Alary Muscles Research Articles

Cardiotoxicity of the diamide insecticide chlorantraniliprole in the intact heart and in isolated cardiomyocytes from the honey bee

In honey bees, circulation of blood (hemolymph) is driven by the peristaltic contraction of the heart vessel located in the dorsal part of the abdomen. Chlorantraniliprole (CHL) is an insecticide of the anthranilic diamide class which main mode of action is to alter the function of intracellular Ca2+ release channels (known as RyRs, for ryanodine receptors). In the honey bee, it was recently found to be more toxic when applied on the dorsal part of the abdomen, suggesting a direct cardiotoxicity. In the present study, a short-term exposure of semi-isolated bee hearts to CHL (0.1–10 µM) induces alterations of cardiac contraction. These alterations range from a slow-down of systole and diastole kinetics, to bradycardia and cardiac arrest. The bees heart wall is made of a single layer of semi-circular cardiomyocytes arranged concentrically all along the long axis of tube lumen. Since the heart tube is suspended to the cuticle through long tubular muscles fibers (so-called alary muscle cells), the CHL effects in ex-vivo heart preparations could result from the modulation of RyRs present in these skeletal muscle fibers as well as cardiomyocytes RyRs themselves. In order to specifically assess effects of CHL on cardiomyocytes, for the first time, intact heart cells were enzymatically dissociated from bees. Exposure of cardiomyocytes to CHL induces an increase in cytoplasmic calcium, cell contraction at the highest concentrations and depletion of intracellular stores. Electrophysiological properties of isolated cardiomyocytes were described, with a focus on voltage-gated Ca2+ channels responsible for the cardiac action potentials depolarization phase. Two types of Ca2+ currents were measured under voltage-clamp. Exposure to CHL was accompanied by a decrease in voltage-activated Ca2+ currents densities. Altogether, these results show that chlorantraniliprole can cause cardiac defects in honey bees.

Insights and perspectives on the enigmatic alary muscles of arthropods.

Three types of muscles, cardiac, smooth and skeletal muscles are classically distinguished in eubilaterian animals. The skeletal, striated muscles are innervated multinucleated syncytia, which, together with bones and tendons, carry out voluntary and reflex body movements. Alary muscles (AMs) are another type of striated syncytial muscles, which connect the exoskeleton to the heart in adult arthropods and were proposed to control hemolymph flux. Developmental studies in Drosophila showed that larval AMs are specified in embryos under control of conserved myogenic transcription factors and interact with excretory, respiratory and hematopoietic tissues in addition to the heart. They also revealed the existence of thoracic AMs (TARMs) connecting to specific gut regions. Their asymmetric attachment sites, deformation properties in crawling larvae and ablation-induced phenotypes, suggest that AMs and TARMs could play both architectural and signalling functions. During metamorphosis, and heart remodelling, some AMs trans-differentiate into another type of muscles. Remaining critical questions include the enigmatic modes and roles of AM innervation, mechanical properties of AMs and TARMS and their evolutionary origin. The purpose of this review is to consolidate facts and hypotheses surrounding AMs/TARMs and underscore the need for further detailed investigation into these atypical muscles.

Twist regulates Yorkie activity to guide lineage reprogramming of syncytial alary muscles.

Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. Here, we provide evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM.

From the knitting shop: the first physical and dynamic model of the taenioglossan radula (Mollusca: Gastropoda) aids in unravelling functional principles of the radular morphology.

The radula is the structure used for food processing in Mollusca. It can consist of a membrane with stiffer teeth, which is, together with alary processus, muscles and odontophoral cartilages, part of the buccal mass. In malacology, it is common practice to infer potential tooth functions from morphology. Thus, past approaches to explain functional principles are mainly hypothesis driven. Therefore, there is an urgent need for a workflow testing hypotheses on the function of teeth and buccal mass components and interaction of structures, which can contribute to understanding the structure as a whole. Here, in a non-conventional approach, we introduce a physical and dynamic radular model, based on morphological data of Spekia zonata (Gastropoda, Paludomidae). Structures were documented, computer-modelled, three-dimensional-printed and assembled to gather a simplistic but realistic physical and dynamic radular model. Such a bioinspired design enabled studying of radular kinematics and interaction of parts when underlain supporting structures were manipulated in a similar manner as could result from muscle contractions. The presented work is a first step to provide a constructional manual, paving the way for even more realistic physical radular models, which could be used for understanding radular functional morphology and for the development of novel gripping devices.

Digestive And Tracheal System of Menopon Gallinae (Phthiraptera: Amblycera) Infesting Poultry Bird (Gallus Gallus Domesticus)

The digestive and tracheal system of a poultry shaft louse, Menopon gallinae (Phthiraptera: Amblycera) has been studied in greater details. Alimentary canal of louse was dissected out along with crop under stereozoom binocular microscope. Entire alimentary canal of M. gallinae was found more or less straight and has three basic parts (fore-gut, mid-gut and hind-gut) while crop-teeth was present in the crop. The posterior end of crop contains 20-30 well developed crop-teeth arranged in a single arced plate, in comb-like fashion. Mid-gut was found as simple tube and contributes nearly one half of the total length, while the hind-gut was marked by the opening of Malpighian tubules in alimentary canal. The heart of M. gallinae is of simplest kind, one chambered bulbous structure having three pairs of laterally placed ostia and supported by four pairs of alary muscles. In the tracheal system there were seven pairs of spiracles occurred on the terga of M. gallinae. The first pair of spiracle was found located close to legs while remaining six abdominal spiracles occur from segment 3rd to 8th. The degree of tracheation of various visceral organs has also been noted.

Alary muscles and thoracic alary-related muscles are atypical striated muscles involved in maintaining the position of internal organs.

Alary muscles (AMs) have been described as a component of the cardiac system in various arthropods. Lineage-related thoracic muscles (TARMs), linking the exoskeleton to specific gut regions, have recently been discovered in Drosophila Asymmetrical attachments of AMs and TARMs, to the exoskeleton on one side and internal organs on the other, suggested an architectural function in moving larvae. Here, we analysed the shape and sarcomeric organisation of AMs and TARMs, and imaged their atypical deformability in crawling larvae. We then selectively eliminated AMs and TARMs by targeted apoptosis. Elimination of AMs revealed that AMs are required for suspending the heart in proper intra-haemocelic position and for opening of the heart lumen, and that AMs constrain the curvature of the respiratory tracheal system during crawling; TARMs are required for proper positioning of visceral organs and efficient food transit. AM/TARM cardiac versus visceral attachment depends on Hox control, with visceral attachment being the ground state. TARMs and AMs are the first example of multinucleate striated muscles connecting the skeleton to the cardiac and visceral systems in bilaterians, with multiple physiological functions.

From a syncytium to mononucleate cells and back: Yki and JNK in symphony.

Alary muscle syncytia in Drosophila larvae undergo a remarkable process of dedifferentiation into single cells that then fuse to become ventral longitudinal muscle in the adult. In this issue, Schaub et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201905048) identify the Hippo and JNK signaling pathways as key regulators of this process of developmental remodeling of cell fate.

Yorkie and JNK revert syncytial muscles into myoblasts during Org-1-dependent lineage reprogramming.

Lineage reprogramming has received increased research attention since it was demonstrated that lineage-restricted transcription factors can be used in vitro for direct reprogramming. Recently, we reported that the ventral longitudinal musculature of the adult Drosophila heart arises in vivo by direct lineage reprogramming from larval alary muscles, a process that starts with the dedifferentiation and fragmentation of syncytial muscle cells into mononucleate myoblasts and depends on Org-1 (Drosophila Tbx1). Here, we shed light on the events occurring downstream of Org-1 in this first step of transdifferentiation and show that alary muscle lineage-specific activation of Yorkie plays a key role in initiating the dedifferentiation and fragmentation of these muscles. An additional necessary input comes from active dJNK signaling, which contributes to the activation of Yorkie and furthermore activates dJun. The synergistic activities of the Yorkie/Scalloped and dJun/dFos transcriptional activators subsequently initiate alary muscle fragmentation as well as up-regulation of Myc and piwi, both crucial for lineage reprogramming.

The Basic Plan of the Adult Heart Is Conserved Across Different Species of Adult Mosquitoes, But the Morphology of Heart-Associated Tissues Varies

The heart is a pivotal organ in insects because it performs a number of different tasks, such as circulating nutrients, hormones, and excreta. In this study, the morphologies of the heart and associated tissues, including pericardial cells (PCs) and alary muscles (AMs), in the hematophagous mosquitoes Anopheles aquasalis Curry (Diptera: Culicidae), Aedes aegypti L. (Diptera: Culicidae), and Culex quinquefasciatus Say (Diptera: Culicidae), and the phytophagous Toxorhynchites theobaldi Dyar & Knab (Diptera: Culicidae) were compared using different microscopy techniques. Mosquito hearts are located across the median dorsal region of the whole abdomen. Paired incurrent openings in the heart wall (ostia) are found in the intersegmental regions (segments 2-7) of the abdomen, while an excurrent opening is located in the terminal cone of Ae. aegypti. The sides of the heart contain PC that are more numerous in An. aquasalis and Th. theobaldi. In these two species, PC form a cord of as closely aggregated cells, but in Ae. aegypti and Cx. quinquefasciatus, PC occur in pairs with two or four PC pairs per intersegmental region. In Th. theobaldi, AM binds to all regions of the heart, whereas in other mosquitoes they only bind in the intersegmental regions. The basic plan of the adult heart was conserved across all the adult mosquitoes investigated in this study. This conserved organization was expected because this organ plays an important role in the maintenance of individual homeostasis. However, the species had different PC and of AM morphologies. These morphological differences seem to be related to distinct physiological requirements of mosquito circulatory system.

Biosynthesis and assembly of the Collagen IV-like protein Pericardin in Drosophila melanogaster.

ABSTRACTIn Drosophila, formation of the cardiac extracellular matrix (ECM) starts during embryogenesis. Assembly and incorporation of structural proteins such as Collagen IV, Pericardin, and Laminin A, B1, and B2 into the cardiac ECM is critical to the maintenance of heart integrity and functionality and, therefore, to longevity of the animal. The cardiac ECM connects the heart tube with the alary muscles; thus, the ECM contributes to a flexible positioning of the heart within the animal's body. Moreover, the cardiac ECM holds the larval pericardial nephrocytes in close proximity to the heart tube and the inflow tract, which is assumed to be critical to efficient haemolymph clearance. Mutations in either structural ECM constituents or ECM receptors cause breakdown of the ECM network upon ageing, with disconnection of the heart tube from alary muscles becoming apparent at larval stages. Finally, the heart becomes non-functional. Here, we characterised existing and new pericardin mutants and investigated biosynthesis, secretion, and assembly of Pericardin in matrices. We identified two new pericardin alleles, which turned out to be a null (pericardin3-548) and a hypomorphic allele (pericardin3-21). Both mutants could be rescued with a genomic duplication of a fosmid coding for the pericardin locus. Biochemical analysis revealed that Pericardin is highly glycosylated and forms redox-dependent multimers. Multimer formation is remarkably reduced in animals deficient for the prolyl-4 hydroxylase cluster at 75D3-4.

Org-1-Dependent Lineage Reprogramming Generates the Ventral Longitudinal Musculature of the Drosophila Heart

Only few examples of transdifferentiation, which denotes the conversion of one differentiated cell type to another, are known to occur during normal development, and more often, it is associated with regeneration processes. With respect to muscles, dedifferentiation/redifferentiation processes have been documented during post-traumatic muscle regeneration in blastema of newts as well as during myocardial regeneration. As shown herein, the ventral longitudinal muscles of the adult Drosophila heart arise from specific larval alary muscles in a process that represents the first known example of syncytial muscle transdifferentiation via dedifferentiation into mononucleate myoblasts during normal development. We demonstrate that this unique process depends on the reinitiation of a transcriptional program previously employed for embryonic alary muscle development, in which the factors Org-1 (Drosophila Tbx1) and Tailup (Drosophila Islet1) are key components. During metamorphosis, the action of these factors is combined with cell-autonomous inputs from the ecdysone steroid and the Hox gene Ultrabithorax, which provide temporal and spatial specificity to the transdifferentiation events. Following muscle dedifferentiation, inductive cues, particularly from the remodeling heart tube, are required for the redifferentiation of myoblasts into ventral longitudinal muscles. Our results provide new insights into mechanisms of lineage commitment and cell-fate plasticity during development.

Thermal effect of blood feeding in the telmophagous fly Glossina morsitans morsitans

During feeding on warm-blooded hosts, haematophagous insects are exposed to thermal stress due to the ingestion of a meal which temperature may highly exceed their own body temperature. In order to avoid overheating and its subsequent deleterious effects, these insects respond by setting up molecular protective mechanisms such as heat shock proteins synthesis or by using thermoregulative strategies. Moreover, the duration of contact with the host depends on the way of feeding displayed by the different species (either telmophagous or solenophagous) and thus also impacts their exposure to heat. Solenophagous insects feed directly on blood vessels and are relatively slow feeders while telmophagous insects by lacerating capillaries, facilitate their access to blood and thus feed more quickly. The aim of this work was to investigate to what extent strictly telmophagous insects such as tsetse flies are exposed to thermal stress during feeding and consequently to evaluate the impact of the feeding strategy on the exposition to overheating in haematophagous insects in general. Real time thermographic analysis during feeding revealed that the flies’ body significantly heat up quite homogeneously. At the end of feeding, however, a marked regional heterothermy occurs as a consequence of the alary muscles warm up that precedes take-off. Feeding strategies, either solenophagy or telmophagy, thus appear to have a great impact on both exposition to predation risks and to thermal stress.

An Org-1-Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila.

The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. We show here that Org-1 and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which we name thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates.

Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila.

BackgroundThe Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles.ResultsUsing an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis.ConclusionsAssembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the β6/β3/β8 interface of the laminin β LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases.

Tip Cells Act as Dynamic Cellular Anchors in the Morphogenesis of Looped Renal Tubules in Drosophila

SummaryTissue morphogenesis involves both the sculpting of tissue shape and the positioning of tissues relative to one another in the body. Using the renal tubules of Drosophila, we show that a specific distal tubule cell regulates both tissue architecture and position in the body cavity. Focusing on the anterior tubules, we demonstrate that tip cells make transient contacts with alary muscles at abdominal segment boundaries, moving progressively forward as convergent extension movements lengthen the tubule. Tip cell anchorage antagonizes forward-directed, TGF-β-guided tubule elongation, thereby ensuring the looped morphology characteristic of renal tubules from worms to humans. Distinctive tip cell exploratory behavior, adhesion, and basement membrane clearing underlie target recognition and dynamic interactions. Defects in these features obliterate tip cell anchorage, producing misshapen and misplaced tubules with impaired physiological function.

The ultrastructure of the Aedes aegypti heart

Comparative structural analyses of the heart and associated tissues in 4th instar larvae (L4), pupae and adults of Aedes aegypti were undertaken using a combination of microscopy techniques. The Ae. aegypti heart consists of cardiomyocytes arranged in a helical fashion, and it is physically associated with intersegmental groups of pericardial cells (PCs) and the alary muscles (AMs). Ramifications commonly present in AMs are more developed in adults than in the immature stages. Pericardial cells absorb and store extracellular components as shown by the uptake of carmine dye fed in larval diet. We also observed that carmine stained inclusions corresponding to electron-dense structures resembling lysosomes that were more abundant and prominent in pupae, suggestive of increase of waste accumulation during pupation. The results presented here expand on previously known aspects of the mosquito heart and describe for the first time comparative aspects of the morphology of the heart in different developmental stages.

The ultrastructure of Drosophila heart cells

The functionality of the Drosophila heart or dorsal vessel is achieved by contributions from several tissues. The heart tube itself is composed of different types of cardiomyocytes that form an anterior aorta and a posterior heart chamber, inflow tracts and intracardiac valves. Herein we present an in-depth ultrastructural analysis of all cell types present in the Drosophila heart at different developmental stages. We demonstrate that the lumen-forming cardiomyocytes reveal a complex subcellular architecture that changes during development. We show that ostial cells, for which it was previously shown that they are specified during embryogenesis, start to differentiate at the end of embryogenesis displaying opening structures that allow inflow of hemolymph. Furthermore we found, that intracardiac valve cells differentiate during larval development and become enlarged during the 3. instar larval stages by the formation of cellular cytoplasmic free cavities. Moreover we were able to demonstrate, that the alary muscles are not directly connected to the heart tube but by extracellular matrix fibers at any stage of development. Our present work will provide a reference for future investigations on normal heart development and for analyses of mutant phenotypes that are caused by defects on the subcellular level.

Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification

Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila.

Morphological features of the heart of six mosquito species as revealed by scanning electron microscopy

The circulatory system in insects is formed by a dorsal vessel (heart) that is a tubular structure that pumps haemolymph in the insect body cavity Up until now, much is known about the structural organization of the heart in certain Brachycera (Diptera) (e.g. Drosophila). In contrast, little information is available regarding members of the Nematocera suborder (e.g. mosquitoes), in spite of their relevance to human health. Considering their importance, details of the heart morphology of six mosquito species (Aedes albopictus, Ae. aegypti, Ae. fluviatilis, Culex quinquefasciatus, Anopheles darlingi and An. aquasalis) were investigated by scanning electron microscopy (SEM). The heart of the adult mosquitoes, both males and females, consists of a dorsal tubular structure in association with pericardial cells located alongside the heart and alary muscles displaying ramifications that extend from the body wall to the heart surface. The SEM analysis also revealed in detail the organization of the heart, including paired valves on the heart wall (ostia). This study provides a detailed analysis of the mosquito heart microanatomy and, in our view, represents a significant contribution towards the understanding of structural-functional relationships associated with the heart of mosquito.

The Proctolin Gene and Biological Effects of Proctolin in the Blood-Feeding Bug, Rhodnius prolixus

We have reinvestigated the possible presence or absence of the pentapeptide proctolin in Rhodnius prolixus and report here the cloning of the proctolin cDNA. The transcript is expressed in the central nervous system (CNS) and some peripheral tissues. The proctolin prepropeptide encodes a single copy of proctolin along with a possible proctolin-precursor-associated peptide. We have biochemically identified proctolin in CNS extracts and shown its distribution using proctolin-like immunoreactivity. Immunostained processes are found on the salivary glands, female and male reproductive tissues, and heart and associated alary muscles. Proctolin-like immunoreactive bipolar neurons are found on the lateral margins of the common oviduct and bursa. Proctolin is biologically active on R. prolixus tissues, stimulating increases in contraction of anterior midgut and hindgut muscles, and increasing heartbeat frequency. Contrary to the previous suggestion that proctolin is absent from R. prolixus, proctolin is indeed present and biologically active in this medically important bug.