Simultaneous coupling of phototaxis and electrotaxis in Volvox algae

In nature, living creatures are affected by several stimuli simultaneously. The response of living creatures to stimuli is called taxis. In order to reveal the principles of taxis behavior in response to complex stimuli, we simultaneously applied photostimulation and electric stimulation perpendicularly to a Volvox algae solution. The probability distribution of the swimming direction showed that a large population of swimming cells moved in a direction that was the result of the composition of phototaxis and electrotaxis. More surprisingly, we uncovered the coupling of signs of taxis, i.e., coupling of phototaxis and electrotaxis induced positive electrotaxis, which did not emerge in the single stimulation experiments. We qualitatively explained the coupling of taxis based on the polarization of the swimming cells induced by the simultaneous photo- and electric stimulation.

Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

Protein-protein interaction between phototaxis receptor sensory Rhodopsin I and its transducer HtrI

The molecular complex containing the seven transmembrane helix photoreceptor S&barbelow;ensory R&barbelow;hodopsin I&barbelow; (SRI) and transducer protein HtrI (H&barbelow;alobacterial Transducer for SRI&barbelow;) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) a repellent response by a two-photon reaction. Three aspects of SRI-HtrI structure/function and the signal transduction pathway were explored. First, the coupling of HtrI to the photoactive site of SRI was analyzed by mutagenesis and kinetic spectroscopy. Second, SRI-HtrI mutations and suppressors were selected and characterized to elucidate the color-sensing mechanism. Third, the signal relay through the transducer-bound histidine kinase was analyzed using an in vitro reconstitution system with known and newly identified taxis components. ^ Twenty-one mutations on HtrI were introduced by site-directed mutagenesis. Several replacements of charged residues perturbed the photochemical kinetics of SRI which led to the finding of a cluster of residues at the membrane/cytoplasm interface in HtrI electrostatically coupled to the photoactive site of SRI. We found by laser-flash kinetic spectroscopy that the transducer and these residues have specific effects on the light-induced proton transfer between the retinal chromophore and the protein. ^ One of the mutations showed an unusual mutant phenotype we called “inverted” signaling, in which the cell produces a repellent response to normally attractant light. Therefore, this mutant (E56Q of HtrI) had lost the color-discrimination by the SRI-HtrI complex. We used suppressor analysis to better understand the phenotype. Certain suppressors resulted in return of attractant responses to orange light but with inversion of the normally repellent response to white light to an attractant response. To explain this and other results, we formulated the Conformational Shuttling model in which the HtrI-SRI complex is poised in a metastable equilibrium of two conformations shifted in opposite directions by orange and white light. We tested this model by behavioral analysis (computerized cell tracking and motion study) of double mutants of inverting and suppressing mutations and the results confirmed the equilibrium-shift explanation. ^ We developed an in vitro system for measuring the effect of purified transducer on the histidine-kinase CheAH that controls the flagellar motor switch. The rate of kinase autophosphorylation was stimulated >2 fold in the reconstitution of the complete signal transduction system from purified components from H. salinarum. The in vitro assay also showed that the kinase activity was reduced in the absence and in the presence of high levels of linker protein CheWH. (Abstract shortened by UMI.) ^

Elevated CO2 affects embryonic development and larval phototaxis in a temperate marine fish

As an effect of anthropogenic CO2 emissions, the chemistry of the world's oceans is changing. Understanding how this will affect marine organisms and ecosystems are critical in predicting the impacts of this ongoing ocean acidification. Work on coral reef fishes has revealed dramatic effects of elevated oceanic CO2 on sensory responses and behavior. Such effects may be widespread but have almost exclusively been tested on tropical reef fishes. Here we test the effects elevated CO2 has on the reproduction and early life history stages of a temperate coastal goby with paternal care by allowing goby pairs to reproduce naturally in an aquarium with either elevated (ca 1400 µatm) CO2 or control seawater (ca 370 µatm CO2). Elevated CO2 did not affect the occurrence of spawning nor clutch size, but increased embryonic abnormalities and egg loss. Moreover, we found that elevated CO2 significantly affected the phototactic response of newly hatched larvae. Phototaxis is a vision-related fundamental behavior of many marine fishes, but has never before been tested in the context of ocean acidification. Our findings suggest that ocean acidification affects embryonic development and sensory responses in temperate fishes, with potentially important implications for fish recruitment.

Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base–Asp-73 interhelical salt bridge

Sensory rhodopsin II (SRII) is a repellent phototaxis receptor in the archaeon Halobacterium salinarum, similar to visual pigments in its seven-helix structure and linkage of retinal to the protein by a protonated Schiff base in helix G. Asp-73 in helix C is shown by spectroscopic analysis to be a counterion to the protonated Schiff base in the unphotolyzed SRII and to be the proton acceptor from the Schiff base during photoconversion to the receptor signaling state. Coexpression of the genes encoding mutated SRII with Asn substituted for Asp-73 (D73N) and the SRII transducer HtrII in H. salinarum cells results in a 3-fold higher swimming reversal frequency accompanied by demethylation of HtrII in the dark, showing that D73N SRII produces repellent signals in its unphotostimulated state. Analogous constitutive signaling has been shown to be produced by the similar neutral residue substitution of the Schiff base counterion and proton acceptor Glu-113 in human rod rhodopsin. The interpretation for both seven-helix receptors is that light activation of the wild-type protein is caused primarily by photoisomerization-induced transfer of the Schiff base proton on helix G to its primary carboxylate counterion on helix C. Therefore receptor activation by helix C–G salt-bridge disruption in the photoactive site is a general mechanism in retinylidene proteins spanning the vast evolutionary distance between archaea and humans.

Phototaxis away from blue light by an Escherichia coli mutant accumulating protoporphyrin IX.

The hemH gene of Escherichia coli encodes ferrochelatase (EC 4.99.1.1), the enzyme that catalyzes the last step in the production of heme, namely the synthesis of heme from protoporphyrin IX plus Fe2+. The behavioral responses to light were studied in E. coli carrying a hemH mutation. It was shown that the hemH mutant displayed a tumbling response upon illumination and a running response upon removal of the light. The most effect light to induce a tumbling response in the hemH mutant was blue light (396-450 nm). The chemotaxis machinery was needed for the light-induced tumbling response in the hemH mutant. The bacterial defect is an analog of the human inherited disease erythropoietic protoporphyria.

Residue replacements of buried aspartyl and related residues in sensory rhodopsin I: D201N produces inverted phototaxis signals.

Residue replacements were made at five positions (Arg-73, Asp-76, Tyr-87, Asp-106, and Asp-201) in the Halobacterium salinarium phototaxis receptor sensory rhodopsin I (SR-I) by site-specific mutagenesis. The sites were chosen for their correspondence in position to residues of functional importance in the homologous light-driven proton pump bacteriorhodopsin found in the same organism. This work identifies a residue in SR-I shown to be of vital importance to its attractant signaling function: Asp-201. The effect of the substitution with the isosteric asparagine is to convert the normally attractant signal of orange light stimulation to a repellent signal. In contrast, similar neutral substitution of the four other ionizable residues near the photoactive site allows essentially normal attractant and repellent phototaxis signaling. Wild-type two-photon repellent signaling by the receptor is intact in the Asp-201 mutant, genetically separating the wild-type attractant and repellent signal generation processes. A possible explanation and implications of the inverted signaling are discussed. Results of neutral residue substitution for Asp-76 confirm our previous evidence that proton transfer reactions involving this residue are not important to phototaxis but that Asp-76 functions as the Schiff base proton acceptor in proton translocation by transducer-free SR-I.

Cytological basis of photoresponsive behavior in a sponge larva

Ontogenetic changes in the photoresponse of larvae from the demosponge Reneira sp. were studied by analyzing the swimming paths of individual larvae exposed to diffuse white light. Larvae swam upward upon release from the adult, but were negatively phototactic until at least 12 hours after release. The larval photoreceptors are presumed to be a posterior ring of columnar monociliated epithelial cells that possess 120-mum-long cilia and pigment-filled protrusions. A sudden increase in light intensity caused these cilia to become rigidly straight. If the light intensity remained high, the cilia gradually bent over the pigmented vesicles in the adjacent cytoplasm, and thus covered one entire pole of the larva. The response was reversed upon a sudden decrease in light intensity. The ciliated cells were sensitive to changes in light intensity in larvae of all ages. This response is similar to the shadow response in tunicate larvae or the shading of the photoreceptor in Euglena and is postulated to allow the larvae to steer away from brighter light to darker areas, such as under coral rubble-the preferred site of the adult sponge on the reef flat. In the absence of a coordinating system in cellular sponges, the spatial organization and autonomous behavior of the pigmented posterior cells control the rapid responses to light shown by these larvae.

Orientation to light of juvenile and adult forms of melanic and albino populations of Biomphalaria glabrata (Say, 1818)

The process of light orientation by the snail Biomphalaria glabrata was studied using the selection technique in a Y-shaped aquarium under vertical or horizontal lighting schemes. Snail behavior was measured on the basis of distance (cm) covered per hour, direction of locomotion, and localization of the animal in the aquarium. A comparison was made of the action of the light stimulus on young and adult animals of albino populations from Santa Luzia (State of Minas Gerais, Brazil) and of a melanic population from Touros (State of Rio Grande do Norte) studied in groups and separately. All groups studied were attracted to light. Analysis of the data suggests the exitence of two orientation mechanisms with respect to light in these animals, i.e. high photo-orthokinesia and positive phototaxis, which influence their motion in the environment. This evidence permitted us to discuss features of the distribution dynamics of these mollusks in the environment and their relationship with the larval phases of Schistosoma mansoni, for which they act as intermediated hosts.

Biology and management of the masked chafer Cyclocephala distincta Burmeister (Melolonthidae, Dynastinae, Cyclocephalini)

Adults of Cyclocephala distincta are flower visitors of Neotropical palms (Arecaceae) and commonly found in the Atlantic Forest of Pernambuco, Brazil. Males and females were collected in the wild and subjected to captive rearing and breeding. The egg hatching rate, the life cycle, longevity of immatures and adults, and oviposition parameters in captivity were analyzed. The average duration of the life cycle of C. distinctawas 108.2 days (n = 45). The egg stage lasted on average 10.9 days, and the egg-hatching rate was 73.9%. The immature stage lasted on average 93.4 days. The larvae stage exhibited negative phototaxis, and the size of their head capsules increased at a constant rate of 1.6 between instars, following Dyar's rule. The average duration of the first instar was 24.8 days (n = 88), whereas the second and third instars lasted for 17.2 (n = 76) and 40.4 (n = 74) days respectively, and survival rates were 21.6%, 86.4% and 97.4%. The pre-pupal stage was recorded, and pupal chambers were built before pupation. The average number of eggs laid per female was 15.5, the total reproductive period lasted for 3.3 days, and the total fertility was 81.2%. Adults that emerged in captivity exhibited an average longevity of 18.9 days. Adult C. distincta exhibited thanatosis behavior upon manipulation, a strategy observed for the first time in Cyclocephala.

Biologia da broca do pedúnculo floral de coqueiro (Homalinotus coriaceus (Gyllenhal, 1836), coleoptera : curculionidae) em três dietas

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Sublethal effects of a common neonicotinoid pesticide, thiamethoxam, on honey bees: impact on locomotion and thermoregulation

Neonicotinoids have been pointed to as a factor responsible for the increased honey bee colony losses in the last decades. Many studies have investigated the effects of the first marketed neonicotinoid, imidacloprid, while fewer have focused on thiamethoxam. One recent study showed that sublethal doses of thiamethoxam lead to colony failure by decreasing forager homing flight success. We thus decided to investigate the mechanism which caused this phenomenon. Our hypothesis was that this effect was caused by impairment of forager locomotion abilities. Therefore we tested the effects of sublethal acute and chronic exposures to thiamethoxam on forager walking (Chapter 2) and flight (Chapter 3) performances. The acute treatment (1.34 ng/bee) affected walking locomotion firstly triggering hyperactivity (30 min post-treatment) and then impairing motor functioning (60 min post-treatment). 2-day continuous exposures to thiamethoxam (32.5, 45 ppb) elicited fewer effects on walking locomotion, however both exposure modes elicited an increased positive phototaxis. Similarly, in flight experiments, the single dose (1.34 ng/bee) elicited hyperactivity shortly after intoxication (increased flight duration and distance), while longer and continuous exposures (32.5, 45 ppb) impaired forager motor functions (decreased flight duration, distance, velocity). It is known that flight muscles temperature needs to be precisely regulated by bees during flight. Therefore, we further hypothesized that the impaired flight performances of neonicotinoid intoxicated bees were caused also by thermoregulation anomalies. We tested the effects that acute thiamethoxam exposures (0.2, 1, 2 ng/bee) elicit on forager thorax temperature (Chapter 4). Foragers treated with high doses exhibited hyperthermia or hypothermia when respectively exposed to high or low environmental temperatures. In summary, we show that sublethal doses of thiamethoxam affected forager walking and flight locomotion, phototaxis and thermoregulation. We also display the intricate mode of action of thiamethoxam which triggered, at different extents, inverse sublethal effects in relation to time and dose.

Different dark conformations function in color-sensitive photosignaling by the sensory rhodopsin I-HtrI complex.

The haloarchaeal phototaxis receptor sensory rhodopsin I (SRI) in complex with its transducer HtrI delivers an attractant signal from excitation with an orange photon and a repellent signal from a second near-UV photon excitation. Using a proteoliposome system with purified SRI in complex with its transducer HtrI, we identified by site-directed fluorescence labeling a site (Ser(155)) on SRI that is conformationally active in signal relay to HtrI. Using site-directed spin labeling of Ser(155)Cys with a nitroxide side chain, we detected a change in conformation following one-photon excitation such that the spin probe exhibits a splitting of the outer hyperfine extrema (2A'(zz)) significantly smaller than that of the electron paramagnetic resonance spectrum in the dark state. The dark conformations of five mutant complexes that do not discriminate between orange and near-UV excitation show shifts to lower or higher 2A'(zz) values correlated with the alterations in their motility behavior to one- and two-photon stimuli. These data are interpreted in terms of a model in which the dark complex is populated by two conformers in the wild type, one that inhibits the CheA kinase (A) and the other that activates it (R), shifted in the dark by mutations and shifted in the wild-type SRI-HtrI complex in opposite directions by one-photon and two-photon reactions.

Attractant and repellent signaling conformers of sensory rhodopsin-transducer complexes.

Attractant and repellent signaling conformers of the dual-signaling phototaxis receptor sensory rhodopsin I and its transducer subunit (SRI-HtrI) have recently been distinguished experimentally by the opposite connection of their retinylidene protonated Schiff bases to the outwardly located periplasmic side and inwardly located cytoplasmic side. Here we show that the pK(a) of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by approximately 1.5 units from that of the inwardly connected conformer. The pK(a) difference enables quantitative determination of the relative amounts of the two conformers in wild-type cells and behavioral mutants prior to photoexcitation, comparison of their absorption spectra, and determination of their relative signaling efficiency. We have shown that the one-photon excitation of the SRI-HtrI attractant conformer causes a Schiff base connectivity switch from inwardly connected to outwardly connected states in the attractant signaling photoreaction. Conversely, a second near-UV photon drives the complex back to the inwardly connected conformer in the repellent signaling photoreaction. The results suggest a model of the color-discriminating dual-signaling mechanism in which phototaxis responses (his-kinase modulation) result from the photointerconversion of the two oppositely connected SRI-HtrI conformers by one-photon and two-photon activation. Furthermore, we find that the related repellent phototaxis SRII-HtrII receptor complex has an outwardly connected retinylidene Schiff base like the repellent signaling forms of the SRI-HtrI complex, indicating the general applicability of macro conformational changes, which can be detected by the connectivity switch, to phototaxis signaling by sensory rhodopsin-transducer complexes.