The effect of l-methionine concentration on juvenile hormone biosynthesis by corpora allata of the cockroach Diploptera punctata

The effect of varying l-methionine (l-met) concentration on rates of juvenile hormone (JH) biosynthesis/release by corpora allata of females of the viviparous cockroach Diploptera punctata has been studied using a radiochemical assay. Both high activity glands (corpora allata from day 5 females) and low activity glands (corpora allata from day 11 females) were used to study the dose dependence of JH biosynthesis on l-met concentrations, under both de novo (spontaneous) conditions of JH biosynthesis and stimulated conditions (in the presence of the exogenous JH III precursor farnesoic acid). Maximal rates of JH biosynthesis/release were observed at l-met concentration of 20 μM (spontaneous) and 40 μM (stimulated). Below these concentrations, rates of JH biosynthesis declined linearly with decreasing l-met concentration. Optimal concentration of l-met appeared to be similar for both high and low activity corpora allata, under spontaneous and stimulated conditions of biosynthesis. Above 40 μM l-met, no increase in rates of JH biosynthesis was observed. It appears that the corpora allata of D. punctata are efficient scavengers of l-met and are able to utilize even low concentrations of the substrate for JH biosynthesis. The corpora allata of D. punctata may prove useful for the biosynthesis of authentic JH III, radiolabelled in the methyl position using as methyl donor, l[methyl-³H]met of high specific activity.     

Improved assay conditions for measurement of corpus allatum activity in vitro in the adult Colorado potato beetle, Leptinotarsa decemlineata

Assay conditions for the short-term, radiochemical, in vitro determination of the spontaneous rate of juvenile biosynthesis by isolated corpora allata from Leptinotarsa decemlineata have been further improved, permitting the measurement of juvenile hormone biosynthesis by individual pairs of corpora allata. The final incubation product has been identified as juvenile hormone III with the aid of High-performance liquid chromatography (HPLC) and juvenile hormone esterase degradation. Using the new assay conditions, the activities of adult corpora allata during maturation were found to be significantly higher in reproductive, long-day animals than in pre-diapause, short-day beetles. During diapause no activity was detectable, whereas corpora allata from post-diapause beetles were reactivated totally after 5 days. Simultaneous determination of the in vitro rates of juvenile hormone biosynthesis and corpus allatum volumes revealed no clear correlation although the results suggest that the volume may be indicative of the maximal capacity for juvenile hormone production. Corpora allata from a population of beetles did not display any synchronous diurnal rhythmicity.     

Precocenes as pro-allatocidins in adult female Diploptera punctata: A functional and ultrastructural study

Adult mated females of the viviparous cockroach Diploptera punctata are moderately sensitive to precocenes. Oöcyte growth is inhibited and oviposition is delayed in insects topically treated with precocene II or precocene III. C₁₆ juvenile hormone release by corpora allata of precocene-treated insects is markedly inhibited when compared to corpora allata of acetone-treated controls. Electron microscopy of the corpora allata reveals that precocene treatment results in a disorganisation of the intracellular organelles. Topically applied precocene II reaches a high concentration in the haemolymph (0.5 mM 2 hr after topical application of 250 μg). C₁₆ juvenile hormone release by isolated corpora allata is inhibited by precocenes in vitro; half-maximal inhibition over a 3 hr period is obtained at 0.4 mM precocene II. In vitro inhibition of corpora allata by precocene II concentrations higher than 1 mM rapidly destroys the glands as evidenced by electron microscopy (total disintegration of cellular organelles) and by the virtual cessation of C₁₆ juvenile hormone synthesis by the corpora allata. Inhibition of C₁₆ juvenile hormone release by precocene is time-dependent and is not reversible over the short-term incubation in vitro. This inhibition does not appear to be related to the spontaneous activity of the glands in vitro, and it can be reduced by two epoxidase inhibitors. Precocenes are pro-allatocidins in this species: they are bioactivated within the corpora allata to cytotoxic epoxides.     

Juvenile hormone III biosynthesis by the larval corpora allata of Manduca sexta

A radioimmunoassay (RIA) for juvenile hormone III has been established which quantifies the biosynthesis of this hormone in vitro by the corpora allata of larvae and pupae of the tobacco hornworm, Manduca sexta. The specificity of the RIA for homologues and metabolites of juvenile hormone III was determined and it was found that the antibody was specific for juvenile hormone III and its acid. The juvenile hormone III RIA activity synthesized in vitro by corpora allata from day-5 last-instar larvae was identified as juvenile hormone III by high pressure liquid chromatography. The kinetics of hormone synthesis by corpora allata from selected stages during larval-pupal development revealed differential rates of synthesis, suggesting that juvenile hormone III may have a hormonal function in the larva and that regulation of its synthesis may occur. The significance of these developmental fluctuations in rates of juvenile hormone III synthesis by the corpora allata is discussed in relation to the haemolymph titres of the hormone.     

Enhancement by farnesol and farnesoic acid of juvenile-hormone biosynthesis in induced low-activity locust corpora allata

Exogenous farnesol or farnesoic acid (FA) stimulates juvenile hormone III (JH III) biosynthesis by isolated corpora allata from Locusta migratoria in a dose-dependent manner. Farnesol and FA also stimulate a dose-dependent accumulation of substantial amounts of methyl farnesoate (MF), identified by gas chromatography-mass spectroscopy (GCMS) analysis, in the corpora allata. Lower quantities of MF were found in the incubation medium. Corpora allata, denervated 2 days prior to assay, showed low spontaneous rates of JH biosynthesis which were stimulated by farnesol and FA. The dose-response curves for control and denervated corpora allata were similar. During oocyte maturation the rate of farnesol and FA stimulation of JH biosynthesis increased gradually. However, after transection of nervus corporis allati 1 (NCA-1), the rate of stimulated JH synthesis was maintained at preoperative levels. Although the spontaneous rate of JH biosynthesis decreased rapidly after NCA-1 transection, denervated glands could still be stimulated by farnesol or FA to produce large amounts of JH. These results suggest that the low spontaneous rate of JH biosynthesis in denervated corpora allata is not caused by inhibition of the final steps of JH biosynthesis.     

Activity of disconnected corpora allata in Locusta migratoria: Juvenile hormone biosynthesis in vitro and physiological effects in vivo

Severance of nervi corporis allati I (NCA I) in day-1 adult female Locusta migratoria resulted in a significant decrease and a loss of the characteristic pattern of juvenile hormone biosynthesis by the corpora allata as determined by radiochemical assay. This decrease in the rate of juvenile hormone biosynthesis was not reflected in basal oöcyte growth. The lengths of the oöcytes were the same in NCA-transectioned and in the sham-operated females. The effect of severance of both NCA I and NCA II on juvenile hormone biosynthesis and ovarian maturation was similar to the effect of NCA I severance only.Rate of juvenile hormone biosynthesis by corpora allata of fourth-instar larvae exhibited a maximum of activity in the middle of the stadium. The severance of NCA I early in the stadium resulted in a very low rate of juvenile hormone biosynthesis and a disappearance of this peak. In NCA I-transectioned larvae, the duration of the stadium was significantly increased although larvae moulted into normal fifth instar.     

Primer effect of queen pheromone on juvenile hormone biosynthesis in adult worker honey bees

Juvenile hormone synthesis in adult worker honey bees was measured by an in vitro corpora allata bioassay. Adult queenless workers exhibit higher rates of juvenile hormone biosynthesis than queenright workers. Hormone synthesis is not correlated with the volume of the glands. Extract of queen mandibular glands, applied to a dummy, reduces juvenile hormone biosynthesis in caged queenless workers to the level of queenright workers. The same result was obtained with synthetic (E)-9-oxo-2-decenoic acid, the principal component of the queen mandibular gland secretion. This pheromonal primer effect may function as a key regulating element in maintaining eusocial colony homeostasis. The presence of brood does not affect the hormone production of the corpora allata.Abbreviations BSA bovine serum albumin - CA Corpora allata - JH juvenile hormone - 9-ODA (E)-9-oxo-2-decnoic acid     

Inhibition of the corpora allata of last-instar male larvae of the cockroach, Diploptera punctata

Normal rates of juvenile hormone synthesis, cell number and volume of corpora allata were measured in penultimate and final-instar male larvae of Diploptera punctata. The rate of juvenile hormone synthesis per corpus allatum cell was highest on the 4th day of the penultimate stadium, declined slowly for the remainder of that stadium, and rapidly after the first day of the final stadium.Regulation of the corpora allata in final-instar males was studied by experimental manipulation of the corpora allata followed by in vitro radiochemical assay of juvenile hormone synthesis. Nervous inhibition of the corpora allata during the final stadium is suggested by the observation that rates of juvenile hormone synthesis increased following denervation of the corpora allata at the start of the stadium; this operation induced a supernumerary larval instar. Juvenile hormone synthesis by corpora allata denervated at progressively later ages in the final stadium and assayed after 4 days decreased with age at operation. This suggests an increasingly unfavourable humoral environment in the final stadium, which was confirmed by the low rate of juvenile hormone synthesis of adult female corpora allata implanted into final-instar larvae. Thus, inhibitory factors or lack of stimulatory factors in the haemolymph may act with neural inhibition to suppress juvenile hormone synthesis in final-instar males.     

The effects of juvenile hormone, 20-hydroxyecdysone and precocene II on activity of corpora allata and the mode of negative-feedback regulation of these glands in the adult Colorado potato beetle

When the titre of juvenile hormone III in female Leptinotarsa decemlineata was elevated by the implantation of supernumerary corpora allata or by the injection of the hormone, the rate of endogenous hormone production by the host glands was significantly restrained, as determined by the short-term in vitro radiochemical assay. From denervation studies, it is suggested that during phases of elevated juvenile hormone titre, the corpus allatum activity is regulated via humoral as well as neural factors requiring intact nerve connections. Restrainment of gland activity appears to be mainly via the neural pathway. Isolated corpora allata were not influenced by 10^?5 M juvenile hormone III added to the incubation medium in vitro.Studies with farnesenic acid revealed that the final two enzymatic steps in the biosynthetic pathway of juvenile hormone are also diminished during prolonged neural inhibition of the corpora allata.20-Hydroxyecdysone and precocene II had no apparent effect on the corpus allatum activity of Leptinotarsa decemlineata.     

Biosynthesis and titre of juvenile hormone during the first gonotrophic cycle in isolated and crowded Locusta migratoria females

Basal oöcyte length, corpus allatum volume and “in vitro” juvenile hormone biosynthesis were measured in isolated and crowded Locusta migratoria females at selected times during the first gonotrophic cycle. Using gas chromatography-mass spectrometry with selected ion monitoring, the juvenile hormone titre in the haemolymph of isolated and crowded females was also determined 1 and 4 days after fledging. The rate of oöcyte growth was more rapid in isolated females and a significant (P < 0.01) difference in mean length was apparent as early as 3 days after fledging. This early manifestation of a difference in rate of oöcyte growth was correlated with a difference in haemolymph juvenile hormone titre between isolated and crowded females. Whilst there was no difference in titre 1 day after fledging, by day 4 the juvenile hormone titre in isolated females was found to be approximately twice that in crowded females. There was no significant difference in the rates of juvenile hormone biosynthesis by corpora allata from isolated and crowded females on days 0 through to 6 after fledging. On day 8, however, the rates of juvenile hormone biosynthesis of corpora allata from isolated females were very high (mean value = 136 pmol/h/pair) and were significantly (P < 0.002) greater than those of corpora allata from crowded females. Day 8 was also the point in the first gonotrophic cycle at which the difference in the mean basal oöcyte length in isolated and crowded females was at a maximum. The mean volume of corpora allata from isolated females was greater than that of corpora allata from crowded females at all points at which measurements were taken during the first gonotrophic cycle.     

Effects of decapitation and ovariectomy on the regulation of juvenile hormone synthesis in the cockroach, Diploptera punctata

Corpora allata from Diploptera punctata females at adult ecdysis or at the end of the last-larval stadium, when implanted into decapitated females, underwent a cycle of juvenile hormone synthesis similar in timing and magnitude to that of glands implanted into control animals which had been starved and allatectomized. Starvation did not alter the cycle in rates of juvenile hormone synthesis of sham-operated animals.Decapitation of ovariectomized animals resulted in no cycle in rates of juvenile hormone synthesis by implanted adult corpora allata; however, implantation of an ovary along with the corpora allata into decapitated, ovariectomized hosts resulted in a cycle of juvenile hormone synthesis. In control animals, which retained their heads but were starved and allatectomized as well as ovariectomized, the implanted corpora allata showed a cycle of juvenile hormone synthesis only when implanted with an ovary. The maximal rates of juvenile hormone synthesis by the corpora allata in both experimental and control conditions were lower than normal, likely due to the repeated trauma of surgery. However, at no time from eclosion to the end of the first gonotrophic period was the brain necessary for the cyclic response of the corpora allata to the presence of the ovary.     

Responsiveness of the adult cricket (Gryllus bimaculatus andAcheta domesticus) retrocerebral complex to allatostatin-1 from a cockroach,Diploptera punctata

Brain-retrocerebral complexes of female crickets,Gryllus bimaculatus andAcheta domesticus, treated with antibody to allatostatin-1 from a cockroach,Diploptera punctata, show extensive immunoreactivity. The results suggest that allatostatins or allatostatin-like molecules are produced in neurosecretory cells of the brain and are delivered to the corpora allata through nervous connections and/or via haemolymph. Radiochemical measurements of juvenile hormone III biosynthesis by isolated corpora cardiaca-corpora allata complexes from adultG. bimaculatus have been used to demonstrate an in vitro sensitivity of these glands to allatostatin-1 fromD. punctata. Allatostatin-1 is a relatively potent inhibitor of juvenile hormone III biosynthesis in corpora allata of both young adult females and males. In glands taken from 3-day virgin females, 50% inhibition of hormone biosynthesis is reached at ca. 3 nmol·l^-1 allatostatin-1. The inhibitory action of allatostatin-1 is rapid, dose-dependent and reversible. Addition of 200 mol·l^-1 farnesol to the incubation medium prevents inhibition of juvenile hormone III biosynthesis by allatostatin-1. Juvenile hormone III biosynthesis by isolated corpora allata of 3-day female house crickets,A. domesticus, is also susceptible to inhibition by 1 mol·l^-1 allatostatin-1.Abbreviations ASB2 Diploptera punctata allatostatin-5 - CA corpora allata - CC corpora cardiaca - Dip A-1 Diploptera punctata allatostatin-1 - HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid - JH juvenile hormone(s) - Mas-AS Manduca sexta allatostatin - MF methyl farnesoate - NCA nervus corporis allati - NCC nervus corporis cardiaci - SEM standard error of mean - TRIS Tris(hydroxymethyl)aminomethane     

The synthetic activity and glandular volume of the corpus allatum during ovarian maturation in the desert locust Schistocdrca gregaria.

We have compared, on an individual basis, the volume of the corpora allata with their ability to synthesize and release juvenile hormone (JH) using glands taken at daily intervals throughout the period of sexual maturation and the first two ovarian cycles in $\text{Schistocerca gregaria}$. A standard $\text{in vitro}$ radiochemical assay was used to measure the rates of both spontaneous JH biosynthesis from [methyl-¹⁴C]-methionine, and of JH biosynthesis stimulated by optimal concentrations of [C-2 ³H]-farnesenic acid. Computation of results showed that there are, during this period, changes of up to 250-fold in the rate of spontaneous JH biosynthesis per unit volume corpora allata. It is concluded that the volume of the corpora allata is of no value as an indicator of the spontaneous synthetic activity of the glands in this species, and that the overall rate of JH synthesis is regulated by mechanisms that do not involve large changes in the volume of the gland cells. However, in the presence of farnesenic acid, there is a corelation between stimulated JH synthesis and glandular volume, suggesting that the volume of the gland reflects the maximum activity of the final two stages in JH biosynthesis.     

Quantitative studies on the activity of the corpora allata in adult male Locusta and Schistocerca

Juvenile hormone has been detected in the haemolymph and corpora allata of adult male Locusta and the haemolymph of adult male Schistocera by a modified Galleria bioassay. The hormone was readily detected in the haemolymph of insects immediately after the final ecdysis, but then became difficult to detect until 2 days prior to the onset of sexual maturation. In sexually mature insects the titre of juvenile hormone was maintained at a constant level. The corpora allata of adult male Locusta increased in size throughout adult life. The juvenile hormone content of the corpora allata was low during the period of somatic growth, but increased at the onset of sexual maturation. Sectioning of the nervi corporis allati I in insects immediately after the final ecdysis prevented the normal increase in size of the corpora allata, but did not render them inactive since juvenile hormone was detected in the haemolymph after the operation. The half life of juvenile hormone in the haemolymph of allatectomized adult male Locusta was 1 to 2 hr.     

Humoral inhibition of the corpora allata in larvae of Diploptera punctata: Role of the brain and ecdysteroids

Juvenile hormone synthesis by adult female corpora allata was inhibited following implantation into final-larval-instar males; inhibition was prevented by decapitation of the larval hosts on day 11 (prior to the head critical period for moulting), but not by decapitation on day 13. Implantation of one larval protocerebrum restored inhibition of implanted corpora allata, demonstrating that the brain releases an inhibitory factor. Corpora allata implanted into larvae decapitated on day 11 were inhibited by injections of 20-hydroxyecdysone. Since treatment of corpora allata with 20-hydroxyecdysone in vitro did not inhibit juvenile hormone synthesis, ecdysteroids probably act indirectly on the corpora allata. Juvenile hormone synthesis and haemolymph ecdysteroid concentration were measured following implantation of corpora allata along with two larval brains into larval hosts. Brain implantation did not affect ecdysteroid concentration, but did inhibit juvenile hormone synthesis, even in animals with low haemolymph ecdysteroid concentration. Incubation with farnesoic acid stimulated juvenile hormone synthesis by corpora allata from males early in the final larval stadium, but not after day 8, showing that one of the final two reactions of juvenile hormone synthesis is rate-limiting in larval corpora allata at this stage. Adult female corpora allata which had been humorally inhibited by implantation into larvae were stimulated by farnesoic acid.     

Partial characterization and isolation of earwig `allatostatins'': biological activities in earwigs and cockroaches

Allatostatins are a family of neuropeptides first isolated from the cockroach, Diploptera punctata, that inhibit juvenile hormone production in that species (but do not do so in earwigs), and inhibit hindgut muscle contractions in some insects, including the earwig, Euborellia annulipes. We examined whether material from earwig brains is similar to cockroach allatostatins biochemically, immunologically and physiologically. Brain extracts from adult female earwigs were separated by high performance liquid chromatography (HPLC), followed by radioimmunoassay using antibodies to cockroach allatostatin (Dip-AST). Fractions that co-eluted with cockroach allatostatins were immunoreactive, and at least two peaks of immunoreactivity were detected. Material from each peak at 10 nM Dip-AST equivalents inhibited juvenile hormone biosynthesis in vitro by corpora allata of 2-day virgin D. punctata cockroaches; 1 nM was less effective, and non-immunoreactive fractions failed to inhibit juvenile hormone biosynthesis. Both crude and Sep-Pak (Waters) purified extracts of brains of earwigs containing 1 nM Dip-AST equivalents failed to suppress hindgut contractions in vitro of 2-day earwigs and of brooding female earwigs. In contrast, 1 nM cockroach allostatin 1 (Dip-AST 7) reversibly inhibited hindgut contractions in vitro. These results suggested the presence of another brain factor, such as proctolin, that counteracts the inhibitory effects of Dip-AST. In support of this hypothesis, proctolin stimulated hindgut contractions in vitro at 1 nM; the effects of equal concentrations of allatostatin and proctolin varied with the stage of the female. Furthermore, HPLC-separated fractions that co-eluted with cockroach allatostatin and were immunoreactive with antibodies to Dip-AST suppressed hindgut contractions in vitro of 2-day female earwigs. Finally, crude brain extracts of earwigs suppressed earwig juvenile hormone biosynthesis in vitro in glands of low, but not in glands of high, activity. Thus, earwig brain extract after HPLC separation has Dip-AST-like material that inhibits cockroach corpora allata and suppresses earwig hindgut contractions. Sep-Pak-extracted earwig brain material, however, does not inhibit earwig gut contraction. Although synthetic Dip-AST 7 does not inhibit juvenile hormone synthesis by earwig corpora allata, there is heat-stable material in earwig brain extract that does have this action.     

The mode of regulation of the corpus allatum activity during starvation in adult females of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

When two-day-old female Leptinotarsa decemlineata were starved, their corpus allatum activity, as measured by the radiochemical in vitro assay, was significantly reduced after 24 hr. Such a reduction was not observed when the nerve connections between the central nervous system and the retrocerebral complex were severed and the beetles starved up to 5 days. In some experiments, the rate of juvenile hormone biosynthesis in vitro, was substantiated by measurement of the juvenile hormone titre in the haemolymph by physico-chemical methods. It is concluded that intact nervous connections between the central nervous system and the corpora allata are essential for restraining the juvenile hormone biosynthesis during the initial stages of starvation.Corpora allata from 1-day starved insects were considerably stimulated in vitro by farnesenic acid indicating that juvenile hormone synthesis is controlled enzymatically at a stage prior to the final two steps in the pathway. However, on day 5 of starvation, rate-limitation may occur after formation of this intermediate, since farnesenic acid stimulation was much less at this time.Corpora allata of adult females newly emerged from the soil were activated within 4 hr regardless of feeding.     

Terminal stages in juvenile hormone biosynthesis in corpora allata of Diploptera punctata: Developmental changes in enzyme activity and regulation by allatostatins

The O-methyltransferase, which is responsible for the methylation of farnesoic acid in the corpora allata of Diploptera punctata, is a cytosolic enzyme. The activity of O-methyltransferase closely parallels JH biosynthesis in last instars and adult females. Because allatostatin 4 (AST 4) from D. punctata and callatostatin 5 (CAST 5) from Calliphora vomitoria can inhibit juvenile hormone biosynthesis, their effects on the activity of O-methyltransferase and epoxidase, the enzymes involved in the final two steps of juvenile hormone biosynthesis, were investigated in vitro. AST 4 can inhibit methyltransferase activity whereas CAST 5 stimulates it. AST 4 inhibits epoxidase activity slightly whereas CAST 5 inhibits it significantly (36%). Treatment of corpora allata with farnesoic acid (40 μM) can reverse the inhibitory effect of AST 4 and CAST 5 on JH release by corpora allata. Thus, allatostatins appear to exert their inhibitory effect on JH biosynthesis at least partially through inhibition of the activity of terminal enzymes. Two biosynthetic pathways for the conversion of farnesoic acid to JH may exist in corpora allata of D. punctata: the predominant pathway is farnesoic acid to methyl farnesoate, then to JH whereas the other, representing about 5–10% of total JH production, is farnesoic acid to JH III acid, then to JH.     

Influence of temperature and carbon dioxide concentration on juvenile hormone titre and dependent parameters of adult worker honey bees (Apis mellifera L.)

It is known that juvenile hormone plays an important role in the regulation of labour division and of the different life spans, and that the microclimate of the bee hive is characterized by its high CO₂ concentration and its varying temperature depending on the presence of brood.We have investigated the influence of microclimates characteristic of breeding and broodless areas on the juvenile hormone titre in the haemolymph and whole body extracts, on the corpora allata in vitro activity, on the degradation of juvenile hormone and on the dry weight of the hypopharyngeal glands using bees of known ages. A microclimate of 35°C and 1.5% CO₂, as observed in the breeding area, induces a rapid and pronounced increase in the juvenile hormone titre. On the other hand, this titre remains low in bees kept at 27°C and 1.5% CO₂, a microclimate associated with broodless combs. Rates of juvenile hormone synthesis by corpora allata in vitro were found to be extremely low, even in the presence of farnesenic acid, and not related to the juvenile hormone titre. In vitro incubation of juvenile hormone in haemolymph revealed no degradation while injected juvenile hormone was found to be degraded and taken up by the gut at rates only weakly correlated with the juvenile hormone titre.We propose a hypothetical model for the regulation of the juvenile hormone titre as well as the course of labour division by the varying microclimates observed in the bee hive.     

Distribution of allatostatin in the adult cockroach,Diploptera punctata and effects on corpora allata in vitro

Direct radiochemical measurements of juvenile hormone synthesis showed that corpora allata from adult female Diploptera punctata can be inhibited in vitro by neuropeptides extracted from several ganglia of the central nervous system of females at many stages of the reproductive cycle. Extracts of protocerebra, corpora cardiaca, suboesophageal, thoracic and ventral ganglia all elicited dose-depedent reductions in juvenile hormone synthesis. On a ‘per organ’ basis, the protocerebrum contains the most extractable material. Inhibitory activity of extracts of suboesophageal, thoracic and 6th abdominal ganglia, like that of protocerebra (Rankin et al., 1986) was trypsin sensitive.Glands of high activity were less sensitive to protocerebral extract than those of low activity. The inhibitory effect on glands of low activity was maximal within 1 h, persisted in the presence of protocerebral extract for at least 46 h, and was abolished within 1 h after corpora allata were placed in normal medium. The inhibitory effect of protocerebral extract was not altered by the addition of magnesium to the medium. The extract had a specific effect on synthetic step(s) prior to methylation and epoxidation as demonstrated by enhanced juvenile hormone synthesis in the presence of inhibitory factor and the juvenile hormone precursor, farnesoic acid.