Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (>200 µm), localization precision of 2-3 nm can be achieved.     

Measuring plasma fatty acid oxidation with intravenous bolus injection of 3H- and 14C-fatty acid

Accurate measures of plasma FA oxidation can improve our understanding of diseases characterized by impaired FA oxidation. We describe and compare the 24 h time-courses of FA oxidation using bolus injections of [1-¹⁴C]palmitate versus [9,10-³H]palmitate under postabsorptive, postprandial, and walking conditions. Fifty-one men and 95 premenopausal women participated in one condition (postabsorptive, postprandial, or walking), one tracer (¹⁴C- or ³H-labeled), and an acetate or palmitate study. Groups were matched for sex, age, and body mass index (BMI). At 24 h, cumulative [³H]acetate recovery as ³H₂O was 80 ± 6%, 78 ± 2%, and 81 ± 6% in the postabsorptive, postprandial, and walking conditions, respectively (not significant). Model-predicted maximum [1-¹⁴C]acetate recovery as expired ¹⁴CO₂ was 59 ± 12%, 52 ± 8%, and 65 ± 10% in the postabsorptive, postprandial, and walking condition, respectively (one way ANOVA, P = 0.12). When corrected with the corresponding acetate recovery factors, 24 h time-courses of FFA oxidation were similar between [1-¹⁴C]palmitate and [9,10-³H]palmitate in all three conditions. In contrast to previous meal ingestion studies, an acetate-hydrogen recovery factor was needed to achieve comparable oxidation rates using an intravenous bolus of [³H]palmitate. In conclusion, intravenous boluses of [9,10-³H]palmitate versus [1-¹⁴C]palmitate gave similar estimates of 24 h cumulative FFA oxidation in age-, sex- and BMI-matched individuals.     

Foraging Behavior of the Parasitoid Encarsia formosa on Gerbera jamesonii Leaves

Insight into the foraging behavior of the parasitoid Encarsia formosa Gahan for whitefly hosts was gained by continuous observation of individual parasitoids on leaves of the ornamental plant Gerbera jamesonii, until females left the leaf. Comparison of the parasitoid behavior on three cultivars gave similar results. Mean searching time on uninfested G. jamesonii leaves of three cultivars was 1 h 30 min and the mean percentage of walking activity of the total observation time on those cultivars was 61%. Both parameters were not influenced by different leaf structures of Gerbera cultivars. Encounters with hosts arrested the parasitoids on the leaves. The walking activity and the percentage of host encounters that resulted in an oviposition decreased with decreasing egg load of the parasitoid. In comparison with tomato, where biological control of whiteflies is successful, only minor differences in the foraging behavior occur, except for the residence time of females, which was about three to four times longer on G. jamesonii leaves, but these leaves are about seven times larger than tomato leaves. The facts that (1) the foraging behavior of E. formosa on G. jamesonii is independant of the cultivar and (2) the foraging behavior is, in many aspects, similar to that on tomato suggest that biological control of whitefly on this ornamental plant is a potential option.     

The effect of uphill and downhill walking on gait parameters: A self-paced treadmill study

It has been shown that gait parameters vary systematically with the slope of the surface when walking uphill (UH) or downhill (DH) (Andriacchi et al., 1977; Crowe et al., 1996; Kawamura et al., 1991; Kirtley et al., 1985; McIntosh et al., 2006; Sun et al., 1996). However, gait trials performed on inclined surfaces have been subject to certain technical limitations including using fixed speed treadmills (TMs) or, alternatively, sampling only a few gait cycles on inclined ramps. Further, prior work has not analyzed upper body kinematics. This study aims to investigate effects of slope on gait parameters using a self-paced TM (SPTM) which facilitates more natural walking, including measuring upper body kinematics and gait coordination parameters.Gait of 11 young healthy participants was sampled during walking in steady state speed. Measurements were made at slopes of +10°, 0° and −10°. Force plates and a motion capture system were used to reconstruct twenty spatiotemporal gait parameters. For validation, previously described parameters were compared with the literature, and novel parameters measuring upper body kinematics and bilateral gait coordination were also analyzed.Results showed that most lower and upper body gait parameters were affected by walking slope angle. Specifically, UH walking had a higher impact on gait kinematics than DH walking. However, gait coordination parameters were not affected by walking slope, suggesting that gait asymmetry, left-right coordination and gait variability are robust characteristics of walking. The findings of the study are discussed in reference to a potential combined effect of slope and gait speed. Follow-up studies are needed to explore the relative effects of each of these factors.     

Sixteen EST-linked microsatellite markers in Günther's walking catfish, Clarias macrocephalus

Twenty-seven new microsatellite sequences were identified by screening 2029 expressed sequence tags from Günther's walking catfish, Clarias macrocephalus. Sixteen loci were polymorphic with the number of alleles ranging from two to 16 per locus and the observed and expected heterozygosities ranging from 0.4667 to 0.9333 and from 0.427 to 0.8819 per locus, respectively. Cross-species amplifications of all 16 primer pairs were tested in four other species of catfish including Clarias gariepinus, Pangasius hypophthalmus, Pangasius larnaudii and Pangasianodon gigas. Eleven loci were found to amplify in other species, with the number of polymorphic loci ranging from one in P. larnaudii to nine in C. gariepinus.     

Toxic temperatures: Bee behaviours exhibit divergent pesticide toxicity relationships with warming

Climate change and agricultural intensification are exposing insect pollinators to temperature extremes and increasing pesticide usage. Yet, we lack good quantification of how temperature modulates the sublethal effects of pesticides on behaviours vital for fitness and pollination performance. Consequently, we are uncertain if warming decreases or increases the severity of different pesticide impacts, and whether separate behaviours vary in the direction of response. Quantifying these interactive effects is vital in forecasting pesticide risk across climate regions and informing pesticide application strategies and pollinator conservation. This multi-stressor study investigated the responses of six functional behaviours of bumblebees when exposed to either a neonicotinoid (imidacloprid) or a sulfoximine (sulfoxaflor) across a standardised low, mid, and high temperature. We found the neonicotinoid had a significant effect on five of the six behaviours, with a greater effect at the lower temperature(s) when measuring responsiveness, the likelihood of movement, walking rate, and food consumption rate. In contrast, the neonicotinoid had a greater impact on flight distance at the higher temperature. Our findings show that different organismal functions can exhibit divergent thermal responses, with some pesticide-affected behaviours showing greater impact as temperatures dropped, and others as temperatures rose. We must therefore account for environmental context when determining pesticide risk. Moreover, we found evidence of synergistic effects, with just a 3°C increase causing a sudden drop in flight performance, despite seeing no effect of pesticide at the two lower temperatures. Our findings highlight the importance of multi-stressor studies to quantify threats to insects, which will help to improve dynamic evaluations of population tipping points and spatiotemporal risks to biodiversity across different climate regions.     

Octopamine effects mimick state-dependent changes in a proprioceptive feedback system

The modulatory actions of the biogenic amine octopamine on the femur tibia (FT) control loop in the stick insect Carausius morosus were examined. The response properties of the FT control loop were determined under open loop conditions. Mechanical stimulation of the femoral chordotonal organ (fCO) was the input and tibial movement and motoneuronal activity were measured as the output of the system. Following octopamine injection into the hemolymph of intact, inactive animals, two consecutive phases occurred at the behavioral level. Octopamine caused initially an activation of the animal. During this first phase (3.5–12 min duration) the response properties of the FT control loop were similar to those found in animals that were activated by tactile stimuli under normal conditions. Afterward, animals became inactive. During this second phase (15–20 min duration), the gain of the control loop was zero and no resistance reflex in the FT joint was generated in response to fCO stimulation. However, active movements of the tibia could still be elicited. As we could show in restrained animals, where dl-octopamine was applied topically onto the undesheated mesothoracic ganglion, the complete suppression of the resistance reflex on the motoneuronal level was dose dependent starting at concentrations of 5 ± 10^?3 M octopamine. We could show that octopamine specifically suppressed the pathways involved in the resistance reflex, while feedback loop responses to fCO stimuli typical for active animals could still be elicited. Our results indicate that an increase in the octopamine concentration mimicks activation of the animal: Properties being characteristic for the control of the FT joint in the inactive animal are inhibited by octopamine, while properties of the FT control loop typical for the active animal appear to be facilitated following octopamine injection. The results clearly demonstrate that different pathways in the neuronal network underlying the FT control loop are involved in the responses of the control loop to fCO stimuli in the inactive and active behavioral states of the stick insect. © 1993 John Wiley & Sons, Inc.     

Walking slope and heavy backpack loads affect torso muscle activity and kinematics

The independent effects of sloped walking or carrying a heavy backpack on posture and torso muscle activations have been reported. While the combined effects of sloped walking and backpack loads are known to be physically demanding, how back and abdominal muscles adapt to walking on slopes with heavy load is unclear. This study quantified three-dimensional pelvis and torso kinematics and muscle activity from longissimus, iliocostalis, rectus abdominis, and external oblique during walking on 0° and ± 10° degree slopes with and without backpack loads using two different backpack configurations (hip-belt assisted and shoulder-borne). Iliocostalis activity was greater during downhill and uphill compared to level walking, but longissimus was only greater during uphill. Rectus abdominis activity was greater during downhill and uphill compared to level, while external oblique activity decreased as slopes progressed from down to up. Longissimus, but not iliocostalis, activity was reduced during both backpack configurations compared to walking with no pack. Hip-belt assisted load carriage required less rectus abdominis activity compared to using shoulder-borne only backpacks; however, external oblique was not influenced by backpack condition. Our results revealed different responses between iliocostalis and longissimus, and between rectus abdominis and external obliques, suggesting different motor control strategies between anatomical planes.     

A chromosomal walk in the region of polytene bands 7C-D of theDrosophila X chromosome

A chromosomal walk on the X chromosome ofDrosophila in the region of polytene bands 7C1 to 7D5 is described. The region is of interest since three olfactory genes have been found to map here in addition to a haplo-inviable locus. Genomic clones spanning 160 kilobases have been isolated and their complete restriction map is presented. The clones have been aligned on the polytene chromosome bands byin situ hybridisation. In addition the end-points of a deficiency and duplication lying in this region have been mapped approximately, showing that an overlap exists between them.