Proximate Mechanisms of Sexual Selection in the Firefly Photinus pyralis (Coleoptera: Lampyridae)

Male competition in courtships of the firefly Photinus pyralis is intense. Morphometric analysis revealed that male size is variable, possibly exhibiting alternative body plans. We examined the outcomes of courtships to determine the proximate mechanisms of selection that may have led to male allometry. Two distinct selective events exist within mate competition: (1) searching and (2) scramble competition. Although many courtships failed, those without rivals were shorter and successful males spent less time in the mate-guarding position than when several males were in competition. Winners outflashed rivals, but flash dialogs occurred at similar frequencies across rival densities, indicating that females require some quantum of information before mating. Female flash preference was examined in choice tests to evaluate female discrimination ability. Females preferred flashes of greater intensity and precedence. This suggests that flash synchronization is a competitive display rather than a cooperative behavior to maintain female responsiveness. Selection for male size was density dependent: females preferred large males in searching, while small males were favored in scrambles. Simulated scrambles showed that small males have superior locomotor abilities, allowing them to reach the female rapidly. Comparison with a species lacking scrambles, P. macdermotti, revealed that selection in P. pyralis is disruptive, targeting traits alternately, and may have led to the evolution of higher allometric ratios in a species with direct male competition. Both male competition and female choice are important determinants of the outcome of P. pyralis courtships.     

Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase

Firefly luciferase is a protein with a large N-terminal and a small C-terminal domain. B-factor analysis shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins have been shown that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on debate. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type although D476N doesn't have any significant difference. Intrinsic and ANS fluorescence studies demonstrate that D476N mutation is brought about by structural changes without significant effect on thermostability and flexibility. Molecular modeling reveals that disruption of a salt bridge between D(474) and K(445) accompanying with some H-bond deletion may be involved in destabilization of D474K mutant.     

Host condition and pathogen identity influence bacterial infection survival in the common eastern firefly,Photinus pyralis

1. The degree to which host, pathogen, and environmental characteristics determine fitness outcomes in response to infection across the tree of life is a key question in ecoimmunology. Because infection experiments require large sample sizes and careful controls, studies have generally been limited to lab-adapted organisms.
2. Photinus pyralis is a charismatic firefly that is abundant during its annual summer emergence in the Eastern United States. Here, we assess the importance of host condition, pathogen identity and dose, and pre-infection environment on survival outcomes in response to bacterial infection in wild-caught adult male P. pyralis.
3. Although survival after sterile saline injection was not statistically different from survival of uninjected controls, survival kinetics support sterile saline injection as the most appropriate control.
4. Serratia marcescens and Providencia rettgeri infections increased mortality, especially at high doses. In contrast to fruit fly studies, Providencia sneebia, Pseudomonas aeruginosa, and Enterococcus faecalis infections did not. Infection with higher doses of P. rettgeri had an increased risk of death for fireflies caught later in the season than those captured earlier, but seasonality did not significantly interact with dose to impact mortality with any of the other bacterial species.
5. These results highlight the importance of considering host, pathogen, and environmental factors when investigating infection outcomes in wild-caught, non-model systems. The differences in survival between fireflies and fruit flies emphasise that further studies in a broad array of organisms are needed to explore the diversity of infection responses across the tree of life.

     

Arachidonic and eicosapentaenoic acids in tissues of the firefly, Photinus pyralis (Insecta: Coleoptera)

We report on the presence of high proportions of arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) in the tissue lipids of adult fireflies, Photinus pyralis. Arachidonic acid typically occurs in very small proportions in phospholipids (PLs) of terrestrial insects, ranging from no more than traces to less than 1% of PL fatty acids, while 20:5n-3 is often missing entirely from insect lipids. Contrarily, 20:4n-6 made up approximately 21% of the PL fatty acids prepared from whole males and females, and from heads and thoraces prepared from males. Proportions of 20:4n-6 associated with PLs varied among tissues, including approximately 8% for male gut epithelia, 13% for testes, and approximately 25% for light organs and body fat from males. Substantial proportions of 20:5n-3 were also associated with PLs prepared from male firefly tissues, including 5% for body fat and 8% for light organs. Because 20:4n-6 and 20:5n-3 are precursors for biosynthesis of prostaglandins and other eicosanoids, we considered the possibility that firefly tissues might produce eicosanoids at exceptionally high rates. Preliminary experiments indicated otherwise. Hence, fireflies are peculiar among terrestrial insects with respect to maintaining high proportions of PL 20:4n-6 and 20:5n-3.     

ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi‐rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an “omega‐loop” motif (T352‐G360) significantly enhanced activity, altered kinetics, reduced Km for D‐luciferin_, altered emission colors, and altered substrate specificity for redshifted analog DL‐infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω‐loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox.     

The expression of the Photinus pyralis luciferase gene in Staphylococcus aureus Cowan I allows the development of a live amplifiable tool for immunodetection.

We expressed the luc gene, encoding luciferase from Photinus pyralis, in Staphylococcus aureus Cowan I downstream of the plasmid-borne promoter for protein A. Constitutive luciferase synthesis did not impair the growth rate of the host nor did it affect the stability of the plasmid. Light production started immediately after addition of luciferin. The kinetic profile is of the glowing rather than the peak type. Because S. aureus Cowan I produces large quantities of protein A, of which a substantial part becomes covalently attached to rigid cell walls, the bacterial cells could be specifically immobilized on a substrate to which immunoglobulin G molecules were adsorbed either directly or as secondary antibodies. Light production from these cells can be used as a reporter tool for the detection of antigen-antibody complexes. Fourfold amplifications of the emitted signals were obtained by in situ incubation of the bound cells in bacterial growth medium.     

Action spectra of the female's response in the firefly Photinus pyralis (Coleoptera: Lampyridae): evidence for an achromatic detection of the bioluminescent optical signal

Behavioural action spectra of the threshold of the Photinus pyralis female response to light stimuli simulating the bioluminescent optical signal of the conspecific male firefly were determined in the laboratory. The action spectra (Fig. 1) were narrow and peaked in the yellow region of the spectrum. The females responded only to stimuli of wavelengths longer than 480 nm and not to stimuli in the blue (420-460 nm) part of the spectrum. The shape of the function corresponds with (a) the electroretinographic spectral sensitivity function in the long wavelength (520-660 nm) region of the spectrum, (b) the action spectrum of the female response (Fig. 1), (c) the species yellow bioluminescence emission spectrum and (d) the action spectrum of the intracellular response from single retinular cells (Fig. 2) of the compound eyes in the firefly. Such a correspondence suggests that the narrow yellow receptors of the female mediate the detection and processing of the optical signal of the conspecific male. Since the bioluminescent optical signal is processed exclusively by a single receptor class, signal detection is achromatic.     

Photic signaling by cryptochrome in the Drosophila circadian system

Oscillations of the period (per) and timeless (tim) gene products are an integral part of the feedback loop that underlies circadian behavioral rhythms in Drosophila melanogaster. Resetting this loop in response to light requires the putative circadian photoreceptor cryptochrome (CRY). We dissected the early events in photic resetting by determining the mechanisms underlying the CRY response to light and by investigating the relationship between CRY and the light-induced ubiquitination of the TIM protein. In response to light, CRY is degraded by the proteasome through a mechanism that requires electron transport. Various CRY mutant proteins are not degraded, and this suggests that an intramolecular conversion is required for this light response. Light-induced TIM ubiquitination precedes CRY degradation and is increased when electron transport is blocked. Thus, inhibition of electron transport may "lock" CRY in an active state by preventing signaling required either to degrade CRY or to convert it to an inactive form. High levels of CRY block TIM ubiquitination, suggesting a mechanism by which light-driven changes in CRY could control TIM ubiquitination.     

Time-Resolved Structural Studies on Insect Flight Muscle after Photolysis of Caged-ATP

The time course of structural changes occurring on ATP-induced relaxation of glycerinated insect flight muscle from the rigor state has been investigated using synchrotron radiation as a source of high intensity x rays and photolysis of caged-ATP to produce a rapid rise in ATP concentration. Temporal resolutions of 1 ms for the strongest equatorial reflections and 5 ms for the 14.5 nm meridional reflection are attainable from single events (i.e., without averaging over several cycles). The equatorial intensity changes completely, the meridional intensity partially, towards their respective relaxed values on a much faster time scale than relaxation of tension. The results suggest that actively cycling bridges present shortly after ATP-release are either too few in number to be detected in the equatorial diffraction pattern or that their structure is different from that of rigor bridges.     

Firefly luciferase exhibits bimodal action depending on the luciferin chirality

Firefly luciferase is able to convert L-luciferin into luciferyl-CoA even under ordinary aerobic luciferin-luciferase reaction conditions. The luciferase is able to recognize strictly the chirality of the luciferin structure, serving as the acyl-CoA synthetase for L-luciferin, whereas d-luciferin is used for the bioluminescence reaction. D-Luciferin inhibits the luciferyl-CoA synthetase activity of L-luciferin, whereas L-luciferin retards the bioluminescence reaction of D-luciferin, meaning that both enzyme activities are prevented by the enantiomer of its own substrate.     

空间诱变高粱突变体的研究

1996年经4返回式卫星搭载处理的纯系高粱品种晋粮5号（CK）的种子经次年播种生,区得少量秆早熟突变体（SP3）。此后连续二年播种,该突变体矮秆早熟性状稳定,该突变体与未经搭载的对照相比多400粒。（3）SP3平均千粒重为34g,对照为27g。（4）SP3叶片变窄,变短,增厚,叶面积减少了43％－22％。（5）SP3穗轴长度比对照增加30％,各节间长度比对照缩短了67％－15％。（6）SP3种子中亮氨酸含量比对照增加15％,可溶性糖含量比对照增加25％,单宁含量降低了30％。     

空间诱变高粱突变体的研究

1996年经返回式卫星搭载处理的纯系高粱品种晋粮5号（CK）的种子经次年播种后,获得少量矮秆早熟突变体（SP3）。此后连续二年播种,该突变体矮秆早熟性状稳定。该突变体与未经搭载的对照相比,有如下不同之处：（1）成熟期缩短15 d左右,株高降低40 cm。（2）穗长增加3.9 cm ,每穗籽粒数比对照多400粒。（3）SP3平均千粒重为34 g,对照为27 g。（4）SP3叶片变窄,变短,增厚,叶面积减少了43%～22%。（5）SP3穗轴长度比对照增加30%,各节间长度比对照缩短了67%～15%。（6）SP3种子中亮氨酸含量比对照增加15%,可溶性糖含量比对照增加25%,单宁含量降低了30%。     

Studies on the Flight Mechanism of Insects : I. The electrophysiology of fibrillar flight muscle

Fibrillar type flight muscle powers the flight machinery of the more phylogenetically advanced groups of flying insects. A comparison of responses from single fibers in insects from various orders having fibrillar muscle reveals fundamental differences. In single fibers of flies and wasps the response to a single threshold stimulus is an all-or-none, uniformly rising, in most cases overshooting action potential. Beetles give variable responses, some of which appear similar to the type mentioned above, and others which summate and facilitate. Some of the latter responses vary with time in a cyclic manner, and some are altered by the intensity of the stimulus. Further differences appear when the two types of muscle are exposed to ether and carbon dioxide. In the wasp and fly ether produces a neuromuscular block, while CO₂ effects a rapid depolarization of the resting fiber membrane. Both reactions are completely reversible. The electrical responses of beetle muscle are somewhat affected but only by massive doses. The implications of these data are discussed relative to the existence of fibrillar muscle "types."     

Effects of Prolonged Exposure to Darkness on Circadian Photic Responsiveness in the Mouse

Circadian rhythms in mammals are generated by an endogenous pacemaker but are modulated by environmental cycles, principally the alternation of light and darkness. Although much is known about nonparametric effects of light on the circadian system, little is known about other effects of photic stimulation. In the present study, which consists of a series of five experiments in mice, various manipulations of photic stimulation were used to dissect the mechanisms responsible for a variation in the magnitude of light-induced phase-shifts that results from prolonged exposure to darkness. The results confirmed previous observations that prolonged exposure to darkness causes an increase in the magnitude of phase shifts (both phase advances and phase delays) evoked by discrete light pulses. The results also indicated that the increase in responsiveness results from the lack of exposure to light per se and not from collateral effects of exposure to constant darkness such as the lack of previous entrainment. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system but with a much slower temporal course. The results suggest that circadian dark adaptation may take place at the retinal level, but it is not clear whether it involves a change in the sensitivity or maximal responsiveness of the system.     

Flight Performance and Dispersal Potential of Red Palm Weevil Estimated by Repeated Flights on Flight Mill

The red palm weevil (Rhynchophorus ferrugineus; Olivier, 1790) (Coleoptera: Curculionidae) has recently become the most severe palm pest in the Mediterranean basin. Its dispersal was initially supported mainly by the acquisition of infested trees, but was further facilitated by the weevils’ flight. Therefore, knowledge of weevils’ flight capacity is a key element in evaluating their dispersal capability and setting preventive actions in advance. We tested the weevils’ flight ability in repeated flights that were 7–10 days apart by computer-monitored flight mill with a seesaw design. Tested flight parameters were: flight distance, duration, and velocity, number of flights, and cumulative flight distance, of virgin and mated weevils of both sexes. Our tests found no differences in flight distance between virgin and mated individuals or between sexes. Weevils showed flight capability between the ages of 2 and 97 days, and covered up to 315 km of cumulative distance during this time. In addition, we tested the effect of age of flight initiation and found that old starters perform fewer flights than young starters, and are thus assumed to possess inferior dispersal capabilities.     

Photic entrainment of circadian rhythms by illumination of implanted brain tissues in the cockroach Blaberus craniifer

The locomotor activity rhythms of ‘blinded’ Blaberus craniifer adults receiving transplants of either an entire brain or both optic lobes of a donor B. craniifer were studied in contrast to the activity rhythms of surgical control and untreated insects. When the implants were located in the abdomen of a ‘host’ insect beneath a small glass ‘window’ and exposed to a 12L : 12D photoperiod entrainment occurred and the activity of such ‘host’ insects receiving either type of implant was comparable to that of control insects. The results provide some substantiating evidence that locomotor activity rhythms may be controlled by a hormonal clock.