Megumi Fuse

Structure-activity relationship of ETH during ecdysis in the tobacco hornworm, Manduca sexta.

In insects, ecdysis or shedding of the old cuticle, consists of a series of behaviors that are regulated by the coordinated actions of a number of neuropeptides, one of which is ecdysis triggering hormone (ETH). ETH acts directly on central pattern generators of the abdominal ganglia to trigger onset of pre-ecdysis behaviors, as well as indirectly to activate release of eclosion hormone, thereby inducing onset of ecdysis behaviors through a cGMP-mediated mechanism. We assessed the minimal C-terminal amino acids required for biological activity of ETH, by assessing: (i) onset of pre-ecdysis and ecdysis behaviors in vivo, after injection of peptide analogs, (ii) onset of fictive pre-ecdysis and ecdysis motor patterns in vitro, as recorded extracellularly, after incubation of the CNS with the peptide analogs, and (iii) accumulation of cGMP within cells of the abdominal ganglia, as assessed immunohistochemically. Amidation of ETH at the C-terminus was required to elicit a biological response in vivo and in vitro, as well as an accumulation of cGMP within the CNS. The five amino acid amidated C-terminus of ETH (NIPRMamide) was the minimal moiety able to induce a robust pre-ecdysis response in vivo and in vitro, while a seven amino acid core (NKNIPRMa) was required for induction of ecdysis, including accumulation of cGMP immunoreactivity within the CNS. Analogs smaller than 12 amino acids in length were only active at very high concentrations in vivo, suggesting that smaller fragments might be susceptible to hemolymph degradation. Some alanine substitutions or removal of internal amino acids altered the activity of ETH, as well as the time of onset of ecdysis behaviors, suggesting that internal amino acids play a role in maintaining proper folding of the peptide for successful binding or activity at the ETH receptor.

Use of von Frey filaments to assess nociceptive sensitization in the hornworm, Manduca sexta.

BACKGROUND The hornworm Manduca sexta exhibits a defensive strike to noxious assaults, a response that is robust and is easily observed by experimenters. Von Frey filaments and methods typical for studying nociception in other animals were used to assess the strike response in M. sexta. NEW METHODS A series of von Frey filaments was applied to the body wall in ascending order and the data generated were used to determine the strike threshold by (i) the up-and-down method, (ii) the first response method, and (iii) the simplified up-and-down order method (SUDO). The effect of a noxious pinch on strike threshold was assessed. COMPARISON WITH EXISTING METHODS To our knowledge none of these methods has been used on M. sexta previously, making the use of the up-and-down and SUDO methods the first in an invertebrate. The use of the first response method has been used in other invertebrates, and the method appears equally suited to M. sexta. RESULTS All three methods were successful in monitoring the threshold sensitivity to touch, which was lowered (sensitized) by tissue damage induced with a pinch. Sensitization lasted 19h. CONCLUSIONS All three methods of assessing nociception were successfully applied to quantify the defensive strike response in M. sexta, although the SUDO method required empirical assessment of which filament to start the test sequence with. The results revealed both short- and long-term sensitization. These methods should prove to be useful for quantifying sensitization in M. sexta.

Classroom sound can be used to classify teaching practices in college science courses

Significance Although the United States needs to expand its STEM (science, technology, engineering, mathematics) workforce, United States postsecondary institutions struggle to retain and effectively teach students in STEM disciplines. Using teaching techniques beyond lecture, such as pair discussions and reflective writing, has been shown to boost student learning, but it is unknown what proportion of STEM faculty use these active-learning pedagogies. Here we describe DART: Decibel Analysis for Research in Teaching, a machine-learning–derived algorithm that analyzes classroom sound to predict with high accuracy the learning activities used in classrooms, and its application to thousands of class session recordings. DART can be used for large-scale examinations of STEM teaching practices, evaluating the extent to which educators maximize opportunities for effective STEM learning. Active-learning pedagogies have been repeatedly demonstrated to produce superior learning gains with large effect sizes compared with lecture-based pedagogies. Shifting large numbers of college science, technology, engineering, and mathematics (STEM) faculty to include any active learning in their teaching may retain and more effectively educate far more students than having a few faculty completely transform their teaching, but the extent to which STEM faculty are changing their teaching methods is unclear. Here, we describe the development and application of the machine-learning–derived algorithm Decibel Analysis for Research in Teaching (DART), which can analyze thousands of hours of STEM course audio recordings quickly, with minimal costs, and without need for human observers. DART analyzes the volume and variance of classroom recordings to predict the quantity of time spent on single voice (e.g., lecture), multiple voice (e.g., pair discussion), and no voice (e.g., clicker question thinking) activities. Applying DART to 1,486 recordings of class sessions from 67 courses, a total of 1,720 h of audio, revealed varied patterns of lecture (single voice) and nonlecture activity (multiple and no voice) use. We also found that there was significantly more use of multiple and no voice strategies in courses for STEM majors compared with courses for non-STEM majors, indicating that DART can be used to compare teaching strategies in different types of courses. Therefore, DART has the potential to systematically inventory the presence of active learning with ∼90% accuracy across thousands of courses in diverse settings with minimal effort.

Different actions of ecdysis-triggering hormone on the brain and ventral nerve cord of the hornworm, Manduca sexta.

Ecdysis, or the shedding of the old cuticle, depends on coordinated stereotyped behaviors, regulated by a number of neuropeptides. In the hornworm, Manduca sexta, two neuropeptides interact, namely ecdysis-triggering hormone (ETH) and eclosion hormone. We looked at the effects of ETH in vivo and in vitro, on the brain and the ventral nerve cord to determine the roles played by these hormones. We monitored ecdysis onset and the presence of cGMP and eclosion hormone immunoreactivity. In vivo, only a fraction of larvae lacking the cell bodies containing eclosion hormone, and injected with ETH, were able to undergo ecdysis, with a delayed response. These animals showed strongest cGMP immunoreactivity in the subesophageal and thoracic ganglia, with concomitant reductions in eclosion hormone immunoreactivity in descending axons in comparison with animals not undergoing ecdysis. Animals lacking the brain showed reduced to no cGMP levels in all ganglia. In vitro, isolated CNS preparations lacking the brain initiated ecdysis motor programs after incubation in ETH, with faster onset times than controls, and with reduced cGMP immunoreactivity. If ETH was applied only to the brain of the isolated CNS, cGMP immunoreactivity was noted primarily in the subesophageal and thoracic ganglia, with a decrease in eclosion hormone immunoreactivity in descending axons. ETH addition to the rest of the nerve cord showed reduced eclosion hormone immunoreactivity but little to no cGMP immunoreactivity in any ganglion. Controls showed strong cGMP immunoreactivity in all ganglia, and even greater reductions in eclosion hormone staining after ETH application. These results support previous suggestions that eclosion hormone is required for a positive feedback loop with ETH as well as onset of an inhibitory component, but also suggest that ETH stimulates eclosion hormone release at multiple spike initiation zones. The resultant up regulation of cGMP does not appear to be required for onset of ecdysis. A new model for ecdysis regulation is considered.

Collectively Improving Our Teaching: Attempting Biology Department–wide Professional Development in Scientific Teaching

A collaborative professional development program that engaged nearly 90% of faculty in a biology department in more than 40 hours of training on scientific teaching was instituted. Participating instructors integrated active learning in their courses, as shown through a variety of methods, and reported positive effects on teaching and departmental community.

Central Neural Alterations Predominate in an Insect Model of Nociceptive Sensitization

Many organisms respond to noxious stimuli with defensive maneuvers. This is noted in the hornworm, Manduca sexta, as a defensive strike response. After tissue damage, organisms typically display sensitized responses to both noxious or normally innocuous stimuli. To further understand this phenomenon, we used novel in situ and in vitro preparations based on paired extracellular nerve recordings and videography to identify central and peripheral nerves responsible for nociception and sensitization of the defensive behavior in M. sexta. In addition, we used the in vivo defensive strike response threshold assayed with von Frey filaments to examine the roles that N‐methyl‐D‐aspartate receptor (NMDAR) and hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels play in this nociceptive sensitization using the inhibitors MK‐801 and AP5 (NMDAR), and ivabradine and ZD7288 (HCN). Using our new preparations, we found that afferent activity evoked by noxious pinch in these preparations was conveyed to central ganglia by axons in the anterior‐ and lateral‐dorsal nerve branches, and that sensitization induced by tissue damage was mediated centrally. Furthermore, sensitization was blocked by all inhibitors tested except the inactive isomer L‐AP5, and reversed by ivabradine both in vivo and in vitro. Our findings suggest that M. sextas sensitization occurs through central signal amplification. Due to the relatively natural sensitization method and conserved molecular actions, we suggest that M. sexta may be a valuable model for studying the electrophysiological properties of nociceptive sensitization and potentially related conditions such as allodynia and hyperalgesia in a comparative setting that offers unique experimental advantages. J. Comp. Neurol. 525:1176–1191, 2017.

Ecdysis behaviors and circadian rhythm of ecdysis in the stick insect, Carausius morosus

Successful ecdysis in insects depends on proper timing and sequential activation of an elaborate series of motor programs driven by a relatively conserved network of neuropeptides. The behaviors must be activated at the appropriate times to ensure successful loosening and shedding of the old cuticle, and can be influenced by environmental cues in the form of immediate sensory feedback and by circadian rhythms. We assessed the behaviors, components of the neural network and the circadian basis of ecdysis in the stick insect, Carausius morosus. C. morosus showed many of the characteristic pre-ecdysis and ecdysis behaviors previously described in crickets and locusts. Ecdysis was described in three phases, namely the (i) preparatory or pre-ecdysis phase, (ii) the ecdysial phase, and (iii) the post-ecdysis or exuvial phase. The frequencies of push-ups and sways during the preparatory phase were quantified as well as durations of all the phases. The regulation of ecdysis appeared to act via elevation of cGMP, as described in many other insects, although eclosion hormone-like immunoreactivity was not noted using a lepidopteran antiserum. Finally, C. morosus showed a circadian rhythm to the onset of ecdysis, with ecdysis occurring just prior to or at lights on. Ecdysis could be induced precociously with mechanical stimulation.

Eigenphenotypes: towards an algorithmic framework for phenotype discovery

Studying the genetic control of molecular, anatomical and/or morphological phenotypes in model organisms is a powerful tool in the functional analysis of a gene. The goal of our research is to develop algorithms that discover phenotypes of behavior in model organisms, which may identify, categorize, and quantify these phenotypes under conditions of minimal a priori information. Starting from a non-invasive video monitoring of a model organism, we propose an eigen-decomposition of the organisms behavior captured in video. Traditional clustering techniques in space, time, and frequency can utilize this decomposition to characterize the categorical behaviors of an animal, and for an analysis of the behavioral repertoire. This supplies a quantified analysis of behavior with minimal assumptions, a crucial first step in the genetic analysis of behavior.

Automated behavioral phenotype detection and analysis using color-based motion tracking

The problem of elucidating the functional significance of genes is a key challenge of modern science. Solving this problem can lead to fundamental advancements across multiple areas such starting from pharmaceutical drug discovery to agricultural sciences. A commonly used approach in this context involves studying genetic influence on model organisms. These influences can be expressed at behavioral, morphological, anatomical, or molecular levels and the expressed patterns are called phenotypes. Unfortunately, detailed studies of many phenotypes, such as the behavior of an organism, is highly complicated due to the inherent complexity of the phenotype pattern and because of the fact that it may evolve over long time periods. In this paper, we propose applying color-based tracking to study Ecdysis in the hornworm - a biologically highly relevant phenotype whose complexity had thus far, prevented application of automated approaches. We present experimental results which demonstrate the accuracy of tracking and phenotype determination under conditions of complex body movement, partial occlusions, and body deformations. A key additional goal of our paper is to expose the computer vision community to such novel applications, where techniques from vision and pattern analysis can have a seminal influence on other branches of modern science.