Morphology of the Antenna of Hermetia illucens (Diptera: Stratiomyidae): An Ultrastructural Investigation.

The black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), is a relevant species in waste and pest management, but is also of forensic and medical importance. A scanning electron microscopy (SEM) investigation of the antennae of both sexes of H. illucens is presented here for the first time. The antenna is composed of three regions: the scape, the pedicel, and the flagellum. The first two regions are single segments, whereas the third region, the longest one, is composed of eight flagellomeres. The scape and pedicel have microtrichia, chaetic sensilla, and rounded perforations. The flagellum is covered by different microtrichia, the morphology of which is described in detail. Two types of sensory pit are found on flagellomeres 1 to 6. An oval depression with trichoid sensilla extends from flagellomeres 4 to 6. On both sides of flagellomere 8 is a lanceolate depression covered by hair-like microtrichia. Morphometric and morphological analyses revealed some sex-related differences. The results of the SEM investigations are compared with those obtained on other species of the family Stratiomyidae and other brachyceran Diptera. The possible role of sensilla in sensory perception is also discussed in comparison with nondipteran species.

Studying the Evolution of the Vertebrate Circadian Clock: The Power of Fish as Comparative Models.

The utility of any model species cannot be judged solely in terms of the tools and approaches it provides for genetic analysis. A fundamental consideration is also how its biology has been shaped by the environment and the ecological niche which it occupies. By comparing different species occupying very different habitats we can learn how molecular and cellular mechanisms change during evolution in order to optimally adapt to their environment. Such knowledge is as important as understanding how these mechanisms work. This is illustrated by the use of fish models for studying the function and evolution of the circadian clock. In this review we outline our current understanding of how fish clocks sense and respond to light and explain how this differs fundamentally from the situation with mammalian clocks. In addition, we present results from comparative studies involving two species of blind cavefish, Astyanax mexicanus and Phreatichthys andruzzii. This work reveals the consequences of evolution in perpetual darkness for the circadian clock and its regulation by light as well as for other mechanisms such as DNA repair, sleep, and metabolism which directly or indirectly are affected by regular exposure to sunlight. Major differences in the cave habitats inhabited by these two cavefish species have a clear impact on shaping the molecular and cellular adaptations to life in complete darkness.

Ultrastructural Morphology of the Antenna and Maxillary Palp of Sarcophaga tibialis (Diptera: Sarcophagidae).

A scanning electron microscopy investigation of the antenna and maxillary palp of the adult of Sarcophaga tibialis Macquart (Diptera: Sarcophagidae), a species of medical, veterinary, and forensic relevance, is presented for the first time. Adults of both sexes used in this study were obtained from larvae collected in a case of traumatic myiasis in a domestic cat in northern Italy. The antenna of S. tibialis is that typical of cyclorrhaphan Diptera, consisting of three segments: the scape, the pedicel, and the postpedicel, bearing the arista. The scape is covered by microtrichia and has a row of long chaetic sensilla. The pedicel is also covered by microtrichia and has three types of chaetic sensilla and a cluster of setiferous plaques. Trichoid, styloconic, clavate, and basiconic sensilla are distributed among the microtrichia on the postpedicel. Invaginated basiconic-like sensilla and olfactory pits are also present, the latter ones more numerous in the female. Our results are compared with those obtained for other calyptrate flies, mainly in the family Sarcophagidae. The data obtained may represent a basis for electrophysiological studies on the sensorial activity of the species related to the search for food sources, mates, and suitable larviposition sites, and for comparative morphological studies with other Diptera.

Ultrafast dynamics. Attosecond band-gap dynamics in silicon.

Electron transfer from valence to conduction band states in semiconductors is the basis of modern electronics. Here, attosecond extreme ultraviolet (XUV) spectroscopy is used to resolve this process in silicon in real time. Electrons injected into the conduction band by few-cycle laser pulses alter the silicon XUV absorption spectrum in sharp steps synchronized with the laser electric field oscillations. The observed ~450-attosecond step rise time provides an upper limit for the carrier-induced band-gap reduction and the electron-electron scattering time in the conduction band. This electronic response is separated from the subsequent band-gap modifications due to lattice motion, which occurs on a time scale of 60 ± 10 femtoseconds, characteristic of the fastest optical phonon. Quantum dynamical simulations interpret the carrier injection step as light-field-induced electron tunneling.

Zebrafish circadian clocks: cells that see light.

In the classical view of circadian clock organization, the daily rhythms of most organisms were thought to be regulated by a central, 'master' pacemaker, usually located within neural structures of the animal. However, with the results of experiments performed in zebrafish, mammalian cell lines and, more recently, mammalian tissues, this view has changed to one where clock organization is now seen as being highly decentralized. It is clear that clocks exist in the peripheral tissues of animals as diverse as Drosophila, zebrafish and mammals. In the case of Drosophila and zebrafish, these tissues are also directly light-responsive. This light sensitivity and direct clock entrainability is also true for zebrafish cell lines and early-stage embryos. Using luminescent reporter cell lines containing clock gene promoters driving the expression of luciferase and single-cell imaging techniques, we have been able to show how each cell responds rapidly to a single light pulse by being shifted to a common phase, equivalent to the early day. This direct light sensitivity might be related to the requirement for light in these cells to activate the transcription of genes involved in DNA repair. It is also clear that the circadian clock in zebrafish regulates the timing of the cell cycle, demonstrating the wide impact that this light sensitivity and daily rhythmicity has on the biology of zebrafish.

Flies and fish: birds of a feather.

The identification of specific clock-containing structures has been a major endeavour of the circadian field for many years. This has lead to the identification of many key components of the circadian system, including the suprachiasmatic nucleus in mammals, and the eyes and pineal glands in lower vertebrates. However, the idea that these structures represent the only clocks in animals has been challenged by the discovery of peripheral pacemakers in most organs and tissues, and even a number of cell lines. In Drosophila, and vertebrates such as the zebrafish, these peripheral clocks appear to be highly autonomous, being set directly by the environmental light/dark cycle. However, a hierarchy of clocks may still exist in mammals. In this review, we examine some of the current views regarding peripheral clocks, their organization and how they are entrained.

A clockwork organ.

The vertebrate circadian clock was thought to be highly localized to specific anatomical structures: the mammalian suprachiasmatic nucleus (SCN), and the retina and pineal gland in lower vertebrates. However, recent findings in the zebrafish, rat and in cultured cells have suggested that the vertebrate circadian timing system may in fact be highly distributed, with most if not all cells containing a clock. Our understanding of the clock mechanism has progressed extensively through the use of mutant screening and forward genetic approaches. The first vertebrate clock gene was identified only a few years ago in the mouse by such an approach. More recently, using a syntenic comparative genetic approach, the molecular basis of the the tau mutation in the hamster was determined. The tau gene in the hamster appears to encode casein kinase 1 epsilon, a protein previously shown to be important for PER protein turnover in the Drosophila circadian system. A number of additional clock genes have now been described. These proteins appear to play central roles in the transcription-translation negative feedback loop responsible for clock function. Post-translational modification, protein dimerization and nuclear transport all appear to be essential features of how clocks are thought to tick.

Asynchronous oscillations of two zebrafish CLOCK partners reveal differential clock control and function.

Most clock genes encode transcription factors that interact to elicit cooperative control of clock function. Using a two-hybrid system approach, we have isolated two different partners of zebrafish (zf) CLOCK, which are similar to the mammalian BMAL1 (brain and muscle arylhydrocarbon receptor nuclear translocator-like protein 1). The two homologs, zfBMAL1 and zfBMAL2, contain conserved basic helix-loop-helix-PAS (Period-Arylhydrocarbon receptor-Singleminded) domains but diverge in the carboxyl termini, thus bearing different transcriptional activation potential. As for zfClock, the expression of both zfBmals oscillates in most tissues in the animal. However, in many tissues, the peak, levels, and kinetics of expression are different between the two genes and for the same gene from tissue to tissue. These results support the existence of independent peripheral oscillators and suggest that zfBMAL1 and zfBMAL2 may exert distinct circadian functions, interacting differentially with zfCLOCK at various times in different tissues. Our findings also indicate that multiple controls may be exerted by the central clock and/or that peripheral oscillators can differentially interpret central clock signals.

Rhythmic transcription: the molecular basis of oscillatory melatonin synthesis.

Pulsatile hormone synthesis and secretion are characteristic features of various oscillatory biological systems. Circadian rhythms are critical in the regulation of most physiological functions, and much interest has been centred on the understanding of the molecular mechanisms governing them. Adaptation to a changing environment is an essential feature of physiological regulation. The day-night rhythm is translated into hormonal oscillations governing the metabolism of all living organisms. In mammals the pineal gland is responsible for the circadian synthesis of the hormone melatonin in response to signals originating from the endogenous clock located in the hypothalamic suprachiasmatic nucleus (SCN). The molecular mechanisms involved in rhythmic synthesis of melatonin involve the CREM gene, which encodes transcription factors responsive to activation of the cAMP signalling pathway. The CREM product, ICER, is rhythmically expressed and participates in a transcriptional autoregulatory loop which also controls the amplitude of oscillations of serotonin N-acetyl transferase, the rate-limiting enzyme of melatonin synthesis. Thus, a transcription factor modulates the oscillatory levels of a hormone.

Light acts directly on organs and cells in culture to set the vertebrate circadian clock.

The expression of clock genes in vertebrates is widespread and not restricted to classical clock structures. The expression of the Clock gene in zebrafish shows a strong circadian oscillation in many tissues in vivo and in culture, showing that endogenous oscillators exist in peripheral organs. A defining feature of circadian clocks is that they can be set or entrained to local time, usually by the environmental light-dark cycle. An important question is whether peripheral oscillators are entrained to local time by signals from central pacemakers such as the eyes or are themselves directly light-responsive. Here we show that the peripheral organ clocks of zebrafish are set by light-dark cycles in culture. We also show that a zebrafish-derived cell line contains a circadian oscillator, which is also directly light entrained.

Phylogenetic relationships of North American damselflies of the genus Ischnura (Odonata: Zygoptera: Coenagrionidae) based on sequences of three mitochondrial genes.

Relationships of North American damselflies of the genus Ischnura (Odonata: Zygoptera: Coenagrionidae) were investigated using a total of 1205 bp from portions of three mitochondrial genes: cytochrome b, cytochrome oxidase II, and 12S ribosomal DNA. Parsimony and neighbor joining analyses reveal a monophyletic group consisting of I. damula, I. demorsa, I. perparva, I. posita posita, I. posita atezca, I. verticalis, and probably I. denticollis, likely reflecting a recent radiation in North America. Ischnura kellicotti, I. barberi, I. prognata, I. hastata, I. ramburii, and I. capreola appear to represent much earlier divergences in the group. Many previous hypotheses of relationships among North American species of Ischnura are not supported by the molecular-based analyses. However, there is agreement in many respects between the results of the molecular phylogenetic analyses and the morphologically based conclusions of Kennedy (1919, "The Phylogeny of the Zygoptera," Ph.D. Dissertation, Cornell University, Ithaca). Although results of single-gene phylogenetic analyses often differ, there are very few cases in which there is strong support for conflicting relationships using different partitions of the data. Combined analysis of all three genes yields trees with stronger support overall than the single-gene analyses, and the combined data trees that result from diverse data treatments are congruent with one another in most respects.

PASting together the mammalian clock.

Over the past year, the first components of the mammalian clock have been identified; Clock, bmal1 and three homologs of Drosophila period have been cloned, all of which encode PAS proteins. Expression of the mammalian period gene oscillates in many tissues in vivo and in immortalized cell cultures in vitro. Now, can we say that every cell has a circadian clock?

Zebrafish Clock rhythmic expression reveals independent peripheral circadian oscillators.

The only vertebrate clock gene identified by mutagenesis is mouse Clock, which encodes a bHLH-PAS transcription factor. We have cloned Clock in zebrafish and show that, in contrast to its mouse homologue, it is expressed with a pronounced circadian rhythm in the brain and in two defined pacemaker structures, the eye and the pineal gland. Clock oscillation was also found in other tissues, including kidney and heart. In these tissues, expression of Clock continues to oscillate in vitro. This demonstrates that self-sustaining circadian oscillators exist in several vertebrate organs, as was previously reported for invertebrates.

Rhythmic transcription: the molecular basis of circadian melatonin synthesis.

Adaptation to a changing environment is an essential feature of physiological regulation. The day/night rhythm is translated into hormonal oscillations governing the physiology of all living organisms. In mammals the pineal gland is responsible for the synthesis of the hormone melatonin in response to signals originating from the endogenous clock located in the hypothalamic suprachiasmatic nucleus (SCN). The molecular mechanisms involved in rhythmic synthesis of melatonin involve the CREM gene, which encodes transcription factors responsive to activation of the cAMP signalling pathway. The CREM product, ICER, is rhythmically expressed and participates in a transcriptional autoregulatory loop which also controls the amplitude of oscillations of serotonin N-acetyl transferase (AANAT), the rate-limiting enzyme of melatonin synthesis. In contrast, chick pinealocytes possess an endogenous circadian pacemaker which directs AANAT rhythmic expression. cAMP-responsive activator transcription factors CREB and ATF1 and the repressor ICER are highly conserved in the chick with the notable exception of ATF1 that possesses two glutamine-rich domains in contrast to the single domain encountered to date in mammalian systems. ICER is cAMP inducible and undergoes a characteristic day-night oscillation in expression reminiscent of AA-NAT, but with a peak towards the end of the night. Interestingly CREB appears to be phosphorylated constitutively with a transient fall occurring at the beginning of the night. Thus, a transcription factor modulates the oscillatory levels of a hormone.

Evidence for a central role of transcription in the timing mechanism of a circadian clock.

The retinal circadian clock in the isolated in vitro eye of the marine mollusc Bulla gouldiana exhibits a phase-dependent requirement for transcription. The transcription-sensitive phase extends through most of the subjective day and therefore is substantially longer than the previously reported translation-sensitive phase. Lower concentrations of transcription inhibitors yield a significant dose-dependent lengthening of circadian period. Clock motion can be stopped by a high concentration of the transcription inhibitor 5,6-dichlorobenz-imidazole riboside (DRB) when applied during the sensitive phase; after withdrawal of the inhibitor, motion resumes from the phase at which it was stopped. In a double-pulse experiment, phase shifts to light pulses applied after DRB pulses, and not during the translation-sensitive phase, indicate that the inhibition of transcription has immediate effects on the phase of the clock. These data suggest that DRB-induced phase shifts are independent of translation, which implies that the rate of transcription itself plays a significant role in the mechanism underlying the generation of the circadian cycle.

Luteal function in unilaterally hysterectomized ewes treated with gonadotropin-releasing hormone.

An experiment was conducted to examine effects of GnRH administered to ewes during metestrus on subsequent luteal and uterine functional interrelationships. Treatments consisted of GnRH (0 or 100 micrograms/d) and uterine status (intact or unilaterally hysterectomized [UHYST]). On d 12 of an estrous cycle, all ewes were unilaterally ovariectomized and one-half of these ewes were subjected to contralateral UHYST. Corpora lutea in the remaining ovary were enucleated. One-half of the intact and UHYST ewes were injected i.v. with 2 mL of GnRH on d 2 and 3 after subsequent estrus, and the remaining ewes were injected similarly with 2 mL of saline. Jugular blood samples were collected at 15-min intervals after GnRH or saline injection and analyzed for serum LH. Caudal vena caval and(or) jugular blood were collected daily from d 5 to 10 and on d 12 and 14 of the cycle and analyzed for progesterone, oxytocin, and prostaglandin F2 alpha (PGF2 alpha). Injection of ewes with GnRH increased serum concentrations of LH within 60 min compared with those of saline-treated ewes (P = .01). Treatment with GnRH did not alter concentrations of oxytocin in intact or UHYST ewes (P > .10) but on d 12 and 14 mean jugular concentrations of oxytocin were greater (P = .01) in intact than in UHYST ewes. Vena cava plasma concentrations of PGF2 alpha did not differ (P > .10) among treatments. Treatment with GnRH did not affect (P > .10) serum concentrations of progesterone, but concentrations of this steroid over the sampling period tended to be greater (P = .09) in UHYST ewes than in intact ewes. In conclusion, treatment of intact and UHYST ewes with GnRH failed to alter systemic concentrations of oxytocin, PGF2 alpha, and progesterone; however, the concentrations of oxytocin were affected by unilateral hysterectomy.

Circadian rhythm generation, expression and entrainment in a molluscan model system.

The Bulla ocular pacemaker provides remarkable opportunities for cellular study of circadian pacemaker systems. The demonstration of circadian oscillations within individual neurons maintained in culture provides us with a first occasion to study the biophysical and biochemical properties of bona fide neuronal circadian pacemakers. The ocular clock is robust and shares formal similarity with other circadian systems. The development of molecular techniques that can be applied to single neurons should allow research on the Bulla retina to continue to progress towards a molecular analysis of circadian timekeeping.

The arrestin superfamily: cone arrestins are a fourth family.

Arrestins constitute a superfamily of regulatory proteins that down-regulate phosphorylated G-protein membrane receptors, including rod and cone photoreceptors and adrenergic receptors. The potential role of arrestin in color visual processes led us to identify a cDNA encoding a cone-like arrestin in Xenopus laevis, the principle amphibian biological model system. Alignment of 18 deduced amino acid sequences of all known arrestins from both invertebrate and vertebrate species reveals five arrestin families. Further analysis identifies 7 variable and 4 conservative arrestin structural motifs that may identify potential functional domains. The adaptive evolutionary relationship of Xenopus cone arrestin to the arrestin gene tree suggests high intrafamily homology and early gene duplication events.

Cellular analysis of a molluscan retinal biological clock.

The eye of the opisthobranch mollusc Bulla gouldiana expresses a circadian rhythm in optic nerve impulse frequency. The circadian rhythm is generated among approximately 100 neurons at the base of the retina referred to as basal retinal neurons. These cells are electrically coupled to one another and fire spontaneous action potentials in synchrony. Basal retinal neurons recorded intracellularly exhibit a circadian rhythm in membrane potential that appears to be driven by a circadian modulation of membrane conductance. Membrane conductance is relatively high during the subjective night and decreases after subjective dawn. Recent experiments in our laboratory indicate that individual basal retinal neurons in culture can express circadian rhythms in membrane conductance. When completely isolated, these cells continue to show circadian conductance changes. These studies provide the first direct demonstration that individual neurons can act as circadian pacemakers. Although the precise details of the mechanism generating the circadian periodicity remain obscure, our research indicates that several transmembrane ionic fluxes are not involved in rhythm generation, but that a transmembrane Ca2+ flux is critical for entrainment. Both transcription and translation appear to play critical roles in generating the circadian cycle.