Large Ventral Lateral Neurons Determine the Phase of Evening Activity Peak across Photoperiods in Drosophila melanogaster

The dual-oscillator model, originally proposed as a mechanism for how vertebrates adapt to seasonal changes, has been invoked to explain circadian entrainment in Drosophila melanogaster. Distinct subsets of neurons have been designated as "morning" and "evening" oscillators that function as regulators of rhythmic activity/rest behavior. Some studies have led to a model in which a subset of 8 "morning" cells (4 bilaterally located small ventral lateral neurons) and another subset of approximately 130 "evening" cells exert different levels of dominance within the circadian circuit in different seasons. However, many studies propose a more integrative neuronal network, with the whole network orchestrating activity/rest rhythms in different seasons, as opposed to hierarchical dominance among neurons. Within the circadian network, our understanding of the role of the large ventral lateral neurons (l-LN(v)) has thus far been limited to conveying light information to the clocks and as light-activated neurons mediating arousal. In support of the framework of a more distributed model, we report an important circadian clock-related role for the l-LN(v) in electrical activity-dependent phasing of the evening peak across a range of photoperiods. Further, we propose a model in which l-LN(v) enable adaptation to seasonal changes by regulating the phase of the evening peak. Additionally, we demonstrate a hitherto unknown role for the small ventral lateral neurons (s-LN(v)) in the arousal circuit, thus showing that neuronal function is flexible such that certain neurons can play more than one role in distinct circuits.     

Expression, purification and ligand binding properties of the recombinant translation initiation factor (PeIF5B) from Pisum sativum

Gene encoding a novel translation initiation factor PeIF5B from Pisum sativum with sequence similarity to eIF5B from H. sapiens, D. melanogaster, S. cerevisiae as well as archaeal aIF5B from M. thermoautotrophicum was earlier reported by us. We now describe the expression and purification of 96 kDa recombinant PeIF5B (rPeIF5B) protein. Using fluorescence and circular dichroism spectra analyses, we show that Mg(2+) binding does not lead to any change in PeIF5B aromatic amino acid micro-environment, whereas GTP binding induces significant changes in the local environment of the aromatic amino acids. However, the protein undergoes changes in secondary structure upon metal ion and nucleotide binding. Charged initiator tRNA binding to PeIF5B is found to be cofactor dependent. PeIF5B binds to GTP in vitro as evident from autoradiography. Based on homology modeling of the catalytic domain of PeIF5B, we could confirm the conformational changes in PeIF5B following ligand binding.     

Estrogenicity of the isoflavone metabolite equol on reproductive and non-reproductive organs in mice

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight](-1) day(-1)) or in the diet (0, 500, or 1,000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 microM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)(-1) day(-1). Dietary equol at 500 or 1,000 ppm produced total serum equol concentrations of 5.9 and 8.1 microM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8-20 mg (kg body weight)(-1) day(-1) and 1,000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor alpha when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.     

Amelioration of genotoxic damage by certain phytoproducts in human lymphocyte cultures

The antigenotoxic effect of some phytoproducts like carotenoid (beta-carotene), curcumin, ascorbic acid and flavonoid (genistein)was demonstrated on the genotoxicity induced by hydrocortisone. Human lymphocyte cultures were studied for the induction of chromosomal aberrations, sister chromatid exchanges and effect on cell cycle kinetics with or without the presence of metabolic activation (S9 mix). The phytoproducts were studied in two most effective doses viz. carotenoid (0.5 and 0.7 microM), curcumin (15 and 25 microM), ascorbic acid (60 and 80 microM) and flavonoid (25 and 40 microM) in 24, 48 and 72 h cultures, and they were found to reduce chromosomal aberrations, sister chromatid exchange and increase replication index. The present study showed that the ascorbic acid and curcumin were more effective than carotenoid and flavonoid, though all provide protection against the genotoxicity of hydrocortisone.     

Oviposition preference for novel versus normal food resources in laboratory populations ofDrosophila melanogaster

Female fecundity, oviposition preference and specificity on one normal and two novel food media were assayed on four laboratory populations ofDrosophila melanogaster, revealing considerable among- and within-population variation in oviposition preference. Overall, there was a significant tendency of females to prefer novel media to their normal banana food as an oviposition substrate. Specificity in the populations was fairly high, implying that a large proportion of females tended to lay the majority of their eggs on the preferred medium. The results showed that oviposition preference for a given food medium could be affected by the alternative provided, and that, consequently, oviposition preference for a given food medium versus another cannot be predicted based upon a knowledge of what the preference for each of the two media was versus a common third medium. Specificity, on the other hand, was not significantly affected by the type of alternative food media provided in a given trial. Moreover, comparison of results from fecundity and oviposition preference assays also showed that the egg laying behaviour ofDrosophila females in response to different food media may be different in choice versus no-choice situations. Thus, a substrate on which fecundity is higher than on another, when assayed in a no-choice situation, may not be preferred over the other substrate when a choice between the two is provided to the ovipositing females. The latter two results point to possible complexity in the responses of females to various oviposition substrates based upon the overall setting of the assay, including the alternative substrates present for egg laying.     

Evolution of reduced pre-adult viability and larval growth rate in laboratory populations of Drosophila melanogaster selected for shorter development time

Four large (n > 1000) populations of Drosophila melanogaster, derived from control populations maintained on a 3 week discrete generation cycle, were subjected to selection for fast development and early reproduction. Egg to eclosion survivorship and development time and dry weight at eclosion were monitored every 10 generations. Over 70 generations of selection, development time in the selected populations decreased by approximately 36 h relative to controls, a 20% decline. The difference in male and female development time was also reduced in the selected populations. Flies from the selected populations were increasingly lighter at eclosion than controls, with the reduction in dry weight at eclosion over 70 generations of selection being approximately 45% in males and 39% in females. Larval growth rate (dry weight at eclosion/development time) was also reduced in the selected lines over 70 generations, relative to controls, by approximately 32% in males and 24% in females. However, part of this relative reduction was due to an increase in growth rate of the controls populations, presumably an expression of adaptation to conditions in our laboratory. After 50 generations of selection had elapsed, a considerable and increasing pre-adult viability cost to faster development became apparent, with viability in the selected populations being about 22% less than that of controls at generation 70 of selection.     

Membrane electrical excitability is necessary for the free-running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free-running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell-autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K(+) channel stops the free-running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free-running larval pacemaker clock-like that of the adult pacemaker neurons they give rise to-requires membrane electrical activity to oscillate. In contrast to the free-running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light-dark) conditions. This specific disruption of the free-running clock caused by targeted K(+) channel expression likely reflects a specific cell-autonomous clock-membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology.     

Circadian pathway: the other shoe drops

Three new papers report the long-awaited functional characterization of the Drosophila receptor for the circadian-rhythm-regulating signalling molecule PIGMENT DISPERSING FACTOR (PDF). The discovery of the PDF receptor heralds progress in understanding the circadian pacemaker circuit and output pathways in insects.     

A case for multiple oscillators controlling different circadian rhythms in Drosophila melanogaster

A population of the fruit fly Drosophila melanogaster was raised in periodic light/dark (LD) cycles of 12:12 h for about 35 generations. Eclosion, locomotor activity, and oviposition were found to be rhythmic in these flies, when assayed in constant laboratory conditions where the light intensity, temperature, humidity and other factors which could possibly act as time cue for these flies, were kept constant. These rhythms also entrained to a LD cycle of 12:12 h in the laboratory with each of them adopting a different temporal niche. The free-running periods (tau) of the eclosion, locomotor activity and oviposition rhythms were significantly different from each other. The peak of eclosion and the onset of locomotor activity occurred during the light phase of the LD cycle, whereas the peak of oviposition was found to occur during the dark phase of the LD cycle. Based on these results, we conclude that different circadian oscillators control the eclosion, locomotor activity and oviposition rhythms in the fruit fly D. melanogaster.