Carbonic anhydrase activity in the vas deferens of the cotton leafworm - Spodoptera littoralis (Lepidoptera: Noctuidae) controlled by circadian clock.

The male reproductive tract of Lepidoptera is an ideal model for the study of the physiological role of peripheral clocks in insects. The latter are significant in the generation and coordination of rhythmic phenomena which facilitate the initial stages of sperm capacitation. This process requires the maintenance of pH in the upper vas deferens (UVD) aided by, among others, H+-ATPase. Our aim was to determine the potential involvement of carbonic anhydrase (CA) in this process, an enzyme tasked with generating protons subsequently utilized by H+-ATPase to acidify the UVD milieu in S. littoralis, during the time when the lumen of this organ is filled with sperm. We attempted to answer the question whether CA activity can be controlled by the biological oscillator present in the male reproductive tract of the cotton leafworm. Using PAGE zymography, the presence of CA was demonstrated in the UVD wall, but not in the luminal fluid nor in the sperm. Using histochemistry, it was shown that CA is active in the UVD epithelium, and that this activity varies throughout the day and is most likely controlled by an endogenous biological clock. Conversely, the application of CA inhibitors, acetazolamide and sodium thiocyanate, in conjunction with an analysis of H+-ATPase activity in the acidification the UVD environment shows that CA most likely does not play a direct role in the regulation of the pH in this organ.

Disruption of sperm release from insect testes by cytochalasin and beta-actin mRNA mediated interference.

Release of sperm bundles from moth testes is controlled by the local circadian oscillator. The mechanism which restricts migration of sperm bundles to a few hours each day is not understood. We demonstrate that a daily cycle of sperm release is initiated by the migration of folded apyrene sperm bundles through a cellular barrier at the testis base. These bundles have conspicuous concentrations of actin filaments at their proximal end. Inhibition of actin polymerization by cytochalasin at aspecific time of day inhibited sperm release from the testis. Likewise, application of double-stranded actin RNA specifically inhibited sperm release. This RNA-mediated interference (RNAi) lowered the pool of actin mRNA in tissues involved in sperm release. The decline in mRNA levels resulted in the selective depletion of F-actin from the tip of apyrene sperm bundles, suggesting that this actin may be involved in the initiation of sperm release. Combined results of RNAi experiments at physiological, cellular and molecular levels identified unique cells that are critically involved in the mechanism of sperm release.

Selection for high diapause incidence in blow flies (Calliphora vicina) maintained under long days increases the maternal critical daylength: some consequences for the photoperiodic clock.

Using a population of Calliphora vicina from southern Scotland (55 degrees N), showing a critical day length for maternal induction of diapause of about 14.5 h per day, strains of flies were selected for a high incidence of larval diapause under long day length (LD 16:8 h). Diapause incidence was raised from under 10% to almost 100% within five or six generations. The selected flies showed an increase in their critical day length to over 16 h per day, and a high incidence of larval diapause under very long photophases. Selected flies, however, showed mean circadian periods for locomotor activity little different from the original stock, or from flies selected for high diapause under LD 12:12 h, and a Nanda-Hamner profile lacking peaks and troughs of diapause incidence at about 24 h intervals. These results are interpreted to show that the photoperiodic regulation of diapause and the control of overt behavioural rhythmicity are 'separate' physiological systems.

Circadian rhythm of glycoprotein secretion in the vas deferens of the moth, Spodoptera littoralis.

BACKGROUND: Reproductive systems of male moths contain circadian clocks, which time the release of sperm bundles from the testis to the upper vas deferens (UVD) and their subsequent transfer from the UVD to the seminal vesicles. Sperm bundles are released from the testis in the evening and are retained in the vas deferens lumen overnight before being transferred to the seminal vesicles. The biological significance of periodic sperm retention in the UVD lumen is not understood. In this study we asked whether there are circadian rhythms in the UVD that are correlated with sperm retention. RESULTS: We investigated the carbohydrate-rich material present in the UVD wall and lumen during the daily cycle of sperm release using the periodic acid-Shiff reaction (PAS). Males raised in 16:8 light-dark cycles (LD) showed a clear rhythm in the levels of PAS-positive granules in the apical portion of the UVD epithelium. The peak of granule accumulation occurred in the middle of the night and coincided with the maximum presence of sperm bundles in the UVD lumen. These rhythms persisted in constant darkness (DD), indicating that they have circadian nature. They were abolished, however, in constant light (LL) resulting in random patterns of PAS-positive material in the UVD wall. Gel-separation of the UVD homogenates from LD moths followed by detection of carbohydrates on blots revealed daily rhythms in the abundance of specific glycoproteins in the wall and lumen of the UVD. CONCLUSION: Secretory activity of the vas deferens epithelium is regulated by the circadian clock. Daily rhythms in accumulation and secretion of several glycoproteins are co-ordinated with periodic retention of sperm in the vas deferens lumen.

Expression of larval hemolymph proteins (Lhp) genes and protein synthesis in the fat body of greater wax moth (Galleria mellonella) larvae during diapause.

When one-day-old, last instar Galleria mellonella larvae are exposed to 18 degrees C they enter diapause and cease further development for several months. During diapause a group of proteins (72-84 kDa) synthesized in the fat body and secreted into the hemolymph is markedly elevated. Partial sequencing of the N-terminus of two proteins from this group confirmed their identity with larval hemolymph proteins (LHP) belonging to the family of hexameric storage proteins. The expression of two Lhp genes of known sequence (Lhp76 and Lhp82) were monitored in both diapausing and non-diapausing individuals. The expression of both genes and subsequent synthesis of the proteins (LHP76 and LHP82) is maintained until at least 90-100 days of diapause.

Temperature-dependent regulatory mechanism of larval development of the wax moth (Galleria mellonella).

The mechanisms underlying larval diapause in the wax moth (Galleria mellonella) is one of the most throughly studied aspects. At the low temperature of 18 degrees C, the last instar larvae did not pupate but transferred to 30 degrees C they initiated development and pupation in a circadian manner. Different types of surgical manipulations including head-ligation, nerve cord-severance, implantation of the brain, prothoracic glands, accompanied with ecdysteroid titre measurements indicated that diapausing arrest of larval development at 18 degrees C might be due to the nervous inhibition of their prothoracic glands.

Circadian regulation of Fos-like expression in the brain of the blow fly Calliphora vicina.

Expression of Fos-like immunoreactivity (FOS-lir) was examined in the brains of the blow fly Calliphora vicina for evidence of circadian regulation by photic stimuli. Fos-lir in various parts of the brain was investigated as a function of light and time of day. Immunohistochemistry demonstrated that photic stimuli have an inductive effect on c-fos expression in the various parts of the brain, but only in the neurons of the pars intercerebralis did the clear photic induction of c-fos expression occur at times when light was capable of phase-shifting circadian locomotor activity rhythms. This suggests that the c-fos gene may play a role in the photic pathway for circadian entrainment and that these neurons may be involved in the transduction of photic signals. Whether changes in c-fos expression are essential components of this pathway remains to be determined.

S-antigen antibody partially blocks entrainment and the effects of constant light on the circadian rhythm of locomotor activity in the adult blow fly, Calliphora vicina.

Injection of S-antigen (arrestin) antibody into the brain of the blow fly, Calliphora vicina, appeared to reduce sensitivity to the photic effects of a light:dark cycle (LD; entrainment) or continuous "bright" light (LL; arrhythmicity). In LD, a proportion of the injected flies evaded entrainment or showed delayed entrainment. In bright LL, flies continued with a free-running rhythm, which remained unchanged (as in continuous darkness) or lengthened (as in "dim" LL). These results focus attention on four groups of arrestin-positive neurons in the fly's brain as potential components of the photoreceptive system.

Vitellogenesis in virgin and mated females of the mealworm beetle, Tenebrio molitor.

A comparison between vitellogenesis in virgin and mated females of Tenebrio molitor showed significant differences at each investigated developmental stage. Yolk protein deposition in oocytes, measured as an increase in their size parameters (length, width, and volume), proceeded much faster and was more efficient in mated females as compared to virgins. In fertilized females the gonadotropic cycle showed a cyclicity with an eight-day period while virgin females finish their vitellogenic stage after the first cycle. These differences were reflected in changes in the rate of protein synthesis in the fat body of females completing vitellogenesis or entering the next oogenetic cycle. In the haemolymph, in addition to a large (158 kDa) and two small (56 kDa and 45 kDa) subunits of vitellogenin, there was an abundance of proteins of 80 kDa and 60 kDa.

Influence of low temperature on the synthesis of some Galleria mellonella proteins.

In developing Galleria mellonella larvae (reared at 30 degrees C) three proteins of 74, 76 and 81/82 kDa were identified. They represent a group of storage proteins (LHP proteins). In Galleria larvae, the development of which is arrested by low temperature (18 degrees C), accumulation of the 74, 76 and 81/82 kDa proteins was detected in the hemolymph. The synthesis of 74 kDa and 76 kDa proteins started after 24 h, and that of about 80 kDa after 96 h following the transfer of larvae from 30 degrees C to 18 degrees C. 20-Hydroxyecdysone inhibited synthesis of the 74 and 76 kDa proteins in larvae exposed to low temperature. The arrest of development of Galleria larvae is associated with the synthesis and accumulation of storage proteins, and ecdysteroids are involved in these processes.

Circadian activity rhythm of the house fly continues after optic tract severance and lobectomy.

Under constant conditions, locomotor activity in about 50% of 63 adult Musca domestica continued to be rhythmic after bilateral severance of optic tracts or bilateral lobectomy. Apparently, the optic lobes of Musca do not contain the oscillator for rhythmic control of locomotor activity as has been proposed for other insects. In 20% of the individuals, several circadian components of activity rhythms were found after operation indicating a role of the optic lobes in the coupling of oscillators. The remaining 30% of the flies with severed optic tracts appeared to be arrhythmic. Most of these flies had vacuolized tissue in the central brain. However, disruption of rhythmicity did not correlate with a common pattern of degeneration. Therefore no conclusions can be drawn as to the localization of the circadian control of locomotor activity in the brain. Flies showing an arrhythmic activity pattern could still be synchronized by LD cycles. Activity did not occur solely during the light period as is the case in controls; but was phase delayed by about 6 hr towards the dark period. Since all flies with severed optic tracts could be synchronized by LD cycles, Musca domestica must possess extraocular photoreceptors.

Annual changes in the greater wax moth (Galleria mellonella L.) development and sensitivity to juvenilizing treatments.

The greater wax moth (Galleria mellonella L.) larvae reared in constant conditions showed endogenous annual changes in the sensitivity to juvenilizing treatments, i.e. cooling and JHA administration. Also control, untreated larvae showed annual changes in normal development. The number of spontaneously appearing extra-larval molts, the number of animals entering the state of permanent larva, as well as the sex-ratio in Galleria population changes with respect to the season of the year. The possible mechanisms involved in these phenomena are discussed.

Graded inhibition of cell disintegration of juvenile hormone.

The proventriculus of Galleria mellonella larvae consists of 260-300 polyploid cells. The cells grow throughout larval development and at each ecdysis produce a new cuticular lining of the proventriculus. Each cell secretes a large sclerotized plaque covered with thorns. At the end of the last larval instar, the cells disintegrate unless the larvae is treated with juvenile hormone or implanted with active corpora allata. Depending on the time of treatment and the hormone dose, these insects preserve their proventricular cells to graded degrees. In some cases, the cells continue to grow or at least preserve their full function, in other instances they secrete but small plaques with fewer thorns or a smooth cuticle without any thorns. Very late treatments preserve healthy cells incapable of secreting the cuticle.