insects: most are restricted to solitary insects, although JH production has been addressed in some social insects, such as honey bees (Huang et al., 1991) and primitive termites (Greenberg and Tobe, 1985). Preliminary studies of JH production must include identification of the type(s) of JH produced as well as some delineation of the pathways of JH synthesis. The identities of several synthesized juvenile hormones have been established (Bergot et al., 1981a,b; Tobe and Stay, 1985; Cusson et al., 1991). For example, in the lepidopteran, Munducu sextu, JH 0, JH I, JH II, JH III and 4-methyl JH I have been reported (Bergot et al., 1981a,b; Peter et al., 1981) as well as acids of three of the JH homologues (Bhaskaran et al., 1986). In Pseuduletiu unipunctu, corpora allata from males also release the acids of the homologues, and perhaps homo- and/or dihomo farnesoic acid, whereas those of females release the three common homologues and an additional esterified product (Cusson et al., 1991). In the lower dipterans, only JH III has been reported (Baker et al., 1983; Guilivard et al., 1984; Readio et al., 1988) whereas in Drosophila melunoguster, the ring glands release both bis-epoxide JH III and methyl farnesoate (Richard et al., 1989) and in Culliphoru vomitoriu, the corpora allata may also release 6,7-epoxide as well (Cusson et al., 1991). In the cockroach Diplopteru punctutu, fourth instar larvae release farnesoic acid as well as JH III (Yagi et al., 1991). Although no information is available for in vitro synthesis of JH by corpora allata of any dermapteran, in the earwig Labidura ripuria, JH III is the primary gonadotropic hormone (Baehr et al., 1982; Vantassel, 1984; Vancassel et al., 1984). We report here our identification of the JH produced by corpora allata in vitro of post-tenera1 adult female earwigs, Euborellia unnulipes using a direct, radiochemical assay. We further establish the degree to which biosynthesized hormone is stored or released from corpora allata in vitro, and the suitability of incubation conditions for incubations of up to 8 hr. We
et
al
assess the correlation between adult female age, basal follicle length, and JH production. Finally, we determine the effects of farnesoic acid on JH production in vitro in glands of varying physiological stages. We thus address the degree to which JH production is tightly coupled to this gonadotrophic cycle in a primitively polytrophic ovary and provide the basis for investigating key regulatory events at the endocrine level that may affect the initiation, maintenance, or termination of subsocial behavior.
Materials and Methods Earwigs Our colony is descended from a founder population collected in College Station, Texas in 1989-1990. Individuals were field-collected as second instar-to-adult animals, and reared subsequently in our laboratories. Generations descended from the founder population were used for our experimental observations. A stock population of animals was groupreared in polypropylene containers (30 x 16 x 8 cm’) with moist paper towels, cotton-stoppered vials and dry catfood ad libitum in incubators at 28°C 12: 12 hr light :dark cycle. For most experiments, O-day adult females were removed from stock cultures and each maintained with one or two post-teneral males in 8 oz polypropylene containers with dry catfood and cotton-stoppered water vials. The moist cotton in each container maintained high humidity and provided oviposition sites. Thus, for these studies, animals were reared as families; however, the adult males were routinely removed on the day of oviposition, since preliminary experiments in which the male remained with the family frequently resulted in the death of the male (presumably by the mother defending her clutch against predation by the male). Similar anecdotal observations of dead males near nests of brooding females or of female aggression have been made for the earwigs E. annulipes 1942; Bharadwaj, 1966) (Klostermeyer, E. cincticollis (Knabke and Grigarick, 1971) Forficulu uuriculuriu (Lamb, 1976; Crumb et al., 1941) and L. ripuriu (Caussanel, 1970). Dissections Surgery was performed on chilled animals. The corpora cardiacacorpus allatum complexes were removed through a dorso-posterior window in the head cuticle. No attempt was made to isolate the corpus allatum from the corpora cardiaca. Earwigs have two corpora cardiaca and a single median corpus allatum in close association with the dorsal aorta (DeLerma, 1942; Cazal, 1948; Ozeki, 1958,
Juvenile
hormone
biosynthesis
1961; Lhoste, 1957; Awasthi, 1975; Juberthie and Caussanel, 1980). Ovaries were dissected from chilled animals following removal of abdominal sternites. Basal follicle lengths were determined using a dissecting microscope with an ocular micrometer, and the number of ovarioles (typically five) was recorded. In vitro incubations
of corpora allata
For identification of the JH produced in this insect, corpora cardiacacorpus allatum complexes were excised from preovulatory females whose ovaries were nearing completion of vitellogenesis (basal follicle length > 0.9 mm), then incubated at 28°C in medium 199 (GIBCO) lacking methionine and supplemented with [methyl-3H]methionine (NEN; 1 PM 3H-met; 80 mCi/pmol), 2% Ficoll and 5 mM CaCl,. The effect of farnesoic acid on JH synthesis by corpora allata from pre-ovulatory females with ovaries in late vitellogenesis was determined in two ways: (1) by incubating glands in medium containing 60 PM farnesoic acid and comparing production with that of glands in normal medium. There was no significant difference in basal follicle length between females of the farnesoic acid-treated groups and those of untreated controls; (2) by incubating glands in normal medium for 3 hr, then transferring the glands to medium supplemented with 60 PM farnesoic acid for an additional 3-hr incubation. Another (control) group was incubated in normal medium for the second 3-hr incubation. The dose-relationship for JH synthesis as a function of L-methionine concentration was determined in medium supplemented with the appropriate concentrations of unlabelled Lmethionine (Sigma Chemical Co., St Louis, MO). Extraction
of biosynthesized
products
High-performance liquid chromatography (HPLC) analysis was performed as described by Cusson et a/. (1991) to identify radiolabelled compounds synthesized by the corpora allata. In this test, six glands with media were extracted as a single sample; following extraction, only one-third of the resulting extract comprised a single application. No difference was seen between the three runs of each sample. Unlabelled standards were applied with our samples to confirm elution times of potential products. Glands were not removed from media prior to extraction; hence, we monitored the total amount of juvenile hormone biosynthesized, and had the potential to determine whether methyl farnesoate which would not ordinarily be released into the medium, was an intermediate in the production of JH III, as has been
243
in earwigs
shown in some other insects [e.g. D. punctata; Feyereisen et al. (198 l)]. Thin-layer chromatography (TLC) was performed (a) to provide independent verification of radiolabelled compounds; (b) to quantify the relationship between the amount of JH released into the medium and the total amount of hormone produced; and (c) to quantify hormone release during the first gonadotrophic cycle. For these analyses, glands and media were extracted and purified separately as described elsewhere (Tobe and Pratt, 1974; Pratt and Tobe, 1974) except that three glands (rather than isolated pairs) were analyzed as single samples to ensure adequate amounts of radiolabelled products. Unlabelled standards were run with our unknown samples to monitor migration of compounds.
Results I. IdentiJication of radiolabelled products and of the efSects of farnesoic production
acid on juvenile
hormone
HPLC analysis (Fig. I), performed on extracts of incubation medium together with corpora allata, demonstrated that JH III was the only JH produced by the corpora allata of adult female earwigs in which ovaries are nearing completion of the gonadotrophic cycle. The major radioactive compound detected after incubation of glands in normal medium (i.e. not supplemented with farnesoic acid) was JH III (73.9%). Not surprisingly, labelled methyl farnesoate was also present (8.1%). Another component (retention time 8.6 min) was also detected that did not correspond to known juvenoids, and comprised 24.3% of the radioactive material. The addition of 60 p M farnesoic acid to the medium resulted in a 4-fold increase in JH production (Fig. 1). In this case, JH III accounted for 92.2 + 0.4% and methyl farnesoate for 7.8 + 0.4%. The unknown component with retention time of 8.6 min was not observed in samples incubated in medium supplemented with farnesoic acid. A second experiment was performed to confirm the results of HPLC analysis and to distinguish between JH production and hormone release into the medium. Samples of three pairs of glands each from 1 l-day females (basal follicle length 0.55 + 0.08 mm) were incubated for 3 hr in normal medium, then transferred to medium supplemented with 60 PM farnesoic acid for a second 3-hr incubation. After incubation, glands were removed from the medium and extracted and processed separately. A typical chromatogram of these results from a single
244
S. M. Rankin
0
2
4
Time
6
er al.
8
after
injection
10
12
14
(min)
Fig. I. High performance liquid chromatography (HPLC) analysis of the radiolabelled products biosynthesized by corpora allata of adult female E. annulipes. Corpora allata from six females per sample were incubated in modified TC 199 medium and ‘H[met], either with (dashed line) or without (solid line) 60 PM farnesoic acid, then assayed according to the methods of Cusson et al. (1991). Retention times of standards indicated by arrows are methyl farnesoate (MF), juvenile hormone I (JH I), juvenile hormone II (JH II) and juvenile hormone III (JH III). Basal follicle lengths from corpus allatum donors were I 17 k 0.18 and 0.96 + 0.18 mm, respectively.
sample is shown in Fig. 2. The major product released into the medium was JH III (Fig. 2B), and farneosic acid again dramatically enhanced JH production. Extraction of glands (Fig. 2A) demonstrated that JH III again was the predominant product, and that methyl farnesoate was also present. In the example shown, the amount of JH III extracted from the glands was only 4.2% of that in the medium, suggesting that the product is released from the glands into the medium. Radiolabelled methyl farnesoate was present in proportionally much greater quantities in the glands than in the medium (for example, the ratio of JH-to-methyl farnesoate in the example shown is 3.3: 1 and 43.6: 1, for glands and medium, respectively). 2. Correlation between juvenile hormone biosynthesis and release There is a linear relationship between JH biosynthesis and release for glands either incubated in normal medium for 6 hr (n = 7) or in medium supplemented with farnesoic acid for the final 3 hr (n = 35) (Fig. 3). For corpora allata in normal medium, radiolabelled JH accounted for 93.6 f 1.6% of the total biosynthesized hormone (r* = 0.994), whereas in supplemented medium, the released radiolabelled hormone accounted for 96.5 ? 0.3% (r* = 1.000). These results are consistent with the TLC analysis presented above (Fig. 2). 3. Time course of earwig juvenile synthesis
hormone
bio-
After establishing that hormone release was highly correlated with JH production in vitro
(Fig. 3) we next determined whether our incubation conditions could sustain JH production in vitro for at least 8 hr. To that end, glands were incubated in medium for 8 hr, but corpora allata were transferred to new medium at 2 hr intervals. Rates of JH production over an 8-hr incubation period were constant for the four samples with varying degrees of activity (r2 = 0.995, 0.925, 0.989, 0.986 for the samples A., 0, a, and 0, respectively) (Fig. 4). These results show that the incubation medium is sufficient to support JH synthesis for at least 8 hr. 4. Correlation between juvenile hormone thesis and the gonadotrophic cycle
syn-
To begin our investigation of the endocrine control of reproduction in this subsocial animal, we documented the relationship between age, basal follicle length, and JH biosynthesis (Fig. 5). For this laboratory population, basal follicle length was small in 3- and 5-day-old females (0.26 + 0.004 and 0.27 + 0.02 mm, respectively), then increased dramatically between days 5 and 13 to 0.84 + 0.05 mm. Oviposition typically occurred on days 15-17 after adult eclosion, over a I-2-day period. By 3 days after oviposition, the lengths of new basal follicle were small (0.40 f 0.06 mm, n = IS), and did not increase (perhaps regressed slightly) for at least 8 days after oviposition (basal follicles were 0.31 + 0.03 mm; n = 15; Fig. 5A). During that post-ovipositional period, females displayed pronounced maternal care.
Juvenile
600
“0
hormone
biosynthesis
245
in earwigs
-
2
4
6
Migration
i
Migration
6
10
distance
(cm)
12
i
distance
(cm)
Fig. 2. Thin-layer chromatographic (TLC) analysis of the biosynthetic products of corpora allata from three 1 l-day-old adult female E. annulipes. Medium (B) was assayed separately from glands (A, assayed after 6 hr) according to the methods of Tobe and Pratt (1974). Glands were incubated in the absence of farnesoic acid (solid line) for 3 hr, then transferred to medium containing 60pM farnesoic acid for an additional 3-hr incubation (dashed line). Migration distances of standards are indicated by arrows for juvenile hormone III (JH III), methyl farnesoate (MF), and JH III bis epoxide (BIS). The addition of farnesoic acid significantly enhanced JH III biosynthesis. The basal follicle length of donor females was 0.55 + 0.08 mm (mean + standard error of the mean, SEM).
JH production by corpora allata from females during the first gonadotrophic cycle was determined by TLC analysis following a 3-hr incuJH production was low bation period. (0.59 + 0.13 fmol hr-‘/corpus allatum) in previtellogenic females, then increased between days 5 and 13 to 1.52 f 0.25 fmol hrr’/corpus allatum as follicles grew rapidly (Fig. 5B,C). JH production remained high on the day of oviposition, likely related to the fact that oviposition spanned a I-2-day interval. Following oviposition, JH production declined to low levels (0.46 + 0.13 fmol hr-‘/corpus allaturn). These experiments also demonstrated (data not shown) that JH III is the major product synthesized by glands of different activities throughout the first reproductive cycle.
We investigated whether the addition of farnesoic acid to incubation medium stimulated JH III production throughout the first gonad0 trophic cycle by incubating glands first for 3 hr in unsupplemented medium, then for 3 hr in medium supplemented with 60 PM farnesoic acid. For glands from females of each physiological stage, farnesoic acid had a dramatic effect on JH III production (Fig. 5B). The cyclic nature of JH production was preserved in glands stimulated with farnesoic acid; however, glands from females on the day of oviposition (near-maximally active glands in unsupplemented medium) were stimulated to a much lesser degree than were corpora allata from females of other physiological states. The ratio between the rates of JH synthesis in
246
S. M. Rankin
et ~1.
80000
60000
0
20000 JH
40000 released
60000
80000
(DPM)
Fig. 3. Relationship between earwig juvenile hormone biosynthesis and release. For this experiment only, biosynthesis is defined as the total amount of juvenile hormone released into the medium and that present in the corpora allata. Glands were either incubated in normal medium for 668 hr (solid circles), or for 3 hr in normal medium followed by 3 hr in medium supplemented with 60 PM farnesoic acid (open circles). Each point represents the measurement of three glands incubated in a single sample.
normal medium and then in the medium enhanced with farnesoic acid, termed the fractional endocrine activity ratio [FEAR; see Tobe and Pratt (1976)], was 0.0440.06 for each physiological state, except ovipositing females, in which the FEAR was 0.13. Thus, the addition of farnesoic acid stimulated JH production by corpora allata in vitro by 7-17-fold. 5. EfSect of L-methionine juvenile hormone biosynthesis on
concentration
from females in late vitellogenesis were used, since they exhibit high rates of hormone biosynthesis in vitro (Fig. 5). At 1 PM, glands released labelled hormone at 1.35 + 0.47 fmol hr ‘/ corpus allatum (confirming relatively high activity of glands). The addition of unlabelled methionine significantly enhanced hormone production (Fig. 6). Rates of JH biosynthesis increased with increasing concentration up to 51 PM, at which concentration corpus allatum activity was 32.8 f 10.9 fmol hr-‘/corpus allaturn. Above this level, no further increase was realized.
on
To assess the dependence of JH biosynthesis methionine concentration, corpora allata
8000
6000
0
2 Incubation
4
6 time
8
10
(hours)
Fig. 4. Time course of earwig juvenile hormone biosynthesis. Each point represents the rate of cumulative biosynthesis from four samples (0, 0, A., A) each containing three glands from vitellogenic stage adult females. Note the variation in rates of synthesis. Total incubation time was 8 hr, but corpora allata were transferred to new medium after 2, 4 and 6 hr of incubation.
0.6
1.6
0.6
1.6
0.6
0.4 ’ u.2 Basal
I
I
0.4
0.6
follicle
length
I 0.6 (mm)
Fig. 5. Relationship between basal follicle length and rate of juvenile hormone synthesis in earwigs, (A) Mean basal follicle lengths (& SEM; n = 12-l 5 females, see below). (B) Juvenile hormone biosynthesis (solid circles) and the effect of farnesoic acid on juvenile hormone biosynthesis (open circles). Assays were determined on corpora allata from the same earwigs for which ovarian basal follicle lengths were determined (n = 5 samples each with corpora allata from three females, except for that of the day of oviposition, wherein four samples of three corpora allata each were assayed). Glands were incubated for 3 hr without farnesoic acid, then transferred to medium containing 60 PM farnesoic acid for an additional 3-hr incubation. (C) Relationship between basal follicle length and juvenile hormone production for females in the first gonadotrophic cycle. Data shown are from glands incubated in medium not supplemented with farnesoic acid. Vertical and horizontal bars represent SEM.
S. M. Rankin
248
1
11
L-methionine
et al.
101
51
concentration
(FM)
Fig. 6. Dose-dependence of juvenile hormone biosynthesis by corpora allata from females with ovaries in late vitellogenesis as a function of L-methionine concentration. Corpora allata (highly active glands) were incubated for 6 hr in medium supplemented with unlabelled L-methionine to the appropriate concentrations. Each sample contained three corpora allata, and each point represents six samples. Vertical bars represent SEM.
Discussion Sufficient radiolabelled product was synthesized and released from short-term incubations of corpora allata from adult female E. annulipes, in medium containing 1 PM [methyl-‘Hlmethionine, to permit identification of the hormone. A combination of HPLC (Fig. 1) and TLC (Fig. 2) has identified the major product to be JH III. We found no incorporation of radiolabel into either JH I or JH II, nor any other known JH-related compound. This finding is in agreement with studies of the hemolymph of post-teneral adult females of the earwig Labidura riparia, using HPLC analysis after derivitization of the extracted JH to the diol form which identified JH III as the principal JH (Vancassel et al., 1984). Presence
of methyl farnesoate
We detected radiolabelled material which comigrated with methyl farnesoate in two instances: (1) using HPLC analysis of samples in which glands were extracted with medium (Fig. I); (2) by TLC analysis of glands alone after incubation (Fig. 2A). These results are consistent with the hypothesis that the production of JH III in earwig corpora allata proceeds along the pathway now attributed to most insects (Cusson et al., 1991): farnesoic acid is likely methylated to form methyl farnesoate, and this product is subsequently epoxidized to form JH III.
Storage/release
of juvenile
hormone
III
JH was not stored to any appreciable extent in corpora allata in vitro (Fig. 3). JH is similarly
released into the medium when produced in vitro by corpora allata from S. gregaria (Tobe and Pratt, 1974), P. americana (Pratt et al., 1975), D. punctata (Feyereisen et al., 1981) and Apis melf@ra (Huang et al., 1991). Thus, in earwigs, as in other insects, JH III titers are likely regulated by synthesis and/or degradation of hormone, rather than by sequestration in or release from the endocrine source. Suitability
of incubation
conditions
JH biosynthesis and release occurred at a constant rate in vitro for at least 8 hr (Fig. 4). Since in our sequential 2-hr incubations, we observed no detectable lag-time in the production of labelled JH, it appears that corpora allata from these earwigs had very low methionine that levels of endogenous were rapidly diluted by the exogenous radiolabelled methionine, as has been suggested for the locust, S. gregaria (Tobe and Pratt, 1974) and the beetle, T. molitor (Weaver et al., 1980). To our knowledge, there are no reports of amino acid concentrations in earwig hemolymph. The linear rate of incorporation of label into newly biosynthesized JH III for 8 hr (Fig. 4) demonstrated that the medium is sufficient to support corpus allatum function for at least that interval. This further suggests that incubations of this duration (or shorter) yield estimates of glandular activity that reflect rates of hormone production by the corpora allata immediately prior to their removal from these adult females.
Juvenile
Correlation of juvenile hormone with age and the gonadotrophic
hormone
biosynthesis
III biosynthesis cycle
This is the first report of rates of JH production in any dermapteran (Fig. 5). Our finding that the rate of JH III production is correlated with the gonadotrophic cycle, and that synthesis remains high during the ovipositional period in this earwig is in agreement with the change in JH III hemolymph titer in L. riparia associated with the gonadotrophic cycle (Vancassel et al., 1984). These findings are consistent with the notion that maturing ovaries might stimulate JH production, as has been shown for the cockroach, D. punctata (Stay et al., 1983; Rankin and Stay, 1984). Variability in egg lengths and corresponding rates of synthesis (Fig. SC) is likely attributable, in part, to incubation of glands from three females (with potentially different basal follicle lengths) as single samples (see above). Our discovery that, in the earwig, JH III production remained high during the ovipositional period suggests that, unlike regulation in D. punctata, JH production in this earwig does not include inhibition by mature ovarian follicles (Rankin and Stay, 1985). Following oviposition (during the brooding period), JH production declined. This period of maternal care may thus be analogous to the situation in pregnant D. punctata (Rankin and Stay, 1985). These results are consistent with the finding in L. riparia that hemolymph titers of JH III are relatively low during the brooding phase (Vancassel et al., 1984). Effects of exogenous farnesoic hormone III biosynthesis
acid on juvenile
Farnesoic acid has been shown to stimulate JH production in vitro in corpora allata from a number of orthopteroids, including adult female D. punctata (Feyereisen et al., 1981; Feyereisen et al., 1984), larval D. punctata (Yagi et al., 1991), P. americana (Pratt et al., 1975) and locusts (Tobe and Pratt, 1974). It is also effective in stimulating corpus allatum activity from the adult female beetles, T. molitor (Weaver et al., 1980) and L. decemlineata (Khan et al., 1982). These findings indicate that in these species, and in the earwig, rate-limitation most likely occurs prior to the conversion of farnesoic acid to juvenile hormone. The degree of stimulation of JH III production by farnesoic acid has been expressed as the ratio between the rates of JH synthesis in normal medium and then in medium supplemented with farnesoic acid [FEAR, see Tobe and Pratt (1976); Feyereisen et al. (1981); Yagi et al. (1991)]. In D. punctata (Feyereisen et al., 1981; Yagi et al., 1991) and in locusts (Tobe and
249
in earwigs
Pratt, 1975a, 1976; Gadot and Applebaum, 1986; Couillaud et al., 1988), glands of low activity have low FEAR values, whereas glands of high spontaneous activity have high FEAR values. If farnesoic acid substantially stimulates JH III biosynthesis (a very low FEAR) as occurs, for example, at the end of last larval stadium in D. punctata males (Yagi et al., 1991), and in adult female earwigs (Fig. 5), the ratelimiting steps very likely occur prior to the O-methyl transferase. Eflects of L-methionine production
concentration
on hormone
The relationship between L-methionine concentration and JH production has been examined in a number of insects; interestingly, the optimal level of methionine is quite variable. For example, for the primitive termite, Z. angusticollis, optimal levels are only 5-10 p M (Greenberg and Tobe, 1985); in the cockroach, D. punctata, optimal levels are 20-40 PM (Tobe and Clarke, 1985); in S. gregaria, the optimum is 100-400 p M (Tobe and Pratt, 1974), similar to that observed for the beetle, T. molitor (300 PM, Weaver et al., 1980). These differences suggest potiential differences in the ability to scavenge L-methionine and in the size of the endogenous pool of L-methionine in glands from different species (Tobe and Clarke, 1985). Our result for this earwig species demonstrating 51 PM as an adequate concentration for methionine clearly places these animals well within the previously observed optimal spectrum of methionine concentrations for insects examined thus far. Conclusions The characterization of the hormone of this earwig as JH III, coupled with the demonstration of constant rates of incorporation for at least 8 hr in vitro, and the strong correlation of JH III production in uitro with the reproductive cycle indicates that the radiochemical assay for JH biosynthesis can be used for the study of the interactions between JH production, egg development and the associated behavioral phenomenon of maternal care. The ability to measure rates of JH biosynthesis in these subsocial insects will also help in our efforts to discover potential physiological linkages of JH and behavior in the evolution of insect sociality. Acknowledgements-This
work was supported
by National
Science Foundation grant no. DCB 9118275 to SMR; support for this research was additionally provided by a grant from the Howard Hughes Medical Institute through the Undergraduate Biological Sciences Education Program awarded by Allegheny College and by a grant from the National Science and Engineering Research Council of Canada. We are also indebted to undergraduate student researchers Heather Dossat and John Lunz for excellent technical assistance.
250
S. M. Rankin
References Awasthi
V. B. (1975) Neurosecretory system of the earwig riparia, and the role of the aorta as a neurohema1 organ. J. Insect Physiol. 21, 1713-1719. Baehr J.-Cl., Cassier P., Caussanel Cl. and Porcheron P. (1982) Activity of corpora allata, endocrine balance and reproduction in female Labidura riparia (Dermaptera). Cell Tiss. Res. 225, 267-282. Baker F. C., Hagedorn H. H., Schooley D. A. and Wheelock G. (1983) Mosquito juvenile hormone: identification and bioassay activity. J. Insect Physiol. 29, 465-470. Bergot B. J., Ratcliff M. and Schooley D. A. (198la) Method for quantitative determination of the four known juvenile hormones in insect tissue using gas chromatography-mass spectroscopy. J. Chromatogr. 204, 231-244. Bergot B. J., Baker F. C., Cerf D. C., Jamieson G. and Schooley D. A. (1981 b) Qualitative and quantitative aspects of juvenile hormone titers in developing embryos of several insect species: Discovery of a new JH-like substance extracted from eggs of Manduca sex&. In Jurenile Hormone Biochemistrv (Edited bv Pratt G. E. and Brooks G. T.), pp. 33-45. Eisevier, Amsterdam. Bharadwaj R. K. (1966) Observations on the bionomics of Euborellia annulipes (Dermaptera: Labiduridae). Ann. Labedura
Entomol.
Sot.
Am. 59, 44-450.
Bhaskaran G., Sparagana S. P., Barrera P. and Dahm K. H. (1986) Change in corpus allatum function during metamorphosis of the tobacco hornworm Manduca sexta: Regulation at the terminal step in juvenile hormone biosynthesis. Arch. Insect Biochem. Physiol. 3, 321-338. Bonhag P. F. (1956) The origin and distribution of periodic acid-Schiff-positive substances in the oocyte of the earwig, Anisolabis maritima (G&n&). J. Morphol. 99, 433- 463. Cazal P. (1948) Les glandes endocrines r&troctrtbrales des lnsectes (ttudes morphologiques). Bull. biol. France et Belgiaue,
spp.
32.
Couilia;d F., Mauchamp B., Girardie A. and DeKort S. (1988) Enhancement bv farnesol and farnesoic acid of juvenile-hormone biosinthesis in induced low activity locust corpora allata. Arch. Insect Biochem. Phy.siol. 7, 133-143.
Cusson M., Yagi K. J.. Ding Q., Duve H., Thorpe A., McNeil J. N. and Tobe S. S. (1991) Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: the search for a unifying arthropod endocrinology. Insect Biochem. 21, l-6. DeLerma B. (1942) Ricerche sul sistema endocrine cefalico (corpora allata e corpi faringei) e sullo stomatogastrico hei Dermatteri. Boll:Sec. Nat. Napoli 52, 25- 42. Edwards J. P.. Chambers J.. Short J. E., Price N. R., Weaver R. J., Abraham L. and Walker C. M. (1990) Endogenous juvenile hormone III levels and in vitro rates of hormone biosynthesis by corpora allata during the reproductive cycle of adult female Periplaneta americana. In Chromatography and Isolation of Insect Hormones and Pheromones (Edited by McCaffery A. R and Wilson 1. D.),
pp. l-8. Plenum Press. Feyereisen R., Friedel T. acid stimulation of C,, corpora allata of adult Biochem.
New York. and Tobe S. S. (1981) Farnesoic juvenile hormone biosynthesis by female Diploptera punctata. Insect
11, 401-409.
Feyereisen R., Ruegg R. P. and Tobe S. S. (1984) Juvenile hormone III biosynthesis: stoichiometric incorporation of 12-‘%I acetate and effects of exogenous farnesol and farneioic acid. Insect Biochem. 14, 657- 661. Gadot M. and Applebaum S. W. (1986) Farnesoic acid and allatotropin stimulation in relation to locust allatal maturation. Molec. Cell. Endocr. 48, 69-76. Greenberg S. and Tobe S. S. (1985) Adaptation of a radiochemical assay for juvenile hormone biosynthesis to study caste differentiation in a primitive termite. J. Insect Phvsiol. 31. 347-352.
et al.
Guilivard D., DeReggi M. and Rioux J. A. (1984) Changes in ecdvsteroid and juvenile hormone titers correlated . tn ._ the iiitiation of Gitellogenesis in two Aedes species (Dintera. Culicidae). Gen. coma. Endocr. 53. 218-223 ,. Hia& Z.-‘Y., Robinson G. E.,*Tobe S. S., ‘Yagi K. J., Strambi C., Strambi A. and Stay B. (1991) Hormonal regulation of behavioural development in the honey he? -__ is-based on changes in the rate of juvenile hormone biosvnthesis. J. Insect Phvsiol. 37. 733-741. Juberthie C. and Caussanel Cl. (1980) Release of brain neurosecretory products from the neurohaemal part of the aorta during egg-laying and egg-care in Labidura riparia (Insecta, Dermaptera) J. Insect Physiol. 26, 427-429.
Khan M. A., Koopmanschap A. B., Privee H. and DeKort C. A. D. (1982) The mode of regulation of the corpus allatum activity during starvation in adult females of the Colorado potato beetle, Leptinotarsa decemlineata (Say). J. Insect Physiol.
28, 791-796.
Klostermeyer E. C. (1942) The life history and habits of the _._. ring-legged earwig Euborellia annulipes (Lucas) (Dermaptera). J. Kansas Entomol. Sot. 15, 13-18. Kramer s. J. (1978) Age-dependent changes in corpus allatum activity in vitro in the adult Colorado potato beetle, Leptinotarsa decemlineata. J. Insect Physiol. 24, 461-464.
Lanzrein B., Gentinetta V. and Liischer M. (1978) Correlation between haemolymph juvenile hormone titre, corpus allatum volume, and corpus allatum in viuo and in vitro activity during oocyte maturation in a cockroach (Naupheota
cinerea).
Gen. Comp. Endocrinol.
36,339%345.
Lanzrein B.. Whilhelm R. and Gentinetta V. (1981) On relations between corpus allatum activity and oocyte maturation in the cockroach Nauphoeta cinerea. In Regulation of Insect Development and Behavior (Edited by Sehnal F., Zabza A., Menn J. J. and Cymborowski B.), pp. 523-534. Wroclaw Technical University Press, Wroclaw. Lhoste J. (1957) Donntes anatomiques et histophysiologiques sur ForJicula auricularia L. (Dermapttre). Arch. 2001.
Erp.
G&n. 95, 75-252.
McCaffery A. R. and McCaffery V. A. (1983) Corpus allatum activity during overlapping cycles of oocyte growth in adult female Melanoplus sanguinipes. J. Insect Physiol.
29, 259-266.
Nath V., Gupta B. L. and Aggarwal S. K. (1959) Histochemistry of vitellogenesis in the earwigs Labidura riparia Pall. and L. bengalensis Dohrn. Res. Bull. Punjab Univ. 10, 315-341. Ozeki K. (1958) Neurosecretion of the earwig, Anisolohi~ -I .._ maritima.
Sci. Pap. Coil. Gen. Ed. 8, 85-92.
Ozeki K. (1961) Secretion of the juvenile hormone in the ...imago 0; the earwig, Anisolabis karitima. Sci. Pap. Coil. Gen. Ed. 11, 101-107. Peter M. G.. Shirk P. D., Dahm K. H. and R(iller H. (19811 On the specificity of juvenile hormone biosynthesis in the male cecropia moth. Z. Naturf 36~. 579-585. Pratt G. E. ‘and Tobe S. S. i1974) Juvenile hormones radiobiosynthesized by farnesoic acid and its analogues. Lancer i, 1270-1273. Pratt G. E., Tobe S. S., Weaver R. J. and Finney J. R. (1975) Spontaneous synthesis and release of C,, juvenile hormone by isolated corpora allata of female locust Schistocerca gregaria and female cockroach Periplaw~~~ --I
america’Aa.VGen.
camp.
Endocr.
26, 478-484.
._‘-
Rankin S. M. and Stay B. (1984) The changing effect of the ovary on rates ofjuvenile hormone synthesis in Diploptera nunctata.
Gen. camp.
Endocr.
54. 382-388.
R&kin S. M. and ‘Stay B. (1985) Ovarian inhibition of juvenile hormone synthesis in the viviparous cockroach, Diploprera punctata. Gen. camp. Endocr. 59, 230-237.
Readio
J., Peck
K., Meola
R. and
Dahm
K. H. (1988)
Juvenile
hormone
biosynthesis
Corpus allatum activity (in vitro) in female C&x pipiens during adult life cvcle. J. Insect Phvsiol. 34, 131-135. Richard-D., Applebaum S. W., Slit& T. J., Baker F. C., Schooley D. A., Reuter C. C., Henrich V. C. and Gilbert L. I. (1989) Juvenile hormone bisepoxide biosynthesis in vitro by the ring gland of Drosophila melanogaster: a putative juvenile hormone in the higher Diptera. Proc. natn. Acad. Sci. U.S.A. 86, 1421-1425. Stay B., Tobe S. S., Mundall E. C. and Rankin S. (1983) Ovarian stimulation of juvenile hormone synthesis in the viviparous cockroach, Diploptera punctata. Gen. camp. Endocr. 52, 34 I-349. Tobe S. S. and Clarke N. (1985) The effect of L-methionine concentration on juvenile hormone biosynthesis by corpora allata of the cockroach, Diploptera punctata. Insect Biochem. 15, 175- 179. Tobe S. S. and Pratt G. E. (1974) The influence of substrate concentrations on the rate of insect juvenile hormone biosynthesis by corpora allata of the desert locust in vitro. Biochem. J. 144, 107-I 13. Tobe S. S. and Pratt G. E. (1975a) Corpus ailatum activity in vitro during ovarian maturation in the desert locust Schistocerca gregaria. J. exp. Biol. 62, 61 l-627. Tobe S. S. and Pratt G. E. (1975b) The synthetic activity and glandular volume of the corpus allatum during ovarian maturation in the desert locust Schistocerca gregaria. Life Sci. 17, 417-422. Tobe S. S. and Pratt G. E. (1976) Farnesenic acid stimulation of iuvenile hormone biosynthesis as an experimental probe-in corpus allatum phisiology. In The’Juvenile Hormones (Edited by Gilbert L. I.), pp. 147-163. Plenum Press, New York.
in earwigs
251
Tobe S. S. and Stay B. (1977) Corpus allatum activity in vitro during the reproductive cycle of the viviparous cockroach, Diploptera puncrata (Eschscholtz). Gen. romp. Endocr. 31, 138-147. Tobe S. S. and Stay B. (1985) Structure and regulation of the corpus allatum. Adv. Insect Physiol. 18, 306-432. Vancassel M. (1984) Plasticity and adaptive radiation of dermapteran parental behavior: results and perspectives. Ad. S/udy Behav. 14, 51-80. Vancassel M., Foraste M., Strambi A. and Strambi C. (1984) Normal and experimentally induced changes in hormonal hemolymph titers during parental behavior of the earwig Labidura ripario. Gen. cornp. Endocr. 56, 444-456. Weaver R. J. and Pratt G. E. (1977) The effect of enforced virginity and subsequent mating on the activity of the corpus allatum of Periplaneta americana measured in vi/ro, as related to changes in the rate of ovarian maturation. Physiol. Entomol. 2, 59-76. Weaver R. J., Pratt G. E. and Finney J. R. (1975) Cyclic activity of the corpus allatum related to gonotrophic cycles in adult female Periplaneta americana. Experientia 31, 597-598. Weaver R. J.. Pratt G. E., Hamnett A. F. and Jennings R. C. (1980) The influence of incubation conditions on the rates of juvenile hormone biosynthesis by corpora allata isolated from adult females of the beetle Tenebrio molitor. Insect Biochem. 10, 245-254. Yagi K. J., Konz K. G., Stay B. and Tobe S. S. (1991) Production and utilization of farnesoic acid in the juvenile hormone biosynthetic pathway by corpora allata of larval Diploptera punctata. Gen. cornp. Endocr. 81, 284-294.