The post-diapause vibrational behavior, motility, and survival of the brown marmorated stink bug Halyomorpha halys (Stål) adults at different temperatures.

Substrate-borne vibrational communication is common in pentatomids. Although several works exist on the vibrational communication of Halyomorpha halys, its vibrational behavior post diapause has not been investigated. In this study, we recorded H. halys overwintered adults using laser doppler vibrometers at three temperatures: 10 °C (inactivity), 18 °C (breaking of diapause), and 25 °C (peak of mating activity). The aim was to assess the effect of temperature on the signaling, motility, and survival of H. halys. The insects were sexed into different cages and recorded separately or joined with a cage of the opposite sex. We calculated the total time spent on signaling and walking per replica. The males predominantly emitted male signal 1 (MS1) throughout the four months of recordings. The females exclusively emitted female signal 2 (FS2) when joined with the opposite sex cage the first two months of recordings. Interestingly, they also started FS2 signaling when recorded separately, after two months. No signaling was recorded at 10 °C. At 25 °C, the signaling latency time before vibrational signaling was 24 h compared to 23 days at 18 °C. The short latency time at 25 °C correlated with a higher death rate in early stages of recording. Male walking activity was significantly higher in joined cages at 18 °C and 25 °C, suggesting the increased searching behavior near the opposite sex. Overwintered H. halys could adapt to different conditions whereas low temperatures maintain the diapause which is characterized by no signaling activity. Our results provide a foundation for bioclimatic modeling of climate change effects on H. halys and insights into the use of vibrational playbacks for mass trapping and monitoring as control techniques.

Assessing crosstalk in simultaneous multicolor single-molecule localization microscopy.

Single-molecule localization microscopy (SMLM) can reach sub-50 nm resolution using techniques such as stochastic optical reconstruction microscopy (STORM) or DNA-point accumulation for imaging in nanoscale topography (PAINT). Here we implement two approaches for faster multicolor SMLM by splitting the emitted fluorescence toward two cameras: simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) that images spectrally separated red and far-red imager strands on each camera, and spectral demixing dSTORM (SD-dSTORM) where spectrally close far-red fluorophores appear on both cameras before being identified by demixing. Using S2C-DNA-PAINT as a reference for low crosstalk, we evaluate SD-dSTORM crosstalk using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then assess if crosstalk can affect the detection of biologically relevant subdiffraction patterns. Extending these approaches to three-dimensional acquisition and SD-dSTORM to three-color imaging, we show that spectral demixing is an attractive option for robust and versatile multicolor SMLM investigations.

Effect of natural abiotic soil vibrations, rainfall and wind on anuran calling behavior: a test with captive-bred midwife toads (Alytes obstetricans).

Anurans are known to detect vibrations, but few studies explore relationships between vibrations and resultant behaviors. We studied the reaction of calling captive-bred male midwife toads (Alytes obstetricans) to the randomized playback of a vibrational crescendo stimulus train. We considered two sources of natural abiotic vibrational stimuli: rainfall and wind. Rainfall was expected to induce calling and wind was expected to inhibit it. Playback experiments with two synthetic tones (200 Hz and 300 Hz) tested the sensitivity to pure tones and could possibly reveal a hearing sensitivity trend between these frequencies. The toads did not increase call rate in response to rainfall vibrations and only one of the five wind stimulus levels caused a significant decrease in call rate. This limited response could be explained, because the tested toads came from a captive population, where emergence may not be mediated by rainfall vibrations. We found that A. obstetricans is highly sensitive to very low frequencies, which could explain the sensitivity observed to vibrational stimuli. Playback of a random crescendo stimulus train proves to be a valid approach for addressing behavioral questions. However, the use of a captive population may have been a limitation in the clarity of the results.

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy.

Fibronectin (Fn1) fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live-imaging and single-molecule localization microscopy data are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing six to eleven Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, in which nanodomains are spaced with an average periodicity of 105±17 nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. Treatment with FUD, a peptide that binds the Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocked the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis. This article has an associated First Person interview with the first author of the paper.

Effect of vibrational mating disruption on flight activity and oviposition to control the grapevine pest, Scaphoideustitanus.

The increasing demand for safe and sustainably produced food is leading to the development of strategies of pest control alternative to chemicals. One innovative method is Vibrational Mating Disruption (VMD) to disrupt insect communication in plants. VMD was proven effective in preventing mating of the grapevine pest Scaphoideus titanus, vector of flavescence dorée. However, the stress induced by VMD on the target species has the potential to influence other crucial aspects of the insect biology and ethology. Therefore, the goal of this study was to understand side effects of VMD on the flight activity and oviposition of S. titanus. The results of our experiments conducted in the greenhouse showed that in the presence of a receptive female, males fly more if exposed to vibrations than in the silent control but not differently from singles males in silence. Surprisingly, we found that also females subjected to VMD fly more than in the silence. Regarding oviposition, we found that mated females exposed to vibrations and single females (unmated) laid significantly fewer eggs than mated females in silence. In conclusion, this study shows the potential of VMD to interfere, besides with mating, with other important biological aspects of the pest species.

Design of ideal vibrational signals for stinkbug male attraction through vibrotaxis experiments.

BACKGROUND: Many groups of insects utilize substrate-borne vibrations for intraspecific communication. This characteristic makes them a suitable model for exploring the use of vibrations as a tool for pest control as an alternative to the use of chemicals. Detailed knowledge of species communication is a prerequisite to select the best signals to use. This study explored the use of substrate-borne vibrations for pest control of the brown marmorated stink bug (BMSB), Halyomorpha halys Stål (Heteroptera: Pentatomidae). For this purpose, we first identified the spectral and temporal characteristics that best elicit male responsiveness. Bioassays were conducted with artificial signals that mimicked the natural female calling signal. Second, we used the acquired knowledge to synthesize new signals endowed with different degrees of attractiveness in single- and two-choice bioassays using a wooden custom-made T stand. RESULTS: The results from this study showed that males were attracted to female signals along a high range of amplitudes, especially starting from a threshold of 100 µm s-1 , a high pulse repetition time (1 s) and frequency peak corresponding to the first harmonic (76 Hz). This resulted in an "optimal" signal for use to attract males, while the choice test in the T arena showed that this signal elicits searching behavior and attracts BMSB males towards a stimulation point. CONCLUSION: We confirm the use of vibrational signals as a strong tool for behavioral manipulation of male BMSB and suggest its possible use in the development of field traps and further management of this pest. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Intrasexual Vibrational Behavior of Philaenus spumarius in Semi-Field Conditions.

Insects that communicate by vibrational signals live in a complex interactive network of communication. Most studies on insect intrasexual behavior, based on plant-borne vibrational signals, have targeted few individuals. Despite their importance, behaviors that occur within groups were often overlooked. The study of multiple individuals, when insects occur in high density could simulate the environment in which they live and provide more reliable information on their behavior. In semi-field conditions, we investigated the intrasexual behavior of the meadow spittlebug, Philaenus spumarius. Vibrational signals exchanged among individuals of the same sex were recorded throughout their adult stage, from late spring to early autumn, and during the day, from the morning to the evening using a laser vibrometer. Males were less active than females throughout the season and their interactions were less frequent compared to females. Intrasexual interactions were characterized by signal overlapping in both unisex groups, in addition to signal alternating only in the case of males. In conclusion, the study of signaling behavior in intrasexual groups contributed to a better understanding of P. spumarius social behavior. We discuss the hypothesis of a possible competitive behavior between males and cooperative behavior between females.

The Oral Bacterial Community in Melanophryniscus admirabilis (Admirable Red-Belly Toads): Implications for Conservation.

Melanophryniscus admirabilis (admirable red-belly toad) is a microendemic and critically endangered species found exclusively along 700 m of the Forqueta River, in a fragment of the Atlantic Forest of southern Brazil. One of the greatest concerns regarding the conservation of this species is the extensive use of pesticides in areas surrounding their natural habitat. In recent years, the adaptation and persistence of animal species in human-impacted environments have been associated with microbiota. Therefore, the present study aimed to characterize the oral bacterial community of wild M. admirabilis and to address the question of how this community might contribute to this toad's adaptation in the anthropogenic environment as well as its general metabolic capabilities. A total of 11 oral samples collected from wild M. admirabilis were characterized and analyzed via high-throughput sequencing. Fragments of the 16S rRNA variable region 4 (V4) were amplified, and sequencing was conducted using an Ion Personal Genome Machine (PGM) System with 316 chips. A total of 181,350 sequences were obtained, resulting in 16 phyla, 34 classes, 39 orders, and 77 families. Proteobacteria dominated (53%) the oral microbiota of toads, followed by Firmicutes (18%), Bacteroidetes (17%), and Actinobacteria (5%). No significant differences in microbial community profile from among the samples were reported, which suggests that the low dietary diversity observed in this population may directly influence the bacterial composition. Inferences of microbiome function were performed using PICRUSt2 software. Important pathways (e.g., xenobiotic degradation pathways for pesticides and aromatic phenolic compounds) were detected, which suggests that the bacterial communities may serve important roles in M. admirabilis health and survival in the anthropogenic environment. Overall, our results have important implications for the conservation and management of this microendemic and critically endangered species.

Advertisement call of two threatened red-bellied-toads, Melanophryniscus cambaraensis and M. macrogranulosus (Anura: Bufonidae), from the Atlantic Rainforest, southern Brazil.

In anuran amphibians, acoustic signals are fundamental mechanisms of mate recognition and mate choice, which makes frog calls a fundamental tool for anuran taxonomy. In this work, we describe the advertisement call of two species for the genus Melanophryniscus, M. cambaraensis and M. macrogranulosus and use the descriptions to try to solve a taxonomic problem between them. We collected data after heavy rains in three different sample sites in Rio Grande do Sul, Brazil, between 2012 and 2013. The advertisement call of both species is composed of two segments. It always begins with part A (about 0.44-6 seconds) composed of single modulated pulses separated by long time intervals. It is followed by part B, a long train of unmodulated pulses with short time intervals, lasting from 9 to 32.2 seconds. Principal Component Analysis (PCA) indicated some variation between temporal parameters of the two species, but Multivariate Analysis of Variance showed no significant differences. Within-individual Coefficient of Variation (CV) showed only two static parameters: pulse rate and peak frequency, both in the part B of the call. Despite intra-male variation in some acoustic parameters, it is not possible to differentiate between M. cambaraensis and M. macrogranulosus species only using bioacoustics.

Myosin 1b flattens and prunes branched actin filaments.

Motile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins, including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Here, by performing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy, we show that this molecular motor flattens (reduces the branch angle) in the Arp2/3-dependent actin branches, resulting in them breaking, and reduces the probability of new branches forming. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and dynamics of actin networks in different cellular regions.This article has an associated First Person interview with the first author of the paper.

Anthropogenic substrate-borne vibrations impact anuran calling.

Anthropogenic disturbance is a major cause of the biodiversity crisis. Nevertheless, the role of anthropogenic substrate vibrations in disrupting animal behavior is poorly understood. Amphibians comprise the terrestrial vertebrates most sensitive to vibrations, and since communication is crucial to their survival and reproduction, they are a suitable model for investigating this timely subject. Playback tests were used to assess the effects of substrate vibrations produced by two sources of anthropogenic activity- road traffic and wind turbines- on the calling activity of a naïve population of terrestrial toads. In their natural habitat, a buried tactile sound transducer was used to emit simulated traffic and wind turbine vibrations, and changes in the toads' acoustic responses were analyzed by measuring parameters important for reproductive success: call rate, call duration and dominant frequency. Our results showed a significant call rate reduction by males of Alytes obstetricans in response to both seismic sources, whereas other parameters remained stable. Since females of several species prefer males with higher call rates, our results suggest that anthropogenically derived substrate-borne vibrations could reduce individual reproductive success. Our study demonstrates a clear negative effect of anthropogenic vibrations on anuran communication, and the urgent need for further investigation in this area.

Actin shells control buckling and wrinkling of biomembranes.

Global changes of cell shape under mechanical or osmotic external stresses are mostly controlled by the mechanics of the cortical actin cytoskeleton underlying the cell membrane. Some aspects of this process can be recapitulated in vitro on reconstituted actin-and-membrane systems. In this paper, we investigate how the mechanical properties of a branched actin network shell, polymerized at the surface of a liposome, control membrane shape when the volume is reduced. We observe a variety of membrane shapes depending on the actin thickness. Thin shells undergo buckling, characterized by a cup-shape deformation of the membrane that coincides with the one of the actin network. Thick shells produce membrane wrinkles, but do not deform their outer layer. For intermediate micrometer-thick shells, wrinkling of the membrane is observed, and the actin layer is slightly deformed. Confronting our experimental results with a theoretical description, we determine the transition between buckling and wrinkling, which depends on the thickness of the actin shell and the size of the liposome. We thus unveil the generic mechanism by which biomembranes are able to accommodate their shape against mechanical compression, through thickness adaptation of their cortical cytoskeleton.

MYO1C stabilizes actin and facilitates the arrival of transport carriers at the Golgi complex.

In this study, we aimed to identify the myosin motor proteins that control trafficking at the Golgi complex. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identified MYO1C as a novel player at the Golgi in a human cell line. We demonstrate that depletion of MYO1C induces Golgi complex fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi complex that colocalize with Golgi-associated actin dots. MYO1C depletion leads to loss of cellular F-actin, and Golgi complex decompaction is also observed after inhibition or loss of the actin-related protein 2/3 complex, Arp2/3 (also known as ARPC). We show that the functional consequence of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes actin at the Golgi complex, facilitating the arrival of incoming transport carriers at the Golgi.This article has an associated First Person interview with the first author of the paper.

Interplay between membrane tension and the actin cytoskeleton determines shape changes.

The ability of mammalian cells to deform their membrane relies on the action of the cytoskeleton. In particular, the dynamics of the actin cytoskeleton, assembling at the plasma membrane, plays a crucial role in controlling cell shape. Many proteins are involved to ensure proper growth of the actin network at the cell membrane. The detailed structure of this network regulates the force that is necessary for membrane deformation. We address here how the presence of capping proteins, which limit the length of actin filaments and thus affects network topology, influences membrane shape. We use a system of liposomes, activated to polymerize actin at their surface, and placed in a mixture of purified proteins that reconstitutes actin dynamics. Our system also allows the variation of membrane tension by deflating the liposomes. We show that membrane deformations are clearly favored in the presence of capping proteins in the actin network. Moreover, in the absence of capping proteins, membrane deformations appear only when the liposomes are deflated. Our results unveil that the interplay between membrane tension and actin network structure and dynamics governs cell shape.

Correction: Effects of traffic noise on the calling behavior of two Neotropical hylid frogs.

[This corrects the article DOI: 10.1371/journal.pone.0183342.].

Are the unken reflex and the aposematic colouration of Red-Bellied Toads efficient against bird predation?

Aposematic signals as well as body behaviours may be important anti-predator defences. Species of the genus Melanophryniscus are characterised by having toxic lipophilic alkaloids in the skin and for presenting a red ventral colouration, which can be observed when they perform the behaviour called the unken reflex. Both the reflex behaviour and the colouration pattern are described as defence mechanisms. However, there are currently no studies testing their effectiveness against predators. This study aimed to test experimentally if both ventral conspicuous colouration and the unken reflex in Melanophryniscus cambaraensis function as aposematic signals against visually oriented predators (birds). We simulated the species studied using three different clay toad models as follows: (a) in a normal position with green coloured bodies, (b) in the unken reflex position with green coloured body and extremities and (c) in the unken reflex position with a green body and red extremities. Models were distributed on a known M. cambaraensis breeding site and in the adjacent forest. More than half of the attacks on the models were from birds; however, there was no preference for any model type. Thus, just the presence of the red colour associated with the motionless unken reflex position does not seem to prevent attacks from potential predators. It is possible that the effective aposematic signal in Melanophryniscus is achieved through the unken reflex movement together with the subsequent exhibition of the warning colouration and the secretion of toxins.

Effects of traffic noise on the calling behavior of two Neotropical hylid frogs.

Anthropogenic disturbance has been pointed to as one of the major causes of the world´s biodiversity crisis. Among them, noise pollution is a potential underestimated threat, projected to increase in the next decades accompanying urban expansion. Rising levels of noise pollution may result in negative impacts on species highly dependent on acoustic communication. Amphibians have long served as model organisms for investigating animal acoustic communication because their reproduction depends on transmitting and receiving acoustic signals. A few studies have investigated the effects of anthropogenic noise on anurans, but there is still limited knowledge on how it affects them. In this study, we test the effect of two intensities of traffic noise on calling males of two Neotropical treefrogs species. We expect to record more changes in call parameters, to avoid masking effect, at higher intensity noise treatments, and in the species with higher call/noise frequency overlap. We performed a set of field playback experiments exposing male frogs to road noise at two different intensities (65dB and 75dB). Focal species are Boana bischoffi (high call/noise frequency overlap) and B. leptolineata (low call/noise frequency overlap). Both species changed acoustic parameters during or after the exposure to traffic noise. Advertisement call rate of B. bischoffi decreased during road noise, and dominant frequency decreased over time. Call length of B. leptolineata increased or decreased, depending on the order of noise intensity. We also observed spatial displacement in both species, which moved away from the noise source. Our results provide evidence that traffic noise affects anuran calling behavior, and noise intensity is an important factor affecting how species respond.

A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation.

Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic "compensation" and congestive "decompensation." Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae.

Myosin regulatory light chain (RLC) phosphorylation change as a modulator of cardiac muscle contraction in disease.

Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.