Single cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identify 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these reveal a highly detailed portrait of cell diversity across the larva, including the identification of neuronal cell types. We then validate important gene regulatory networks driving sea urchin development and reveal new domains of activity within the larval body. Focusing on neurons that co-express Pdx-1 and Brn1/2/4, we identify an unprecedented number of genes shared by this population of neurons in sea urchin and vertebrate endocrine pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we show that Pdx1 is necessary for the acquisition of the neuronal identity of these cells. We hypothesize that a network similar to the one orchestrated by Pdx1 in the sea urchin neurons was active in an ancestral cell type and then inherited by neuronal and pancreatic developmental lineages in sea urchins and vertebrates.

Development of Chrysomya albiceps (Wiedemann, 1819) (Diptera: Calliphoridae) from the Jazan region of Southwest Saudi Arabia under different laboratory temperatures: applications in forensic entomology

Background Chrysomya albiceps (Wiedemann, 1819) (blowflies), family Calliphoridae, is important in forensic entomology, where the minimum and maximum postmortem intervals (PMI) are estimated on the basis of the developmental stages of Diptera larvae that consume dead tissue. The present study was designed to estimate the effects of different ambient temperatures (20, 25, and 30 °C) under controlled laboratory conditions on the developmental stages of C. albiceps from the Jazan region, Saudi Arabia. Results The present study showed that the larval body weight and length were significantly increased when larvae were reared at 30 °C compared with corresponding values at 24 h, 48 h, and 72 h at rearing temperatures of 20 °C and 25 °C; however, the weight and length were significantly decreased compared with corresponding values at 96 h at 20 °C and 25 °C. The pupation time was inversely related to the rearing temperature, occurring at 144, 124, and 120 h at rearing temperatures of 20 °C, 25 °C, and 30 °C, respectively. The pupal weight and length were significantly increased in larvae reared at 30 °C compared with those reared at 20 °C and 25 °C. At 20 °C, 25 °C, and 30 °C, larval durations of 5.00, 5.00, and 4.00 days were recorded, respectively. Pupae and adults showed gradual decreases in life stage durations, at 6.00, 5.30, and 4.80 days in pupae and 20.00, 18.70, and 16.90 days in adults, with increasing rearing temperatures. Average adult longevity at 30 °C (194.40 h) was significantly less than adult longevity at 20 °C (216.00 h) and 25 °C (204.60 h). The results showed an inverse relation between durations of developmental stages and rearing temperatures. Conclusions Insect laboratory colonization for the determination of biological characteristics of insects is economically viable for forensic entomology and as a technique for evaluating insect evidence.

In Silico and In Vivo Analysis of Amino Acid Substitutions That Cause Laminopathies

Mutations in the LMNA gene cause diseases called laminopathies. LMNA encodes lamins A and C, intermediate filaments with multiple roles at the nuclear envelope. LMNA mutations are frequently single base changes that cause diverse disease phenotypes affecting muscles, nerves, and fat. Disease-associated amino acid substitutions were mapped in silico onto three-dimensional structures of lamin A/C, revealing no apparent genotype–phenotype connections. In silico analyses revealed that seven of nine predicted partner protein binding pockets in the Ig-like fold domain correspond to sites of disease-associated amino acid substitutions. Different amino acid substitutions at the same position within lamin A/C cause distinct diseases, raising the question of whether the nature of the amino acid replacement or genetic background differences contribute to disease phenotypes. Substitutions at R249 in the rod domain cause muscular dystrophies with varying severity. To address this variability, we modeled R249Q and R249W in Drosophila Lamin C, an orthologue of LMNA. Larval body wall muscles expressing mutant Lamin C caused abnormal nuclear morphology and premature death. When expressed in indirect flight muscles, R249W caused a greater number of adults with wing posturing defects than R249Q, consistent with observations that R249W and R249Q cause distinct muscular dystrophies, with R249W more severe. In this case, the nature of the amino acid replacement appears to dictate muscle disease severity. Together, our findings illustrate the utility of Drosophila for predicting muscle disease severity and pathogenicity of variants of unknown significance.

Hormonal Regulation of Programmed Cell Death in Sea Urchin Metamorphosis

Programmed cell death (PCD) has been identified as a key process in the metamorphic transition of indirectly developing organisms such as frogs and insects. Many marine invertebrate species with indirect development and biphasic life cycles face the challenge of completing the metamorphic transition of the larval body into a juvenile when they settle into the benthic habitat. Some key characteristics stand out during this transition in comparison to frogs and insects: (1) the transition is often remarkably fast and (2) the larval body is largely abandoned and few structures transition into the juvenile stage. In sea urchins, a group with a drastic and fast metamorphosis, development and destruction of the larval body is regulated by endocrine signals. Here we provide a brief review of the basic regulatory mechanisms of PCD in animals. We then narrow our discussion to metamorphosis with a specific emphasis on sea urchins with indirect life histories and discuss the function of thyroid hormones and histamine in larval development, metamorphosis and settlement of the sea urchin Strongylocentrotus purpuratus. We were able to annotate the large majority of PCD related genes in the sea urchin S. purpuratus and ongoing studies on sea urchin metamorphosis will shed light on the regulatory architecture underlying this dramatic life history transition. While we find overwhelming evidence for hormonal regulation of PCD in animals, especially in the context of metamorphosis, the mechanisms in many marine invertebrate groups with indirect life histories requires more work. Hence, we propose that studies of PCD in animals requires functional studies in whole organisms rather than isolated cells. We predict that future work, targeting a broader array of organisms will not only help to reveal important new functions of PCD but provide a fundamentally new perspective on its use in a diversity of taxonomic, developmental, and ecological contexts.

Behavior and body size modulate the defense of toxin-containing sawfly larvae against ants

The sawfly larvae of most Argidae and Pergidae (Hymenoptera: Symphyta) species contain toxic peptides, and these along with other traits contribute to their defense. However, the effectiveness of their defense strategy, especially against ants, remains poorly quantified. Here, five Arge species, A. berberidis, A. nigripes, A. ochropus, A. pagana, A. pullata, plus three Pergidae species, Lophyrotoma analis, Lophyrotoma zonalis, Philomastix macleaii, were tested in laboratory bioassays on ant workers mainly of Myrmica rubra. The experiments focused on short-term predator–prey interactions, sawfly survival rate after long-term interactions, and feeding deterrence of the sawfly hemolymph. The larvae of Arge species were generally surrounded by few ants, which rarely bit them, whereas larvae of Pergidae, especially P. macleaii, had more ants around with more biting. A detailed behavioral analysis of Arge-ant interactions revealed that larval body size and abdomen raising behavior were two determinants of ant responses. Another determinant may be the emission of a volatile secretion by non-eversible ventro-abdominal glands. The crude hemolymph of all tested species, the five Arge species and L. zonalis, was a strong feeding deterrent and remained active at a ten-fold dilution. Furthermore, the study revealed that the taxon-specific behavior of ants, sting or spray, impacted the survival of A. pagana but not the large body-sized A. pullata. The overall results suggest that the ability of Arge and Pergidae larvae to defend against ants is influenced by the body size and behavior of the larvae, as well as by chemicals.

Effects of Preservative Concentrations on Larval Cephalopharyngeal Skeleton of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae), an Alternative Indicator to Larval Body Length for mPMI Estimation

Introduction: In forensic entomology, dipterous larval specimens found feeding on decomposing corpses are usually preserved in a range of 70-95% ethanol before being subjected to minimum postmortem interval (mPMI) assessment. However, larval body size, which is commonly used to infer mPMI, can be affected by preservatives resulting miscalculation of estimation. This study compared the effect of ethanol concentrations on larval body and cephalopharyngeal skeleton, a potential substitute to estimate larval age for mPMI estimation. Methods: Experiments were conducted on homogenised colony of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) third instar larvae reared in similar conditions. They were fixed with hot water (»80ºC) and preserved in 70% and 90% ethanol. After seven days in preservatives, larvae were measured for total body length from furthest part of the head to the last abdominal segment. Cephalopharyngeal skeleton was subsequently extracted from the body and measured based on morphometric landmarks on the pharyngeal sclerite. Centroid size of the cephalopharyngeal skeleton was also calculated based on the configuration of five morphometric landmarks. Results: In all four study replicates, pairwise comparisons with the original size indicated that larval body length was significantly affected by ethanol concentration (p<0.001) whilst only two of the study replicates showed cephalopharyngeal skeleton maintained its size when preserved in different ethanol concentration. Conclusion: Possible causes of variations are discussed herein with the results clearly indicated cephalopharyngeal skeleton should be considered as alternative growth parameter for mPMI estimation.

Gelsolin and dCryAB act downstream of muscle identity genes and contribute to preventing muscle splitting and branching in Drosophila

A combinatorial code of identity transcription factors (iTFs) specifies the diversity of muscle types in Drosophila. We previously showed that two iTFs, Lms and Ap, play critical role in the identity of a subset of larval body wall muscles, the lateral transverse (LT) muscles. Intriguingly, a small portion of ap and lms mutants displays an increased number of LT muscles, a phenotype that recalls pathological split muscle fibers in human. However, genes acting downstream of Ap and Lms to prevent these aberrant muscle feature are not known. Here, we applied a cell type specific translational profiling (TRAP) to identify gene expression signatures underlying identity of muscle subsets including the LT muscles. We found that Gelsolin (Gel) and dCryAB, both encoding actin-interacting proteins, displayed LT muscle prevailing expression positively regulated by, the LT iTFs. Loss of dCryAB function resulted in LTs with irregular shape and occasional branched ends also observed in ap and lms mutant contexts. In contrast, enlarged and then split LTs with a greater number of myonuclei formed in Gel mutants while Gel gain of function resulted in unfused myoblasts, collectively indicating that Gel regulates LTs size and prevents splitting by limiting myoblast fusion. Thus, dCryAB and Gel act downstream of Lms and Ap and contribute to preventing LT muscle branching and splitting. Our findings offer first clues to still unknown mechanisms of pathological muscle splitting commonly detected in human dystrophic muscles and causing muscle weakness.

The Drosophila gene smoke alarm regulates nociceptor-epidermis interactions and thermal nociception behavior

The detection and processing of noxious sensory input depends on the proper growth and function of nociceptor sensory neurons in the peripheral nervous system. In Drosophila melanogaster, the class IV (cIV) multidendritic dendritic arborization (md-da) neurons detect noxious stimuli through their highly branched dendrites that innervate the epidermis of the larval body wall. Here, we describe requirements of a previously uncharacterized gene named smoke alarm (smal), a discoidin domain receptor, in cIV md-da dendrite morphogenesis and nociception behavior. We find that smal mutant larvae exhibit thermal hyperalgesia that is fully rescued with a BAC transgene containing smal. Consistent with this phenotype, a smal reporter gene was expressed in nociceptors and other peripheral sensory neurons. Smal::GFP protein localized to punctate structures throughout the cIV md-da neurons. We further show that smal loss-of-function results in reduced nociceptor dendrite branching. Interestingly, mammalian homologues of smal act as collagen receptors, and we find that smal mutant dendrites showed an increase in epidermal cell ensheathment relative to animals that are wild type for smal. Based on this phenotype we propose that Smal protein function is required for attachment of dendrites to the extracellular matrix (ECM) and the loss of activity results in thermal hyperalgesia.

Contrasting Manual and Automated Assessment of Thermal Stress Responses and Larval Body Size in Black Soldier Flies and Houseflies

Within ecophysiological and genetic studies on insects, morphological and physiological traits are commonly assessed and phenotypes are typically obtained from manual measurements on numerous individuals. Manual observations are, however, time consuming, can introduce observer bias and are prone to human error. Here, we contrast results obtained from manual assessment of larval size and thermal tolerance traits in black soldier flies (Hermetia illucens) and houseflies (Musca domestica) that have been acclimated under three different temperature regimes with those obtained automatically using an image analysis software (Noldus EthoVision XT). We found that (i) larval size estimates of both species, obtained by manual weighing or by using the software, were highly correlated, (ii) measures of heat and cold tolerance using manual and automated approaches provided qualitatively similar results, and (iii) by using the software we obtained quantifiable information on stress responses and acclimation effects of potentially higher ecological relevance than the endpoint traits that are typically assessed when manual assessments are used. Based on these findings, we argue that automated assessment of insect stress responses and largescale phenotyping of morphological traits such as size will provide new opportunities within many disciplines where accurate and largescale phenotyping of insects is required.