Ecdysteroids in the haemolymph and the ovaries of the firebrat Thermobia domestica (Packard) (Insecta,Thysanura): Correlations with integumental and ovarian cycles

Ecdysteroids were analysed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) in females of the apterygotous insect Thermobia domestica, which ahs overlapping moulting and reproductive cycles. During each moulting cycle, a peak in the haemolymph concentration of ecdysteroids occurs at day 9 (in the 11-day standard cycle), which can be correlated with apolysis and the beginning of new cuticle deposition. The ovaries show a peak of ecdysteroids at day 5 (i.e. one day before egg-laying), which suggests that these hormones are also involved in the reproductive cycle. In both cases, HPLC analysis combined with RIA suggests that the main ecdysteroid is 20-hydroxyecdysone. This duality in the function of ecdysteroids is discussed.     

Treatment of larvae with repeated doses of precocene II induces precocious metamorphic changes in Spodoptera mauritia (Lepidoptera: Noctuidae)

In Spodoptera mauritia repeated daily treatments of larvae with 40 μg of precocene II throughout the fifth and sixth instar larval period had no effect on the larval-larval period but prolonged larval-pupal ecdysis. The resulting pupae showed precocious adult differentiation of mouth parts, wings, eyes, legs, and fat body.     

Effects of precocene I and II on flight behaviour in Oncopeltus fasciatus, the migratory milkweed bug

Treatment of newly emerged adult Oncopeltus fasciatus with the corpus allatum inhibitors. 7-methoxy-2,2-dimethyl chromene or 6,7-dimethoxy-2,2-dimethyl chromene (preocene I and II) results in an inhibition of long-term flight (presumed migratory) behaviour in both males and females and inhibition of oögenesis in females. Treatment of reproductive females with precocene briefly stimulates flight behaviour (which ceases in such females as oviposition begins) and then subsequently inhibits it. Oviposition also ceases in such females and oöcyte resorption occurs. Topical application or injections of JH III to precocenetreated animals results in immediate restoration of the tendency to make long tethered flights while corpora cardiaca extract injections, corpora cardiaca implantation, sham implantation, sham injections and sham topical applications were ineffective in restoring prolonged flight behaviour to precocene-treated animals.Restoration of flight by JH III injection was observed within 1 hr after treatment with the hormone. These results indicate that JH is necessary for prolonged flight and presumably migratory behaviour in this species and its stimulatory effect on flight behaviour is immediate. Possible mechanisms of action of the hormone on flight behaviour are discussed.     

Inhibition of reproduction and embryogenesis in the firebrat, Thermobia domestica, by juvenile hormone analogues

Application of compounds with juvenile hormone (JH) activity to reproducing females may result in shortening of the ovipositor and occasionally in some other changes in appearance. Severe defects occur in the ovaries: the differentiation of oögonia and prefollicular cells seems to be hindered and the mature oöcytes are resorbed. The affected ovarioles diminish and in extreme cases tear into the germarium, which remains attached to the terminal filament, and the rest of the ovariole, which shrinks into a small rudiment adjacent to the oviduct. The development of eggs deposited by treated females is often lethally affected. Disorders in embryogenesis also occur in the eggs contaminated with active substances at any time between deposition and 2 days before hatching. The substances interrupt the normal course of embryogenesis but the embryos survive for some time and may develop into strange creatures; in one case, two embryos were found within a single egg shell. One out of 27 tested substances was active in amounts lower than 1 ng/egg.     

20-Hydroxyecdysone and ovarian maturation in the firebrat Thermobia domestica (Thysanura: Lepismatidae)

The effects of 20-hydroxyecdysone on ovary maturation in the firebrat Thermobia domestica were investigated by in vivo injections of different doses of the hormone on various days of the reproductive cycle. Several females were neck-ligated or treated with precocene II before injection. Experiments were also performed on ovarioles incubated in vitro. It was demonstrated that 20-hydroxyecdysone has direct and dose-dependent effects on the ovary, inducing growth of all the previtellogenic follicles whatever the day of the reproductive cycle, except at times when the ovaries contain follicles undergoing choriogenesis. The major effect of the hormone is the stimulation of young-follicle formation in the anterior part of the previtellarium and the accelerated growth of a set of basal previtellogenic follicles, which reach the critical size required for yolk deposition. However, 20-hydroxyecdysone did not induce cellular differentiation, in particular, the enlargement of the perioocyte space and the development of oocyte microvilli, which normally occur before yolk precursor incorporation. The present results give a better understanding of the temporal relationships between molting and reproductive cycles and also explain the periodicity of ovarian maturation.     

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.     

Mitochondrial-driven sustained active water vapour absorption (WVA) in the firebrat, Thermobia domestica (Packard), during development and the moulting cycle

Rapid pre-functional mitochondrial biogenesis in the short-lived first-instar nymph occurs in the cells of the developing posterior rectal sacs which when mature are the organs believed to be responsible for sustained active water vapour absorption (WVA) in the firebrat Thermobia domestica (Packard). During the second instar, the mitochondria migrate apically and begin to associate with deep portasome-studded infolds of the apical plasma membrane (apm), just as WVA starts sporadically. By mid to late in the third instar the mature mitochondria-apm apical complex is fully developed, with elongated tubular mitochondria vertically packed hexagonally in transverse section and adpressed to the deeply pleated infolds of the apm, giving the greatest known concentration of mitochondria. This coincides with peak mass-specific WVA. During moults, WVA temporarily ceases as the sac cells secrete a new cuticle overlying the sac epithelium. Their apical complex fully regresses. Mitochondria migrate perinuclearly. The apm is pinched-off into numerous small portasome-studded pouches which remain sequestered within the cytoplasm. Towards the end of the moult the apm pouches reassemble into deeply pleated infolds into which the mitochondria migrate and elongate, rapidly re-establishing the hexagonal array of the apical complex. This coincides with the recommencement of WVA.     

The morphological and behavioural effects of precocene II on Locusta

The precocious adultiform larvae produced by topical applications of precocene II to early 4th instar Locusta were found to be intermediate between normal 5th instar larvae and normal adults on behavioural as well as morphological criteria. Precocious adultiforms walked significantly smaller distances in 2 hr than normal larvae, but slightly more than normal adults. The adultiforms jumped significantly greater distances without wing movements than normal larvae, but they also made frequent, short, adult-type jumps with wing movements. Most advanced adultiforms made jumps of both types whereas normal adults made only a small percentage of the larval-type jumps. Apparently the central nervous organisation of the adultiforms provides equally and separately for both larval and adult behaviour patterns, rather than compounding the two.Increasing doses of precocene above 100 μg produced more advanced adultiforms, and there was some indication that morphologically similar types produced by different doses may differ in their behaviour.     

Effects of juvenile hormone I, precocene I and precocene II on the progeny of Microplitis rufiventris Kok. female when administered via its host, Spodoptera littoralis (Boisd.)

Reproductive biologies of Microplitis rufiventris Kok. females resulting from topically treated Spodoptera littoralis (Boisd.) larvae with constant effective doses of juvenile hormone I (JHI, 1 μg), precocene I (PI, 25 μg) or PII (25 μg) were investigated. Although the female wasps were treated during their presence as eggs or larvae in their hosts, the complete effects of the test compound were not apparent until the wasps had become adults. On the bases of the obtained results, the reproductive inhibition activity caused by the test compounds comprises of two categories: (1) reduction in progeny production, and (2) induction of significant proportion of imperfect ‘non‐functional’ parasitoid progeny. Whereas, the adverse effect of JHI is only restricted to the second category, the adverse effects of PI or PII fall into both categories. Thus, workers should be aware of the delayed effects of new generations of pesticides which may occur in later stages of the non‐target insects.     

Development of physogastry in the queen of the fungus-growing termite Macrotermes michaelseni (Isoptera: Macrotermitinae)

Female reproductives of the fungus-growing termite Macrotermes michaelseni never showed signs of physogastry if they were kept in petri dishes to establish an incipient colony. During the 200 days of observation, their corpora allata volume, juvenile hormone titre and number of active ovarioles remained on a more or less constant level, and their gut was filled with soil. In contrast, these parameters are enhanced in partially and especially in fully physogastric queens, and these females had only a transparent liquid in the gut.Application of the juvenile hormone mimic ZR 515 to young females accelerated yolk incorporation, increased the number of active ovarioles and brought about the release of the soil from the gut. The same changes were induced by adding workers to increase the population of incipient colonies from about 45 to 400. This also resulted in an increase of the corpus allatum volume and of the juvenile hormone titre in young females. Their intestine was in this case filled with transparent liquid associated with the increased population of workers. It was, therefore, concluded that the development of physogastry depends on a positive feedback: the more workers are with the young female, the more nutrients are available and the more juvenile hormone the female produces, the more eggs are laid and the more workers can again develop to nurture the queen.     

Vitellogenins in the firebrat,Thermobia domestica (Packard): Immunological quantification during reproductive cycles and in relation to insemination (Thysanura: Lepismatidae)

An antiserum raised against the major fractions (240 + 300 Kd) of the vitellins extracted from maturing oöcytes of Thermobia domestica, allowed the immunological relationship between these proteins and their haemolymph precursors (vitellogenins) to be established, as well as the female specificity of these protein fractions. Using rocket immunoelectrophoresis, the vitellogenin concentration in the female haemolymph was measured on each day of a reproductive cycle. The changes in vitellogenin titre appear to be correlated with ovarian maturation: very low levels during the previtellogenic phase and increasing levels during the vitellogenic growth phase of the basal oöcytes. Also, these changes are synchronized with the moulting cycles which persist in the adults of these primitive insects. An insemination at the beginning of the interecdysial period causes a large increase in the vitellogenin titre synchronized with accelerated oöcyte growth.     

The hormonal regulation of the larval diapause in the codling moth, Laspeyresia pomonella (Lep. Tortricidae)

The hormonal control of the facultative diapause of the codling moth has been investigated. The diapause can be divided into 4 phases or periods: (1) diapause induction by short-day conditions (SD) in young larvae, (2) initiation of the diapause in the early last larval instar by a high titre of juvenile hormone, (3) onset and maintenance of diapause with inactivity of the neuroendocrine system, as evidenced by the results of neck-ligation experiments, (4)termination of diapause by the production of ecdysteroid.Diapause-induced larvae pupated after spinning the cocoon, if the state of induction was changed by injection with the anti-juvenile hormone precocene II at the beginning of the last larval instar and subsequent results of neck-ligation experiments, (4) termination of diapause by the production of ecdysteroid. treated with juvenile hormone during the first 1.5 days after the last larval moult and subsequently reared under SD. Under LD, continuous application of juvenile hormone during the last larval instar and after spinning did not prevent the insects from moulting to either a supernumerary larva, a pupa or a larval-pupal intermediate. Termination of diapause, i.e. pupation, was achieved by injecting diapausing larvae with 20-hydroxyecdysone. Although juvenile hormone was found to have a prothoractropic effect in diapausing larvae, no pupal moult could be induced by the application of the hormone. Contrary to the hormonal situation before pupation of nondiapausing larvae, no juvenile hormone could be detected before or during the pupation of larvae after diapause.     

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.     

The role of exogenous JH I,JH III and Anti-JH (precocene II) on queen induction of 4.5-day-old worker honey bee larvae

Worker larvae at an age of 4½ days were fed one of several mixtures of reconstituted royal jelly adjusted to a refractive index of 1.3825 and supplemented with JH I, JH III or Anti-JH (precocene II). In addition, juvenile hormone was topically applied to larvae of the same age. It was readily apparent that caste induction is concentration-dependent and that 4?-day-old worker larvae can still develop into queens under laboratory conditions, providing that they have not stopped feeding or can be induced to commence feeding again. These findings are contrary to the general belief that queen induction is not possible after a socalled sensitive period of 3–3½ days. Queens resulted only from honey bee larvae exposed to royal jelly containing 1 μg of JH I. In addition, oral application at this concentration resulted in the only case in which the normal mean weights of worker honey bees were exceeded. All other concentrations of juvenile hormone were not sufficient to initiate queen induction, although its lower concentration may have influenced the production of intercastes.Precocene II did not play a role in queen induction and it also did not interfere with the growth of developing larvae or adults. In addition, the lack of malformations in honey bees treated with precocene II indicates that the use of such a compound as a control agent in insect populations will probably not be detrimental to honey bee larvae that are at least 4½ days old. However, large doses of precocene will quickly kill most 3½-day-old honey bee larvae.The evidence presented here clearly indicates that caste determination is regulated by the endocrine system in honey bee larvae. Food intake in honey bee larvae may well be regulated by the endocrine system. Thus, an apparently inhibited corpus allatum (C.A.) could be reactivated by food intake coupled with juvenile hormone. The food intake restriction that worker larvae normally encounter in the hive probably results in a cessation of C.A. activity. The increase in food intake by queen larvae, on the other hand, carries an increase in growth and accompanying morphological changes necessary for queen development. This concept may also explain the development of intercastes encountered in in vitro studies. Only those larvae that follow a normal food intake sequence, i.e. moderate during the first 3–4 days or so, will develop into queens. Conversely, those larvae that take in too much food during the early portion of development may achieve incomplete development of the neurosecretory system and, thus, develop into intercastes.     

Lipoxygenase conversion of arachidonic acid in males and inseminated females of the firebrat,Thermobia domestica (Thysanura)

This is the first investigation concerning prostaglandin-like compounds in the primitive insect, Thermobia domestica. The incubation of homogenates of reproductive tissues in the presence of [U-¹⁴C]arachidonic acid yielded several compounds which have been characterized by their chromatographic mobilities as well as by the enzyme systems involved in their formation. The three major compounds (I to III) had R_f values very different from those of several prostaglandin standards (PGE₂, PGF_2α and 6-keto PGF_1α). As the addition of aspirin or indomethacin had no effect on the conversion of arachidonic acid, a cyclo-oxygenase pathway leading to prostaglandins seems to be excluded. However, another compound (noted V), present in very small quantities, could be a prostaglandin, owing to its chromatographic mobility near that of the PGE₂ standard. By contrast, compounds I and II co-migrated with 8- and 5-hydroxyeicosatetraenoic acid standards, respectively, and the addition of 4,7,10,13-eicosatetraynoic acid (ETYA) or nordihydroguaiaretic acid (NDGA) showed a pronounced and dose-dependent inhibition of arachidonic acid conversion. These data demonstrate lipoxygenase activity. Such a pathway in the metabolism of arachidonic acid had not, as yet, been reported in insects. This enzyme system can be demonstrated in the genital tract of the male and also in the seminal receptacle of the female, especially after insemination. So the enzyme system is probably transferred from male to female during mating.     

Changes in the size of corpora allata,in the juvenile hormone III titer in the hemolymph,and in the protein content of terminal oocytes throughout the first gonadotropic cycle in Aiolopus thalassinus (Insecta: Orthoptera: Acrididae)

In Aiolopus thalassinus (Fabr.), the changes in the volume of the corpora allata (CA), in the concentration of juvenile hormone III (JH III) in the hemolymph, and in protein content in the terminal (t) oocytes were studied during the first gonadotropic cycle. These parameters could be better related to the volume of t-oocytes than to age after emergence. The JH III titer curve was maximum (2.9 pmol/10 μl) at an oocyte volume of 1.2 mm³. Before oviposition (days 10–16) the JH III titer decreased to 0.65 pmol/10 μl hemolyph. The increase in JH III titer reflected a period of high protein storage in the t-oocytes. The largest volume of the CA was reached at the beginning of yolk storage in the t-oocytes. The highest JH III titer did not correspond with the largest volume of CA, which occurred much earlier. © 1993 Wiley-Liss, Inc.     

Allatotropic and nervous control of corpora allata in the adult male loreyi leafworm, Mythimna loreyi (Lepidoptera: Noctuidae)

Allatotropic activity is found in methanolic extracts of the brain–suboesophageal ganglion (SOG)–corpora cardiaca (CC) complex from virgin males of Mythimna loreyi Duponchel (Lepidoptera, Noctuidae). Corpora allata (CA) from 6‐day‐old virgin males exhibit low rates of release of Juvenile Hormone (JH) acid (JHA) in vitro. Release of JHA can be activated by the addition of an extract of brain–SOG–CC complex in a dose‐dependent manner, and this allatotropic activation can be sustained consistently in the continuous presence of such extracts. Based on its trypsin sensitivity and heat stability, the allatotropic factor is most likely a peptide. The allatotropic activity is dependent on the concentration of calcium ions in the medium, with the highest activation achieved beyond 2 m m . The results of nerve transection experiments suggest that both nervi corporis allati I (NCA I) and NCA II are involved in mediating the allatotropic control of CA in vitro. Isolated CA alone show significantly higher rates of release of JHA than the intact brain–SOG–CC–CA complex during the first 3 h of incubation, but the release of JHA reaches almost the same range in both groups by the end of the fourth hour of incubation.     

Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle,Tenebrio molitor (Coleoptera,Tenebrionidae)

The genome of Tribolium castaneum encodes two allatostatin [AS type B; W(X)₆Wamide and AS type C; PISCF‐OH] and one allatotropin (AT) precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing Consortium, 2008: Nature 452:949–955). Here we studied the activity (in vitro) of peptides derived from these precursors on the synthesis/release of juvenile hormone (JH) III. The corpora cardiaca‐corpora allata (CC‐CA) complexes of adult females of another tenebrionid beetle, the mealworm Tenebrio molitor, were used. Incubating the gland complexes in a medium containing Trica‐AS B3 peptide, we showed that the peptide has allatostatic function in T. molitor. The activity of the type C AS depended on the age of the test animals and their intrinsic rate of JH III biosynthesis. The Trica‐AS C peptide inhibited the JH release from CA of 3‐day‐old females with a high intrinsic rate of JH synthesis, but activated JH release from the CA of 7‐day‐old females with a lower intrinsic rate of JH production. The allatotropin peptide (Trica‐AT) also activated the JH release from the CA of 7‐day‐old females in a dose‐dependent and reversible manner. Unexpectedly, a type A AS derived from the precursor of the American cockroach Periplaneta americana (Peram‐AS A2b) inhibited the JH release from the CA of younger and older females in the concentration range of 10^?8 to 10^?4 M, and the effects were fully reversible in the absence of peptide. These data suggest a complex role of allatoactive neuropeptides in the regulation of JH III biosynthesis in beetles. © 2010 Wiley Periodicals, Inc.     

Correlation between larval weights,endocrine gland activities and competence period during differentiation of workers and soldiers in Macrotermes michaelseni (Isoptera:Termitidae)

Correlations were established between corpora allata and prothoracic gland activities, changes in fresh weights and competence period for soldier formation in a higher termite species, Macrotermes michaelseni. There was a time-course differential increase in fresh weights observed in pairs of female third (final)-instar larvae of the same age previously adopted by a pair of female reproductives from incipient colonies. A bimodal distribution of these larvae according to weights was evident from the first day of the instar. Heavier larvae were associated with higher corpora allata and prothoracic gland volumes (activities), and differentiation into presoldiers, while lighter larvae had considerably less-active corpora allata and prothoracic glands and developed into workers. The peak in the activities of these glands in both cases marked the end of the competence period.The chain of events that may precede caste differentiation is discussed briefly and the significance of the findings outlined.