Differentiation and function of cardiac valves in the adult Drosophila heart.

Drosophila, like all insects, has an open circulatory system for the distribution of haemolymph and its components. The circulation of the haemolymph is essentially driven by the pumping activity of the linear heart. The heart is constructed as a tube into which the haemolymph is sucked and pumped forward by rhythmic contractions running from the posterior to the anterior, where it leaves the heart tube. The heart harbours cardiac valves to regulate flow directionality, with a single heart valve differentiating during larval development to separate the heart tube into two chambers. During metamorphosis, the heart is partially restructured, with the linear heart tube with one terminal wide-lumen heart chamber being converted into a linear four-chambered heart tube with three valves. As in all metazoan circulatory systems, the cardiac valves play an essential role in regulating the direction of blood flow. We provide evidence that the valves in adult flies arise via transdifferentiation, converting lumen-forming contractile cardiomyocytes into differently structured valve cells. Interestingly, adult cardiac valves exhibit a similar morphology to their larval counterparts, but act differently upon heart beating. Applying calcium imaging in living specimens to analyse activity in valve cells, we show that adult cardiac valves operate owing to muscle contraction. However, valve cell shape dynamics are altered compared with larval valves, which led us to propose our current model of the opening and closing mechanisms in the fly heart.

Tailup expression in Drosophila larval and adult cardiac valve cells.

In Drosophila larvae, the direction of blood flow within the heart tube, as well as the diastolic filling of the posterior heart chamber, is regulated by a single cardiac valve. This valve is sufficient to close the heart tube at the junction of the ventricle and the aorta and is formed by only two cells; both are integral parts of the heart tube. The valve cells regulate hemolymph flow by oscillating between a spherical and a flattened cell shape during heartbeats. At the spherical stage, the opposing valve cells close the heart lumen. The dynamic cell shape changes of valve cells are supported by a dense, criss-cross orientation of myofibrils and the presence of the valvosomal compartment, a large intracellular cavity. Both structures are essential for the valve cells' function. In a screen for factors specifically expressed in cardiac valve cells, we identified the transcription factor Tailup. Knockdown of tailup causes abnormal orientation and differentiation of cardiac muscle fibers in the larval aorta and inhibits the formation of the ventral longitudinal muscle layer located underneath the heart tube in the adult fly and affects myofibrillar orientation of valve cells. Furthermore, we have identified regulatory sequences of tup that control the expression of tailup in the larval and adult valve cells.

Silencing Transglutaminase Genes TGase2 and TGase3 Has Infection-Dependent Effects on the Heart Rate of the Mosquito Anopheles gambiae.

Transglutaminases are pleiotropic enzymes that in mosquitoes participate in the formation of the mating plug and the wound-induced antimalarial response. Moreover, one transglutaminase, TGase3, negatively regulates the infection-induced aggregation of hemocytes on the heart. Given that TGase3 is an inhibitor of periostial hemocyte aggregation, we used RNAi-based gene silencing followed by intravital video imaging to scrutinize whether any of the three transglutaminases encoded in the genome of the mosquito, Anopheles gambiae, play a role in modulating the heart rate of uninfected and infected mosquitoes. Initially, we confirmed that an infection decreases the heart rate. Then, we uncovered that silencing TGase1 does not impact heart physiology, but silencing TGase2 results in a constant heart rate regardless of infection status, eliminating the infection-induced decrease in the heart rate. Finally, silencing TGase3 decreases the heart rate in uninfected mosquitoes but increases the heart rate in infected mosquitoes. We conclude that TGase2 and TGase3 modulate heart physiology and demonstrate that factors not classically associated with insect circulatory physiology are involved in the functional integration of the immune and circulatory systems of mosquitoes.

Enabled/VASP is required to mediate proper sealing of opposing cardioblasts during Drosophila dorsal vessel formation.

INTRODUCTION: The Drosophila dorsal vessel (DV) is comprised of two opposing rows of cardioblasts (CBs) that migrate toward the dorsal midline during development. While approaching the midline, CBs change shape, enabling dorsal and ventral attachments with their contralateral partners to create a linear tube with a central lumen. We previously demonstrated DV closure occurs via a "buttoning" mechanism where specific CBs advance ahead of their lateral neighbors, and attach creating transient holes, which eventually seal. RESULTS: Here, we investigate the role of the actin-regulatory protein enabled (Ena) in DV closure. Loss of Ena results in DV cell shape and alignment defects. Live analysis of DV formation in ena mutants shows a reduction in CB leading edge protrusion length and gaps in the DV between contralateral CB pairs. These gaps occur primarily between a specific genetic subtype of CBs, which express the transcription factor seven-up (Svp) and form the ostia inflow tracts of the heart. In WT embryos these gaps between Svp+ CBs are observed transiently during the final stages of DV closure. CONCLUSIONS: Our data suggest that Ena modulates the actin cytoskeleton in order to facilitate the complete sealing of the DV during the final stages of cardiac tube formation.

Improved cardiac contraction imaging in live Drosophila embryos.

Drosophila melanogaster is a powerful model organism in which to address the genetics of cardiac patterning and heart development. This system allows the pairing of live imaging with the myriad available genetic and transgenic techniques to not only identify the genes that are critical for heart development, but to assess their impact on heart function in living organisms. There are several described methods to assess cardiac function in Drosophila. However, these approaches are restricted to imaging of mid- to late-instar larval and adult hearts. This technical hurdle therefore does not allow for the recording and analysis of cardiac function in embryos bearing strong mutations that do not hatch into larvae. Our technical innovation lies in transgenically labeling the cells of the Drosophila heart and using line scan-based confocal imaging to repeatedly image the walls of the heart. By plotting this line scan as a kymograph, heart contractions can be visualized and assayed, thereby allowing for quantification of physiological defects. This method can be used to obtain physiological data from known mutations that affect cardiac development yet are incapable of hatching into larvae for conventional analysis.•Use transgenic methods to label heart proper walls•Use high-speed line scanning to capture position of heart proper walls•Create X vs. time plot to visualize and quantify contractions over imaging period.

Three-dimensional analysis of the heart function and effect cholinergic agonists in the cockroach Gromphadorhina portentosa.

Many relevant aspects of mammal's cardiac physiology have been mainly investigated in insect models such as Drosophila melanogaster and Periplaneta americana. Cardiac function has been poorly studied in the cockroach Gromphadorhina portentosa, which has some advantages for experimental purposes such as an easier culture, bigger organs and a robust physiology. On the other hand, the study of cardiac physiology in insects has been largely improved since the arrival of digital imaging technologies for recording purposes. In the present work, we introduce a methodology of video recording coupled to an isotonic transducer for a three-dimensional analysis of the heart and intracardiac valves of G. portentosa. We used this methodology for assessing the physiological responses of the cockroach heart upon the application of different cholinergic neurotransmitters (acetylcholine, nicotine and muscarine). We recorded in detail the relationship between intracardiac valves movement, hemolymph flow, diastole and systole. Acetylcholine and nicotine induced a biphasic effect on the cardiac frequency. Acetylcholine increased the diastolic opening. Nicotine at high concentration caused paralysis. Muscarine induced no major effects. These findings suggest a combined action of cholinergic agonists for a finely tuned the cardiac frequency, intracardiac valves function and cardiac cycle.

Nitric oxide produced by periostial hemocytes modulates the bacterial infection-induced reduction of the mosquito heart rate.

The circulatory and immune systems of mosquitoes are functionally integrated. An infection induces the migration of hemocytes to the dorsal vessel, and specifically, to the regions surrounding the ostia of the heart. These periostial hemocytes phagocytose pathogens in the areas of the hemocoel that experience the highest hemolymph flow. Here, we investigated whether a bacterial infection affects cardiac rhythmicity in the African malaria mosquito, Anopheles gambiae We discovered that infection with Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis, but not Micrococcus luteus, reduces the mosquito heart rate and alters the proportional directionality of heart contractions. Infection does not alter the expression of genes encoding crustacean cardioactive peptide (CCAP), FMRFamide, corazonin, neuropeptide F or short neuropeptide F, indicating that they do not drive the cardiac phenotype. Infection upregulates the transcription of two superoxide dismutase (SOD) genes, catalase and a glutathione peroxidase, but dramatically induces upregulation of nitric oxide synthase (NOS) in both the heart and hemocytes. Within the heart, nitric oxide synthase is produced by periostial hemocytes, and chemically inhibiting the production of nitric oxide using l-NAME reverses the infection-induced cardiac phenotype. Finally, infection induces the upregulation of two lysozyme genes in the heart and other tissues, and treating mosquitoes with lysozyme reduces the heart rate in a manner reminiscent of the infection phenotype. These data demonstrate an exciting new facet of the integration between the immune and circulatory systems of insects, whereby a hemocyte-produced factor with immune activity, namely nitric oxide, modulates heart physiology.

The Insect Circulatory System: Structure, Function, and Evolution.

Although the insect circulatory system is involved in a multitude of vital physiological processes, it has gone grossly understudied. This review highlights this critical physiological system by detailing the structure and function of the circulatory organs, including the dorsal heart and the accessory pulsatile organs that supply hemolymph to the appendages. It also emphasizes how the circulatory system develops and ages and how, by means of reflex bleeding and functional integration with the immune system, it supports mechanisms for defense against predators and microbial invaders, respectively. Beyond that, this review details evolutionary trends and novelties associated with this system, as well as the ways in which this system also plays critical roles in thermoregulation and tracheal ventilation in high-performance fliers. Finally, this review highlights how novel discoveries could be harnessed for the control of vector-borne diseases and for translational medicine, and it details principal knowledge gaps that necessitate further investigation.

Pericardin, a Drosophila collagen, facilitates accumulation of hemocytes at the heart.

Hematopoietic cell lineages support organismal needs by responding to positional and systemic signals that balance proliferative and differentiation events. Drosophila provides an excellent genetic model to dissect these signals, where the activity of cues in the hemolymph or substrate can be traced to determination and differentiation events of well characterized hemocyte types. Plasmatocytes in third instar larvae increase in number in response to infection and in anticipation of metamorphosis. Here we characterize hemocyte clustering, proliferation and transdifferentiation on the heart or dorsal vessel. Hemocytes accumulate on the inner foldings of the heart basement membrane, where they move with heart contraction, and are in proximity to the heart ostia and pericardial nephrocytes. The numbers of hemocytes vary, but increase transiently before pupariation, and decrease by 4 h before pupa formation. During their accumulation at the heart, plasmatocytes can proliferate and can transdifferentiate into crystal cells. Serrate expressing cells as well as lamellocyte-like, Atilla expressing ensheathing cells are associated with some, but not all hemocyte clusters. Hemocyte aggregation is enhanced by the presence of a heart specific Collagen, Pericardin, but not the associated pericardial cells. The varied and transient number of hemocytes in the pericardial compartment suggests that this is not a hematopoietic hub, but a niche supporting differentiation and rapid dispersal in response to systemic signals.

Complement-like proteins TEP1, TEP3 and TEP4 are positive regulators of periostial hemocyte aggregation in the mosquito Anopheles gambiae.

The mosquito immune and circulatory systems are functionally integrated. During an infection, hemocytes aggregate around the ostia (valves) of the dorsal vessel - areas of the heart called the periostial regions - where they phagocytose live and melanized pathogens. Although periostial hemocyte aggregation is an immune response that occurs following infection with bacteria and malaria parasites, the molecular basis of this process remains poorly understood. Here, we show that the thioester-containing proteins, TEP1, TEP3 and TEP4 are positive regulators of periostial hemocyte aggregation in the African malaria mosquito, Anopheles gambiae. RNAi-based knockdown of TEP1, TEP3 and TEP4 resulted in fewer periostial hemocytes following Escherichia coli infection, without affecting the adjacent population of non-periostial, sessile hemocytes. Moreover, knockdown of TEP1, TEP3 and TEP4 expression resulted in reduced bacterial accumulation and melanin deposition at the periostial regions. Finally, this study confirmed the role that TEP1 plays in reducing infection intensity in the hemocoel. Overall, this research shows that the complement-like proteins, TEP1, TEP3 and TEP4, are positive regulators of the functional integration between the immune and circulatory systems of mosquitoes.

Quantifying Tissue-Specific Overexpression of FOXO in Drosophila via mRNA Fluorescence In Situ Hybridization Using Branched DNA Probe Technology.

While the highly conserved FOXO transcription factors have been studied in Drosophila melanogaster for decades, the ability to accurately control and measure their tissue-specific expression is often cumbersome due to a lack of reagents and to limited, nonhomogeneous samples. The need for quantitation within a distinct cell type is particularly important because transcription factors must be expressed in specific amounts to perform their functions properly. However, the inherent heterogeneity of many samples can make evaluating cell-specific FOXO and/or FOXO load difficult. Here, we describe an extremely sensitive fluorescence in situ hybridization (FISH) approach for visualizing and quantifying multiple mRNAs with single-cell resolution in adult Drosophila cardiomyocytes. The procedure relies upon branched DNA technology, which allows several fluorescent molecules to label an individual transcript, drastically increasing the signal-to-noise ratio compared to other FISH assays. This protocol can be modified for use in various small animal models, tissue types, and for assorted nucleic acids.

Identification and In Vivo Characterisation of Cardioactive Peptides in Drosophila melanogaster.

Neuropeptides and peptide hormones serve as critical regulators of numerous biological processes, including development, growth, reproduction, physiology, and behaviour. In mammals, peptidergic regulatory systems are complex and often involve multiple peptides that act at different levels and relay to different receptors. To improve the mechanistic understanding of such complex systems, invertebrate models in which evolutionarily conserved peptides and receptors regulate similar biological processes but in a less complex manner have emerged as highly valuable. Drosophila melanogaster represents a favoured model for the characterisation of novel peptidergic signalling events and for evaluating the relevance of those events in vivo. In the present study, we analysed a set of neuropeptides and peptide hormones for their ability to modulate cardiac function in semi-intact larval Drosophila melanogaster. We identified numerous peptides that significantly affected heart parameters such as heart rate, systolic and diastolic interval, rhythmicity, and contractility. Thus, peptidergic regulation of the Drosophila heart is not restricted to chronotropic adaptation but also includes inotropic modulation. By specifically interfering with the expression of corresponding peptides in transgenic animals, we assessed the in vivo relevance of the respective peptidergic regulation. Based on the functional conservation of certain peptides throughout the animal kingdom, the identified cardiomodulatory activities may be relevant not only to proper heart function in Drosophila, but also to corresponding processes in vertebrates, including humans.

Mosquito Hemocytes Associate With Circulatory Structures That Support Intracardiac Retrograde Hemolymph Flow.

A powerful immune system protects mosquitoes from pathogens and influences their ability to transmit disease. The mosquito's immune and circulatory systems are functionally integrated, whereby intense immune processes occur in areas of high hemolymph flow. The primary circulatory organ of mosquitoes is the dorsal vessel, which consists of a thoracic aorta and an abdominal heart. In adults of the African malaria mosquito, Anopheles gambiae, the heart periodically alternates contraction direction, resulting in intracardiac hemolymph flowing toward the head (anterograde) and toward the posterior of the abdomen (retrograde). During anterograde contractions, hemolymph enters the dorsal vessel through ostia located in abdominal segments 2-7, and exits through an excurrent opening located in the head. During retrograde contractions, hemolymph enters the dorsal vessel through ostia located at the thoraco-abdominal junction, and exits through posterior excurrent openings located in the eighth abdominal segment. The ostia in abdominal segments 2 to 7-which function in anterograde intracardiac flow-are sites of intense immune activity, as a subset of hemocytes, called periostial hemocytes, respond to infection by aggregating, phagocytosing, and killing pathogens. Here, we assessed whether hemocytes are present and active at two sites important for retrograde intracardiac hemolymph flow: the thoraco-abdominal ostia and the posterior excurrent openings of the heart. We detected sessile hemocytes around both of these structures, and these hemocytes readily engage in phagocytosis. However, they are few in number and a bacterial infection does not induce the aggregation of additional hemocytes at these locations. Finally, we describe the process of hemocyte attachment and detachment to regions of the dorsal vessel involved in intracardiac retrograde flow.

Structural and functional characterization of the contractile aorta and associated hemocytes of the mosquito Anopheles gambiae.

The primary pump of the circulatory system of insects is a dorsal vessel that traverses the length of the insect. The anterior portion, located in the head, neck and thorax, is the aorta, and the posterior portion, located in the abdomen, is the heart. Here, we characterize the structure and function of the aorta and conical chamber of the mosquito, Anopheles gambiae The aorta begins in the head with an excurrent opening located above the dorsal pharyngeal plate and ends at the thoraco-abdominal junction where it joins the conical chamber of the heart. The aorta lacks ostia, and based on the diameter of the vessel as well as the density and helical orientation of muscle, consists of three regions: the anterior aorta, the bulbous chamber, and the posterior aorta. The aorta contracts in the anterograde direction, but these contractions are independent of heart contractions and do not play a major role in hemolymph propulsion. Intravital imaging of the venous channels, the first abdominal segment and the neck revealed that hemolymph only travels through the aorta in the anterograde direction, and does so only during periods of anterograde heart flow. Furthermore, hemolymph only enters the thoraco-abdominal ostia of the conical chamber when the heart contracts in the retrograde direction, propelling this hemolymph to the posterior of the body. Finally, very few hemocytes associate with the aorta, and unlike what is seen in the periostial regions of the heart, infection does not induce the aggregation of hemocytes on the aorta.

Drosophila pericardial nephrocyte ultrastructure changes during ageing.

Here we show that a labyrinth channel compartment and slit diaphragms, which are the histological structures enabling insect nephrocytes ultrafiltration, are established during embryogenesis first by the garland nephrocytes (GCNs). The later pericardial nephrocytes, which represent the majority of functional nephrocytes in larvae and adults, lack these characteristic features at the embryonic stage. During larval development, a subpopulation of the pericardial cells survives and matures into functional nephrocytes (PCNs) displaying a fully differentiated slit diaphragm and a labyrinth channel compartment. Likely the embryonic pericardial cells have primary functions other than ultrafiltration (e.g. in production and secretion of ECM constituents). We also show, for the first time, that PCNs in the adult fly undergo dramatic histological degeneration upon ageing. The slit diaphragms disappear, the labyrinth channel system degenerates and the lysosomal compartment becomes highly enriched with electron-dense material. When using nephrocytes as a model for genetic screening purposes or to investigate the specific role of genes involved in endocytosis, histological changes occurring upon ageing need to be taken into account when interpreting structural data.

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

In 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.

Eater and draper are involved in the periostial haemocyte immune response in the mosquito Anopheles gambiae.

Haemocytes respond to infection by phagocytosing pathogens, producing the enzymes that drive the phenoloxidase-based melanization cascade, secreting lytic factors, and producing other humoral proteins. A subset of haemocytes, called periostial haemocytes, aggregate on the surface of the heart of mosquitoes and kill pathogens in areas of high haemolymph flow. Periostial haemocytes are always present, but an infection induces the recruitment of additional haemocytes to these regions. Here, we tested whether members of the Nimrod gene family are involved in the periostial immune response of the African malaria mosquito, Anopheles gambiae. Using organismal manipulations, RNA interference (RNAi) and microscopy, we show that, following an infection with Escherichia coli, nimrod - the orthologue of Drosophila NimB2 - is not involved in periostial responses. At 4 h postinfection, however, RNAi-based knockdown of draper results in a marginal increase in the number of periostial haemocytes and a doubling of E. coli accumulation at the periostial regions. Finally, at 24 h postinfection, knockdown of eater decreases the number of periostial haemocytes and decreases the phagocytosis of E. coli on the surface of the heart. Phagocytosis of bacteria is more prevalent in the periostial regions of the mid abdominal segments, and knockdown of draper, nimrod or eater does not alter this distribution. Finally, knockdown of Nimrod family genes did not have a meaningful effect on the accumulation of melanin at the periostial regions. This study identifies roles for eater and draper in the functional integration of the mosquito immune and circulatory systems.

Automatic non-invasive heartbeat quantification of Drosophila pupae.

The importance of studying model organisms such as Drosophila melanogaster has significantly increased in recent biological research. Amongst others, Drosophila can be used to study heart development and heartbeat related diseases. Here we propose a method for automatic in vivo heartbeat detection of Drosophila melanogaster pupae based on morphological structures which are recorded without any dissection using FIM imaging. Our approach is easy-to-use, has low computational costs, and enables high-throughput experiments. After automatically segmenting the heart region of the pupa in an image sequence, the heartbeat is indirectly determined based on intensity variation analysis. We have evaluated our method using 47,631 manually annotated frames from 29 image sequences recorded with different temporal and spatial resolutions which are made publicly available. We show that our algorithm is both precise since it detects more than 95% of the heartbeats correctly as well as robust since the same standardized set of parameters can be used for all sequences. The combination of FIM imaging and our algorithm enables a reliable heartbeat detection of multiple Drosophila pupae while simultaneously avoiding any time consuming preparation of the animals.

Mosquito aging modulates the heart rate and the proportional directionality of heart contractions.

Mosquito aging impacts a myriad of physiological processes, including digestion, flight, mating, reproductive success, and immunity. In the present study, we conducted intravital video imaging in 1, 3, 5, 10, 15 and 20-day-old Anopheles gambiae female adults to assess whether aging impacts mosquito heart physiology. We found that the heart contraction rate increases over the first 15days of adulthood and then decreases. These changes occur for both contraction directions, although aging results in a relative change in the anterograde versus retrograde contraction rates. That is, whereas for the first 5days of life the anterograde and retrograde contraction rates are similar, from day 10 to day 20 the retrograde contraction rate is higher than the anterograde contraction rate. Aging also biases the proportional directionality of heart contractions, from approximately two thirds of the time being spent contracting in the anterograde direction and two thirds of the contractions propagating anterograde during the first 5days of life to an approximately even split between anterograde and retrograde when the mosquitoes have reached 10 to 20days of age. Transcriptional analyses of crustacean cardioactive peptide (CCAP), FMRFamide, calcium-calmodulin dependent kinase II (CaMKII), pygopus, manganese-iron superoxide dismutase (MnSOD1) and vinculin by quantitative RT-PCR revealed age-associated changes in gene expression, with MnSOD1 and vinculin expression showing a declining trend with age. RNAi-based knockdown of MnSOD1 or vinculin resulted in heart physiology that trended toward the aging phenotype for every parameter that was measured, suggesting that these two genes are involved in cardiac aging.

Hemolymph circulation in insect flight appendages: physiology of the wing heart and circulatory flow in the wings of the mosquito Anopheles gambiae.

The wings of insects are composed of membranes supported by interconnected veins. Within these veins are epithelial cells, nerves and tracheae, and their maintenance requires the flow of hemolymph. For this purpose, insects employ accessory pulsatile organs (auxiliary hearts) that circulate hemolymph throughout the wings. Here, we used correlative approaches to determine the functional mechanics of hemolymph circulation in the wings of the malaria mosquito Anopheles gambiae Examination of sectioned tissues and intravital videos showed that the wing heart is located underneath the scutellum and is separate from the dorsal vessel. It is composed of a single pulsatile diaphragm (indicating that it is unpaired) that contracts at 3 Hz and circulates hemolymph throughout both wings. The wing heart contracts significantly faster than the dorsal vessel, and there is no correlation between the contractions of these two pulsatile organs. The wing heart functions by aspirating hemolymph out of the posterior wing veins, which forces hemolymph into the wings via anterior veins. By tracking the movement of fluorescent microspheres, we show that the flow diameter of the wing circulatory circuit is less than 1 µm, and we present a spatial map detailing the flow of hemolymph across all the wing veins, including the costa, sub-costa, ambient costa, radius, media, cubitus anterior, anal vein and crossveins. We also quantified the movement of hemolymph within the radius and within the ambient costa, and show that hemolymph velocity and maximum acceleration are higher when hemolymph is exiting the wing.