Correlation among corpus allatum volume,cell size,and juvenile hormone biosynthesis in ovariectomized adult Blattella germanica

The corpora allata (CA) of both intact and ovariectomized Blattella germanica adult females exhibited a high degree of bilateral symmetry in the rate of juvenile hormone (JH) biosynthesis, the mean size of CA cells, and gland volume (81.3%, 98.3%, and 100% respectively with less than a twofold difference between the two glands in CA pairs). This permitted us to split each CA pair randomly, measure JH biosynthesis in one gland, and dissociate the other gland into a cell suspension in which the size of CA cells was measured. In ovariectomized females, changes in CA volume and the spontaneous and farnesoic acid (FA)-stimulated rates of JH biosynthesis, measured from the same glands, were well correlated (r = 0.78, for both correlations). Similarly, the mean volume of CA cells in one gland increased in relation to increases in both the spontaneous and FA-stimulated rates of JH biosynthesis by the contralateral member of the pair (r = 0.83 and r = 0.91, respectively). Concurrent changes in CA cell size and activity suggest that in the CA of B. germanica cellular growth and degradation are involved in the regulation of JH biosynthesis.     

Expression of juvenile hormone acid O‐methyltransferase and juvenile hormone synthesis in Blattella germanica

Juvenile hormone (JH), a sesquiterpenoid synthetized by the insect corpora allata (CA), plays critical roles in metamorphosis and reproduction. Penultimate or last step of JH synthesis is catalyzed by juvenile hormone acid O‐methyltransferase (JHAMT). Here we report the cloning and expression analysis of the JHAMT orthologue in the cockroach, Blattella germanica (L.) (BgJHAMT). BgJHAMT is mainly expressed in CA, with only expression traces in ovary. Three different isoforms, differing in the 3′‐UTR sequence, were identified. Isoform A shows between 35 and 65 times higher expression than B and C in CA from penultimate nymphal instar and adult females. RNAi‐triggered knock down of BgJHAMT produces a dramatic reduction of JH synthesis, concomitant with a decrease of fat body vitellogenin expression and basal follicle length. BgJHAMT mRNA levels in CA of females along the gonadotrophic cycle parallel, with a slight advancement, JH synthesis profile. BgJHAMT mRNA levels were reduced in starved females and in females in which we reduced nutritional signaling by knocking down insulin receptor and target of rapamycin (TOR). Results show that conditions that modify JH synthesis in adult B. germanica females show parallel changes of BgJHAMT mRNA levels and that the JH‐specific branch of the JH synthesis pathway is regulated in the same way as the mevalonate branch. Furthermore, we demonstrate that nutrition and its signaling through the insulin receptor and TOR pathways are essential for activating BgJHAMT expression, which suggests that this enzyme can be a checkpoint for the regulation of JH production in relation to nutritional status.     

Differential adaptation to water deprivation in first-instar nymphs of the German cockroach (Blattella germanica) and the brown-banded cockroach (Supella longipalpa)

This study compares the relative abilities of rist-instar nymphs of the german cockroach (Blattella germanica L.) and the brown-banded cockroach (Supella longipalpa F.) to survive prolonged periods of food and/or water deprivation.S. longipalpa nymphs survived significantly longer thanB. germanica nymphs when drinking water was absent, but the advantage ofS. longipalpa overB. germanica disappeared if food was also absent. These results are consistent with the hypothesis thatS. longipalpa is adapted to drier habitats than isB. germanica, and suggest thatS. longipalpa nymphs may be more capable of producing and utilizing extra metabolic water from food thanB. germanica nymphs. Evidence from the literature suggests that this species difference applies mainly to juveniles, since adults ofB. germanica andS. longipalpa do not differ in ability to withstand water deprivation, regardless of food availability.     

Effects of ovariectomy and mating on the activity of the corpora allata in adult female Blattella germanica (L.) (Dictyoptera: Blattellidae)

In adult female cockroaches, the ovary greatly affects the synthesis of Juvenile Hormone (JH) by the corpora allata, and in females of some cockroach species, removal of the ovaries results in a permanent depression of JH synthesis. We report that the corpora allata in ovariectomised, adult virgins of the German cockroach, Blattella germanica (L.), increase and then decrease in activity, as they do in intact females. Moreover, the distal tubules in the left colleterial glands of ovariectomised females accumulate abundant protein, the production of which is regulated by JH. In both ovariectomised and sham‐operated females, the activity of the corpora allata more than tripled between days 1 and 4 of adulthood, during which the oöcytes of sham‐operated females grew considerably in length. The corpora allata of sham‐operated females produced even more JH on day 7, but very little on day 10, by which time all females had oviposited. The glands of ovariectomised females, by constrast, produced a similar amount of JH on day 7 as on day 4, but much less on day 10. Beginning on day 13, the activity of the corpora allata increased again in ovariectomised females, an increase that did not occur until day 22 in sham‐operated females. Mating of ovariectomised females on day 6 resulted in a significant increase in the activity of the corpora allata by day 10. We conclude that both the ovary and mating stimulate the synthesis of JH early in the reproductive cycle, but that neither is needed for the occurrence of a complete cycle of JH synthesis.     

Biochemistry and regulation of pheromone production in Blattella germanica (L.) (Dictyoptera,Blattellidae)

Females of the German cockroach, Blattella germanica, produce a contact sex pheromone consisting of the methyl ketones 3,11-dimethyl-2-heptacosanone, 3,11-dimethyl-2-nonacosanone, 29-hydroxy- and 29-oxo-3,11-dimethyl-2-nonacosanone. We review evidence in support of the hypothesis that in adult females the hydrocarbon 3,11-dimethylnonacosane is oxidized to the corresponding methyl ketone pheromone. Recent studies on the precursors and directionality of synthesis of the methyl-branched alkane indicate that it is formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation. The hydrocarbon is then hydroxylated and oxidized at the 2-position to form the methyl ketone pheromone. The in vivo synthesis of pheromone and its accumulation on the cuticle are correlated to the synthesis of juvenile hormone (JH) by the corpora allata (CA) in vitro and to oocyte development, suggesting common regulation by JH of pheromone production as well as other reproductive events. The patterns of pheromone and hydrocarbon production in starved, allatectomized and head-ligated females, as well as in females rescued with hormone-replacement therapy, suggest two mechanisms of regulation of pheromone production: a JH-induced conversion of hydrocarbon to pheromone is related to the CA cycle and to oocyte development, while a JH-independent mechanism, which is probably related to feeding, supplies precursors for hydrocarbon biosynthesis.     

In vitro biosynthesis of JH III by the corpora allata of adult females of Blattella germanica (L)

A radiochemical assay which fulfills the required validation criteria has been used for quantification of the in vitro biosynthesis of JH III by the corpora allata of adult females of Blattella germanica throughout the 7 days of the first reproductive cycle. The presence of JH III has been independently confirmed by HPLC and mass spectrometry. Results indicate that rates of JH release increase repidly from day 3 to day 6, which is correlated with oocyte growth. The highest levels of JH release (2.58 ± 1.11 pmol/hr per pair) were obtained from day-6 females. The time course of JH production by CA from day-6 females showed that CA released JH at a linear rate for at least 9 hr. From these results, it can be concluded that titers at high production ages and linearity ranges are satisfactory enough to be used in studies on the regulation of JH production in this species.     

Effects of chilling stress on allatal growth and juvenile hormone synthesis in the cockroach, Diploptera punctata

During the ovarian cycle of the cockroach, Diploptera punctata, a mitotic wave occurs in the corpora allata before an increase in gland volume and juvenile hormone (JH) synthesis. Previous studies have demonstrated that the brain inhibits mitosis and JH synthesis in corpus allatum (CA) cells until adult females have mated. Herein, we report that chilling stress effectively suppresses mating induced proliferation of CA cells. In mated females, chilling on melting ice for 0.5-3 hours caused a strong, dose-dependent decrease in mitotic activity. In insects chilled for 3 hours, although the mitotic wave in the CA was practically abolished, CA volume and JH synthesis finally reached peak levels typical of unchilled insects, despite a 2-day delay. Consequently, oocyte maturation and oviposition were also delayed by 2 days, yet in both chilled and unchilled insects, peak values of basal oocyte length were the same. By allowing virgin females to mate on different days after chilling, we found that the chilling effect could be retained in the insect body for at least 2 days. During this period, signals from mating could not effectively remove inhibition of CA cell proliferation. Unilaterally disconnecting the CA from the brain revealed that chilling stress mediated CA cell proliferation via the brain, and did not directly affect the CA.     

Ultrastructural changes induced by precocene II and 3,4-dihydroprecocene II in the corpora allata of Blattella germanica

Summary Ultrastructural studies on corpora allata (CA) from different stages during the first gonadotropic cycle of the cockroach Blattella germanica have shown well defined changes which have a correspondence with oocyte length, CA volume and juvenile hormone (JH) biosynthesis. The most significant variations concern the mitochondria and the endoplasmic reticulum. Topically applied precocene II (P II) at a dose of 200 g induced a transient arrest of CA function, although cytotoxic effects were occasionally observed. When CA were maintained in vitro with 10^-3 M of P II, a relationship between the time of treatment (3, 6 or 9 h) and the intensity of the effects was apparent. The 9-h treatment led to an irreversible inhibition of JH production which parallels the severe damages observed in the CA (membrane lysis, nuclear pyknosis, vacuolization). Equivalent studies performed with the chroman derivative 3,4-dihydroprecocene II (DHP II) showed that it is less active than P II. Only treatments as severe as 12 h of incubation with a 10^-3 M concentration elicited cytotoxic effects which could be due to radical species involved in the in situ oxidative bioactivation of DHP II. Thus, this compound could be regarded as a new type of pro-allatocidin.     

Modulation of juvenile hormone release and oocyte growth following (RS)-hydroprene treatment in virgin female Diploptera punctata

ABSTRACT. The effect of (flS)-hydroprene treatment (2, 20, 200 μ g) on JH release was assessed in virgin females of D. punctata (Eschscholtz) during the first 10 days of adult life as was basal oocyte length and number of cells in the CA. At a dose of 2 μ g hydroprene, JH release was stimulated slightly and, on days 4 and 6, oocyte growth was significantly greater than that of acetone-treated controls. A similar but more striking enhancement of JH release and basal oocyte growth was observed at a dose of 20 μ g and a significant inhibition of JH release, in concert with a rapid growth of basal oocytes, was observed at a dose of 200 μ g. During the observation period, the mean number of cells in the CA decreased in a dose-dependent fashion, with a highly significant reduction in numbers in 20 and 200 μ g-treated animals. Reimplantation of vitellogenic ovarioles (three or six) into ovariectomized virgin females also resulted in an enhancement of JH release; this indicates that virgin female CA can respond to the stimulatory action of the ovary and is consistent with a model for ( RS )-hydroprene action in which the 'positive feedback' effect (stimulation of JH release) observed with low doses of the analogue occurs as a consequence of the action of the analogue on the ovary. ( RS )-hydroprene treatment stimulates basal oocyte growth to the point at which the previously unstimulatory virgin oocytes are able to enhance JH release by a feedback loop involving the CA and probably the brain.     

Juvenile Hormone inhibition in corpora allata from ovariectomized Blattella germanica

Abstract. Ovariectomy has been used to study the role of the ovary in endocrine homeostasis. Our studies on young virgin adults of the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) show that the cytological development of the corpora allata (CA) in ovariectomized females proceeds as in intact specimens, whereas the rates of Juvenile Hormone (JH) synthesis are lower. Stimulation of the CA from ovariectomized females in vitro by mevalonolactone suggests that enzymatic mechanisms which follow mevalonate formation in the biosynthetic pathway are functional. The synthetic capabilities of these CA are also illustrated by the kinetics of JH production in vitro , because hormonal release increases with time to reach 'normal' levels after 8h of incubation. Our data suggest that the absence of ovaries leads to effective inhibition of JH biosynthesis rather than to an impairment of the developmental process in the CA cells.     

Behavioural responses of diverse insect groups to electric stimuli

Anecdotal evidence suggests that cockroaches respond to electrical appliances or outlets. Our objectives were to determine the effect of field‐inducing sources and field attributes on attraction of German cockroaches, Blattella germanica (L.) (Blattodea: Blattellidae), and to test those parameters found effective for attraction of B. germanica for attraction of other groups of insects. In two‐choice, large‐arena experiments, significantly more female, but not nymphal, B. germanica settled in or near electrified coils with static or fluctuating electromagnetic fields produced by low‐level direct current (DC) or alternating current (AC) sources than in control coils without current. Electromagnetic fields with the magnetic, but not the electric, component of the field nulled still attracted B. germanica, suggesting that the electric component of the field may contribute to the attraction or arrestment response of B. germanica. DC‐powered coils with static electromagnetic fields also attracted/arrested brown‐banded cockroaches, Supella longipalpa (Fabricius) (Blattodea: Blattellidae), common silverfish, Lepisma saccharina (L.), firebrats, Thermobia domestica (Packard) (both Thysanura: Lepismatidae), and European earwigs, Forficula auricularia (L.) (Dermaptera: Forficulidae), but they repelled American cockroaches, Periplaneta americana (L.) (Blattodea: Blattidae). If proven in field experiments, electrified coils as trap baits may offer non‐toxic alternatives to pesticides for selective insect control in urban environments.     

Laboratory and field evaluation of Beauveria bassiana bait against two cockroach species (Dictyoptera: Blattellidae,Blattidae) in Jinan City,East China

A Beauveria bassiana bait formulation was evaluated for its efficacy in the laboratory and field against German cockroaches, Blattella germanica (L.), and American cockroaches, Periplaneta americana (L.). The LT₅₀ values were 46 and 119?h, respectively, for B. germanica and P. americana. The cockroach species were lured and killed effectively when linear distance was about 5?m between B. bassiana bait and fresh mouse food. In the field, more than 90% relative decline in density was found 14?d after treatment, which lasted for 90?d. In conclusion, laboratory and field studies demonstrated the high efficacy of a B. bassiana bait formulation against common cockroach species.     

Identification of juvenile hormone III in the hemolymph of Blattella germanica adult females by gas chromatography-mass spectrometry

Juvenile hormone III was identified in purified hemolymph extracts of adult females of the cockroach Blatella germanica by gas chromatography-mass spectrometry with chemical ionization and selected ion monitoring. Under these conditions, juvenile hormones I and II were not detected within the sensitivity ranges of this analytical method. For quantification purposes a 5,5-bisdeuterated analog of juvenile hormone III was synthesized and used as an internal standard. In general, juvenile hormone III titers obtained correlated with data on oocyte growth and with hormone titers found from in vitro corpora allata incubations along the first gonotrophic cycle.     

Juvenile hormone biosynthesis and oocyte development in adult female Blattella germanica: Effects of grouping and mating

The roles of grouping and mating in modulating the activity of the corpora allata (CA) in adult female cockroaches were investigated using the in vitro radiochemical assay of juvenile hormone (JH) biosynthesis. Isolated virgin females have longer, asynchronous cycles of CA activity and oocyte maturation than do isolated females mated on day 8. Three factors were identified as the major contributors to this difference: (1) an experimental artifact of selection for sexually receptive females, (2) a positive effect of grouping on JH synthesis and oocyte maturation, and (3) a positive effect of copulation on oviposition and retention of the ootheca. Mated females constitute a subpopulation of receptive females that differ significantly from other females by having higher rates of JH synthesis prior to mating. The relative importance of such selection is substantial when the rate of mating is low, as in experiments with isolated females that are exposed to males for a short period of time. Long-term exposure of females to males introduces a grouping effect, which obscures any additional effect of mating on CA activity and oocyte development. However, mating influences ootheca formation and its retention. The effect of grouping can be mimicked in isolated females by transection of the nerves connecting the CA–corpora cardiaca complex to the brain, suggesting that in this insect isolation causes brain inhibition of the CA, and grouping provides disinhibitory stimuli that release the CA from brain inhibition.     

Isolation of cockroach Phe-Gly-Leu-amide allatostatins from the termite Reticulitermes flavipes and their effect on juvenile hormone synthesis

Immunoreactivity to cockroach Diploptera punctata allatostatin-7 (Dippu AST-7) has been demonstrated previously in axons innervating the corpora allata of the termite Reticulitermes flavipes. This peptide and Dippu AST-11 inhibited juvenile hormone (JH) synthesis by corpora allata (CA) of brachypterous neotenic reproductives (secondary reproductives) of termites. The present study shows that R. flavipes CA are also inhibited by Dippu AST-2, AST-5, AST-8, and AST-9 at approximately the same rank order of potency as demonstrated in D. punctata. Another allatostatin from Periplaneta americana (Peram AST-12) also inhibits JH synthesis by R. flavipes CA. Sensitivity to the allatostatins is higher in glands with low rates of JH synthesis than in those with relatively high JH synthetic rates as has been demonstrated in CA from male and female secondary reproductives as well as in those from non-egg-laying and egg-laying females. The identical inhibitory effects of R. flavipes brain extract on CA from both D. punctata and R. flavipes and the isolation and identification of five cockroach allatostatins (Dippu AST-1, AST-2, AST-5, AST-8, and Peram AST-12) from termite brain extract reflect the close relationship between cockroaches and termites.     

The effect of enforced virginity and subsequent mating on the activity of the corpus allatum of Periplaneta americana measured in vitro, as related to changes in the rate of ovarian maturation

ABSTRACT. Female P. americana, reared with males from the time of adult emergence, mated on the 4th–5th day after metamorphosis, produced the first ootheca on the 8th or 9th day, and then produced successive oothecae at intervals of 3.0 days, whereas, only 50% of virgin females had produced their first ootheca by the 28th day after adult emergence. Examination of the ovaries indicated that oocyte development is normal in virgins until shortly after the time when they first become receptive to males. When mating was not allowed there was a dramatic reduction in the rate of vitellogenic growth of the terminal batch of oocytes which persisted until mating was allowed, and was often accompanied by resorption of a percentage of the oocytes. Short-term, in vitro, radiochemical assay of juvenile hormone (JH III) biosynthesis by corpora allata (CA) showed that, in females reared with males, the cycles of ovarian development are accompanied by regular pulses of CA activity. There is a small, possibly preparatory peak of JH III biosynthesis before vitellogenesis of the first wave of oocytes, followed by a larger peak of JH III production during vitellogenesis of this batch of eggs and one peak of CA activity between ovulation of each subsequent wave of oocytes. Activities as low as 0.25 pmol C₁₆JH/CA pair/h and as high as 48.38 pmol/CA pair/h were observed in CA from mated females after the onset of cyclic activity. Stimuli received during mating are somehow responsible for the cyclic activity of the CA, for when females were subjected to enforced virginity the first small peak was normal but the second peak was not fully realized and there was then a gradual decline in CA activity until approximately 2 weeks post-emergence. Thereafter the glands exhibited a more or less constant rate of JH biosynthesis (mean = 3.45 ± 0.32 pmol/CA pair/h.) When females were mated after 21 days of enforced virginity the activity of the CA was enhanced. By 48 h after mating the mean glandular activity was at least four times that found in virgins of the same age, and by 72 h rates as high as 40 pmol/CA pair/h were observed. This was followed by normal cyclic activity of the CA. The increase in rate of JH biosynthesis appears to result in a recommencement of oocyte development in these ‘delayed-mated’ females.     

Time-course haemolymph juvenile hormone titres in solitarious and gregarious adults of Schistocerca gregaria, and their relation to pheromone emission, CA volumetric changes and oocyte growth

The haemolymph JH III titres in solitarious and gregarious adult desert locusts, Schistocerca gregaria, were examined in relation to corpus allatum (CA) volumes, aggregation-maturation pheromone production in males and oocyte growth in females. The JH titres of gregarious females were generally higher than those of solitarious females at all ages studied. The titre patterns, however, were similar: relatively high on day 10, dropping to low levels between days 20 and 25, before rising again by day 25. In the solitarious males, the JH titre was very low on day 10 after fledging, but increased gradually and reached a maximal amount on day 30. The JH titre in gregarious males was low on day 10, elevated on day 15 coinciding with the start of the production of the pheromone, and dropped to a relatively low level on day 20 around the time of maximal pheromone production, then rising again by day 25. These results suggest that biosynthesis of the pheromone is associated with a high JH titre peak in the haemolymph. Although a clear relationship was found during the first gonadotropic cycle between JH titres, on one hand, and CA volume and oocyte growth, on the other, in both phases, no such correlation could be discerned in the second cycle.     

Juvenile hormone and methyl farnesoate production in cockroach embryos in relation to dorsal closure and the reproductive modes of different species of cockroaches

Juvenile hormone (JH), produced by the corpora allata (CA), is first detectable after dorsal closure, a conspicuous event in embryogenesis. The present research found that the timing of dorsal closure was consistently at about 45% of the total embryonic development time across most of the oviparous and ovoviviparous cockroach species examined. These included the ovoviviparous cockroaches Blaberus discoidalis, Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, Phoetalia pallida, Schultesia lampyridiformis, and Panchlora nivea, as well as the oviparous cockroaches Blatta orientalis, Periplaneta americana, Eurycotis floridana, and Supella longipalpa. However, the only known viviparous cockroach Diploptera punctata completed dorsal closure at 20.8% of embryo development time. Methyl farnesoate (MF), the immediate precursor of JH III, is considered a functional molecule in crustaceans; however, in insects its function is still unclear. To understand the role of JH and MF in cockroach embryos, I compared JH and MF biosynthesis and release in several cockroach species of known phylogenetic relationships. Using a radiochemical assay, the present research showed that cockroach embryos representing all three reproductive modes produced and released both JH and MF, as previously shown for B. germanica, N. cinerea, and D. punctata. Members of a pair of embryonic CA from B. discoidalis, B. fumigata, R. maderae, and D. punctata were incubated with and without farnesol. MF accumulated in large amounts only in CA of R. maderae in the presence of farnesol, which indicates that control of the last step of biosynthesis of JH, conversion of MF into JH by MF epoxidase, is probably a rate-limiting step in this species.     

Action of a juvenile hormone analogue on the activity of Periplaneta americana corpora allata in vitro and on juvenile hormone III levels in vivo

The effects of the juvenile hormone (JH) analogue fenoxycarb (ethyl[2-(4-phenoxyphenoxy)-ethyl]carbamate) on the activity of corpora allata (CA) from adult female Periplaneta americana have been investigated. The in vitro biosynthesis of JH III by isolated CA was inhibited by about 85% in the presence of a high concentration (1 × 10⁻⁴ M) of fenoxycarb. However, at lower concentrations (1 × 10⁻⁶ M and 1 × 10⁻⁸ M) no inhibition of JH biosynthesis was apparent. Topical treatment of adult female cockroaches with fenoxycarb (100 μg/insect) did not reduce the subsequent rate of JH III biosynthesis by CA in vitro. By contrast, the same treatment markedly reduced the titre of endogenous JH III in intact cockroaches. These results suggest that CA activity in adult female P. americana may be controlled by negative feedback, and that this system of control is dependent on the maintenance of contact between the CA and nervous or humoral factors in the intact insect. Alternatively, it is possible that treatment with fenoxycarb increases the rate at which endogenous JH is metabolized.     

Identification and quantification of juvenile hormones from different developmental stages of the cockroach Nauphoetacinerea

By the combined use of high-pressure liquid chromatography, $\text{Galleria}$ bioassay and gas chromatography/ chemical ionization/mass spectrometry we were able to isolate and identify the three known natural juvenile hormones (JHs) from haemolymph extracts of larval and adult females of the cockroach $\text{Nauphoeta}$$\text{cinerea}$. This is the first demonstration of the simultaneous occurrence of the three JHs in the same insect and the first time JH I and II have been identified in a hemimetabolous insect. Quantitative investigations show that the composition of the three JHs is different at different developmental stages. The haemolymph of larvae contains a high percentage of JH I and II, whereas the haemolymph of adult females in the oocyte maturation stage contains mostly JH III. This suggests more juvenilizing functions for JH I and II and more gonadotropic functions for JH III.