60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression.

Radiation is considered as a promising insect pest control strategy for minimizing postharvest yield losses. Among various techniques, irradiation is a method of choice as it induces lethal biochemical or molecular changes that cause a downstream cascade of abrupt physiological abnormalities at the cellular level. In this study, we evaluated the effect of 60Co-γ radiation on various developmental stages of Zeugodacus cucurbitae Coquillett and subsequent carry-over effects on the progeny. For this purpose, we treated eggs with 30- and 50-Gy radiation doses of 60Co-γ. We found that radiation significantly affected cellular antioxidants, insect morphology, and gene expression profiles. Our results indicate that in response to various doses of irradiation reactive oxygen species, catalase, peroxidase, and superoxide dismutase activities were increased along with a significant increase in the malondialdehyde (MDA) content. We observed higher mortality rates during the pupal stage of the insects that hatched from irradiated eggs (50 Gy). Furthermore, the life span of the adults was reduced in response to 50 Gy radiation. The negative effects carried over to the next generation were marked by significantly lower fecundity in the F1 generation of the irradiation groups as compared to control. The radiation induced morphological abnormalities at the pupal, as well as the adult, stages. Furthermore, variations in the gene expression following irradiation are discussed. Taken together, our results signify the utility of 60Co-γ radiation for fruit fly postharvest management.

The Two-Photon Reversible Reaction of the Bistable Jumping Spider Rhodopsin-1.

Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore-attached to the protein through a protonated Schiff base-and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography.

Effect of antibiotic treatment and gamma-irradiation on cuticular hydrocarbon profiles and mate choice in tsetse flies (Glossina m. morsitans).

BACKGROUND: Symbiotic microbes represent a driving force of evolutionary innovation by conferring novel ecological traits to their hosts. Many insects are associated with microbial symbionts that contribute to their host's nutrition, digestion, detoxification, reproduction, immune homeostasis, and defense. In addition, recent studies suggest a microbial involvement in chemical communication and mating behavior, which can ultimately impact reproductive isolation and, hence, speciation. Here we investigated whether a disruption of the microbiota through antibiotic treatment or irradiation affects cuticular hydrocarbon profiles, and possibly mate choice behavior in the tsetse fly, Glossina morsitans morsitans. Four independent experiments that differentially knock down the multiple bacterial symbionts of tsetse flies were conducted by subjecting tsetse flies to ampicillin, tetracycline, or gamma-irradiation and analyzing their cuticular hydrocarbon profiles in comparison to untreated controls by gas chromatography - mass spectrometry. In two of the antibiotic experiments, flies were mass-reared, while individual rearing was done for the third experiment to avoid possible chemical cross-contamination between individual flies. RESULTS: All three antibiotic experiments yielded significant effects of antibiotic treatment (particularly tetracycline) on cuticular hydrocarbon profiles in both female and male G. m. morsitans, while irradiation itself had no effect on the CHC profiles. Importantly, tetracycline treatment reduced relative amounts of 15,19,23-trimethyl-heptatriacontane, a known compound of the female contact sex pheromone, in two of the three experiments, suggesting a possible implication of microbiota disturbance on mate choice decisions. Concordantly, both female and male flies preferred non-treated over tetracycline-treated flies in direct choice assays. CONCLUSIONS: While we cannot exclude the possibility that antibiotic treatment had a directly detrimental effect on fly vigor as we are unable to recolonize antibiotic treated flies with individual symbiont taxa, our results are consistent with an effect of the microbiota, particularly the obligate nutritional endosymbiont Wigglesworthia, on CHC profiles and mate choice behavior. These findings highlight the importance of considering host-microbiota interactions when studying chemical communication and mate choice in insects.

Comparison of the trapping effect and antioxidant enzymatic activities using three different light sources in cockchafers.

Light traps have been widely used for controlling underground pests. However, very little is known regarding the relationship between trapping effect and antioxidant enzymatic activities using light irradiation in underground pests. Thus, we determined the trapping effect of three light sources of the frequoscillation pest-killing lamp on two species of cockchafers, Serica orientalis Motschulsky (Coleoptera: Melolonthidae) and Anomala corpulenta Motschulsky (Coleoptera: Rutelidae), and evaluated the effect of the same three light sources on the activities of their antioxidant enzymes. The catches of S. orientalis were significantly higher compared to A. corpulenta using light source A in peanut fields in China. After irradiation by light source A, the malondialdehyde (MDA) contents and activities of superoxide dismutase (SOD) and glutathione S-transferases (GST) in S. orientalis were significantly and marginally significantly lower compared to A. corpulenta. Taken together, these results indicated a weaker antioxidant enzyme activity response to light stress and a larger quantity of trapping catches using light irradiation in cockchafers. Thus, we proposed a potential negative relationship between trapping effect and antioxidant enzymatic activities in response to light irradiation in cockchafers.

Polarizing properties and structure of the cuticle of scarab beetles from the Chrysina genus.

The optical properties of several scarab beetles have been previously studied but few attempts have been made to compare beetles in the same genus. To determine whether there is any relation between specimens of the same genus, we have studied and classified seven species from the Chrysina genus. The polarization properties were analyzed with Mueller-matrix spectroscopic ellipsometry and the structural characteristics with optical microscopy and scanning electron microscopy. Most of the Chrysina beetles are green colored or have a metallic look (gold or silver). The results show that the green-colored beetles polarize reflected light mainly at off-specular angles. The gold-colored beetles polarize light left-handed near circular at specular reflection. The structure of the exoskeleton is a stack of layers that form a cusplike structure in the green beetles whereas the layers are parallel to the surface in the case of the gold-colored beetles. The beetle C. gloriosa is green with gold-colored stripes along the elytras and exhibits both types of effects. The results indicate that Chrysina beetles can be classified according to these two major polarization properties.

Differential sensitivity of Chironomus and human hemoglobin to gamma radiation.

Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study.

Unexpected behavior of irradiated spider silk links conformational freedom to mechanical performance.

Silk fibers from Argiope trifasciata and Nephila inaurata orb-web weaving spiders were UV irradiated to modify the molecular weight of the constituent proteins. Fibers were characterized either as forcibly silked or after being subjected to maximum supercontraction. The effect of irradiation on supercontraction was also studied, both in terms of the percentage of supercontraction and the tensile properties exhibited by irradiated and subsequently supercontracted fibers. The effects of UV exposure at the molecular level were assessed by polyacrylamide gel electrophoresis and mass spectrometry. It is shown that UV-irradiated fibers show a steady decrease in their main tensile parameters, most notably, tensile strength and strain. The combination of the mechanical and biochemical data suggests that the restricted conformational freedom of the proteins after UV irradiation is critical in the reduction of these properties. Consequently, an adequate topological organization of the protein chains emerges as a critical design principle in the performance of spider silk.

The expression of three opsin genes from the compound eye of Helicoverpa armigera (Lepidoptera: Noctuidae) is regulated by a circadian clock, light conditions and nutritional status.

Visual genes may become inactive in species that inhabit poor light environments, and the function and regulation of opsin components in nocturnal moths are interesting topics. In this study, we cloned the ultraviolet (UV), blue (BL) and long-wavelength-sensitive (LW) opsin genes from the compound eye of the cotton bollworm and then measured their mRNA levels using quantitative real-time PCR. The mRNA levels fluctuated over a daily cycle, which might be an adaptation of a nocturnal lifestyle, and were dependent on a circadian clock. Cycling of opsin mRNA levels was disturbed by constant light or constant darkness, and the UV opsin gene was up-regulated after light exposure. Furthermore, the opsin genes tended to be down-regulated upon starvation. Thus, this study illustrates that opsin gene expression is determined by multiple endogenous and exogenous factors and is adapted to the need for nocturnal vision, suggesting that color vision may play an important role in the sensory ecology of nocturnal moths.

Resilin-like protein in the clamp sclerites of the gill monogenean Diplozoon paradoxum Nordmann, 1832.

Resilin is a soft and elastic protein, which is found in many exoskeletal structures of arthropods. Proteins with similar chemical properties have been described for other invertebrates including monogenean fish parasites. However, for the latter taxon no clear microscopic evidence for a resilin-like protein has been shown so far. Here, we present the results of detailed microscopic analyses of the clamp sclerites (attachment devices) of the monogenean Diplozoon paradoxum. Toluidine blue, which is known to stain resilin, selectively stained the material in the clamp sclerites. In addition, when exposed to UV light, this material exhibited an intense blue autofluorescence. The emission spectrum of this autofluorescence has its maximum at 424 nm and is nearly identical to emission spectra of blue autofluorescences observed in 2 well-studied structures containing high proportions of resilin in the exoskeleton of the locust Schistocerca gregaria. The results strongly indicate that the sclerite material of D. paradoxum contains a resilin-like protein. The presence of such a protein likely enhances the attachment efficiency of the clamp sclerites and increases their lifetime.

Transmission X-ray microscopy of spider dragline silk.

We have investigated the structure of spider silk fibers from two different Nephila species and three different Araneus species by transmission X-ray microscopy (TXM). Single fibers and double fibers have been imaged. All images are in agreement with a homogenous density on length scales between the fiber diameter and the resolution of the instrument, which is about 25 nm.

Structural evolution of regenerated silk fibroin under shear: combined wide- and small-angle x-ray scattering experiments using synchrotron radiation.

The structural evolution of regenerated Bombyx mori silk fibroin during shearing with a Couette cell has been studied in situ by synchrotron radiation small- and wide-angle x-ray scattering techniques. An elongation of fibroin molecules was observed with increasing shear rate, followed by an aggregation phase. The aggregates were found to be amorphous with beta-conformation according to infrared spectroscopy. Scanning x-ray microdiffraction with a 5 microm beam on aggregated material, which had solidified in air, showed silk II reflections and a material with equatorial reflections close to the silk I structure reflections, but with strong differences in reflection intensities. This silk I type material shows up to two low-angle peaks suggesting the presence of water molecules that might be intercalated between hydrogen-bonded sheets.

The elasticity of single kettin molecules using a two-bead laser-tweezers assay.

Kettin is a high molecular mass protein of insect muscle associated with thin filaments and alpha-actinin in the Z-disc. It is thought to form a link between thin and thick filaments towards its C-terminus, contributing significantly to passive sarcomere stiffness. Here the elastic properties were characterised by mechanical stretches on an antibody-delimited region of the single molecule using two independent optical traps capable of exerting forces up to 150 pN. Step-like events were observed in the force-extension relationships consistent with the unfolding of Ig domains at moderate force and refolding of these domains at significantly higher forces than have been observed for related modular proteins.

Photophysical, photochemical, and thermodynamic properties of shikimic acid derivatives: calycin and rhizocarpic acid (lichens).

Photophysical and photochemical parameters of the lichen metabolites calycin and rhizocarpic acid were determined. Experiments were carried out in micellar solutions of 3% Brij 35, at pH 2 and 12, and in acetonitrile. Both metabolites absorb in the UV-A and UV-B regions, and emit fluorescence in the visible region of the solar spectrum. Shifts were not observed in the absorption spectra, at pH 2 and 12. The low phi(c), between 10(-5) and 10(-2), shows that both compounds are photostable in the experimental conditions. For rhizocarpic acid, two values of pK(a) were obtained: 5.1 corresponding to the hydroxyl group, and 9.0 corresponding to the protonated nitrogen. Calycin presents only one value of pK(a): 4.9, that is attributed to the hydroxyl group. L-(+)-Gluconic-gamma-lactonic acid was used as a reference model; the compound showed greater photoinstability, demonstrating that the photodegradation observed occurs mainly in the oxolane carbonylic ring.

The kinetics of radiation damage to the protein luciferase and recovery of enzyme activity after irradiation.

Experimental observations are reported which follow the bioluminescence intensity of luciferase during irradiation by a 5 MeV proton beam. Bioluminescence is a measure of the protein enzyme activity and provides an assay of the enzyme rate of reaction in real time. Transient responses after a pulse of protons show recovery of the reaction rate with two time constants of 0.3 s(-1) and 0.01 s(-1). Changes in the reaction rate are due to radiation damage to the active form of the protein luciferase. Quantitative analysis of the radiation damage and recovery of the protein shows that products of the radiolysis of water play major part in the process of enzyme damage at room temperature. A few minutes after the pulse of protons, most of the enzyme activity has recovered. We attribute the fast recovery to the removal of charged ions, while the slow recovery involves refolding of denatured protein.

[The interaction of the rad201 gene with mei-9 and mei-41 genes in germline cells of Drosophila female]. / Vzaimodeistvie gena rad201 s genami mei-9 i mei-41 v polovykh kletkakh samok drozofily.

The effect of Drosophila mutation rad201G1 together with mutations mei-41D5 and mei-9a on the sensitivity of oocytes to induction of dominant lethals (DLs) was studied. To this end, the frequencies of spontaneous and gamma-radiation-induced DLs in consecutive egg batches of females carrying double or single mutations were estimated. Since the effects of the mutations examined are expressed only at the previtellogenetic stages of oogenesis, only newly hatched (0-5-hour-old) females, whose oocytes did not develop farther than stage 7, were irradiated. The results obtained indicated that in intact and irradiated oocytes of double mutants mei-9a rad201G1 and mei-41D5 rad201G1, mutation rad201G1 epistatically suppresses the mutations of the both mei genes.

X-ray diffraction on spider silk during controlled extrusion under a synchrotron radiation X-ray beam.

The structure of a single thread of Nephila edulis silk has been studied by in situ X-ray diffraction from a living spider. A systematic increase of orientational order with increasing silking speed up to 40 mm s-1 was observed. Within a few mm from the spinnerets exit, crystalline domains with a beta-poly(L-alanine) structure were observed. The data also suggest an increase in crystalline fraction in the immediate vicinity of the spigot exit.

Observations on the effects of low frequency electromagnetic fields on cellular transcription in Drosophila larvae reared in field-free conditions.

Drosophila larvae reared inside a micro-metal box with an internal field strength 0.004 microT, were treated with a magnetic field of 50 Hz, 8 microT. for 20 min. Control experienced 0.004 microT. Cellular transcript levels were assessed using slot blots and quantified using a Phosphorimager. Blots were hybridised using probes against HSP 70a, Histone 1.9, and Copia. The low frequency EMFs very significantly decreased transcript levels, indicating that experimental responses may be influenced by previous exposure or lack of previous exposure.

Response of the timeless protein to light correlates with behavioral entrainment and suggests a nonvisual pathway for circadian photoreception.

The period (per) and timeless (tim) genes are required for circadian behavioral rhythms in Drosophila. The current model for how these rhythms entrain to light is based upon the light induced decrease in timeless protein (TIM) levels. We show here that the TIM response to light correlates with the effect of light on the behavioral rhythm. To identify components of the entrainment pathway, we also assayed the TIM response in flies with mutant visual systems. Flies that lacked eyes displayed a normal response in lateral neurons. The TIM response to a light pulse was attenuated in flies that were mutant for the transient receptor potential (trp) and trp-like (trpl) genes, which are required for calcium conductance in the visual transduction cascade. The reduced TIM response was accompanied by a reduced phase shift in the behavioral rhythm, but neither response was completely eliminated, and the trpl;trp flies entrain to light-dark cycles, suggesting that these genes perturb some aspect of circadian entrainment when mutated but are not essential for it. The TIM response was also unaffected in ninaE flies that lack the rhodopsin protein (rh1). These results support the hypothesis that circadian entrainment does not rely on the visual system and likely involves a dedicated pathway for photoreception.

Evidence that the TIM light response is relevant to light-induced phase shifts in Drosophila melanogaster.

Light is a major environmental signal for the entrainment of circadian rhythms. In Drosophila melanogaster, recent experiments suggest that photic information is transduced to the clock through the timeless gene product, TIM. We provide genetic and spectral evidence supporting the relevance of TIM light responses to clock resetting. A missense mutant TIM, TIM-SL, exhibits greater sensitivity to light in both TIM protein disappearance and locomotor activity phase shifting assays. We show that the wavelength dependence of light-induced decreases in TIM levels and that of light-mediated phase shifting are virtually identical. Analysis of dose response of TIM disappearance in a variety of mutant genotypes suggests cell-autonomous light responses that are largely independent of the canonical visual transduction pathway.

Differential effects of light and heat on the Drosophila circadian clock proteins PER and TIM.

Circadian (approximately 24-h) rhythms are governed by endogenous biochemical oscillators (clocks) that in a wide variety of organisms can be phase shifted (i.e., delayed or advanced) by brief exposure to light and changes in temperature. However, how changes in temperature reset circadian timekeeping mechanisms is not known. To begin to address this issue, we measured the effects of short-duration heat pulses on the protein and mRNA products from the Drosophila circadian clock genes period (per) and timeless (tim). Heat pulses at all times in a daily cycle elicited dramatic and rapid decreases in the levels of PER and TIM proteins. PER is sensitive to heat but not light, indicating that individual clock components can markedly differ in sensitivity to environmental stimuli. A similar resetting mechanism involving delays in the per-tim transcriptional-translational feedback loop likely underlies the observation that when heat and light signals are administered in the early night, they both evoke phase delays in behavioral rhythms. However, whereas previous studies showed that the light-induced degradation of TIM in the late night is accompanied by stable phase advances in the temporal regulation of the PER and TIM biochemical rhythms, the heat-induced degradation of PER and TIM at these times in a daily cycle results in little, if any, long-term perturbation in the cycles of these clock proteins. Rather, the initial heat-induced degradation of PER and TIM in the late night is followed by a transient and rapid increase in the speed of the PER-TIM temporal program. The net effect of these heat-induced changes results in an oscillatory mechanism with a steady-state phase similar to that of the unperturbed control situation. These findings can account for the lack of apparent steady-state shifts in Drosophila behavioral rhythms by heat pulses applied in the late night and strongly suggest that stimulus-induced changes in the speed of circadian clocks can contribute to phase-shifting responses.