Mosquitoes were anesthetized for less than a minute by placing them in a Petri dish resting on ice. Immediately prior to use, a mosquito was injected at the thoracic anepisternal cleft with 0. This restraint prevented mosquito movement whilst preserving a natural body position during imaging.
Following video acquisition, each wing heart contraction was manually counted by visualizing the spatial shift of microspheres that aggregated at the periphery of the pulsatile diaphragm. Similarly, each heart contraction was manually counted by visualizing the spatial shift of microspheres that aggregated on the surface of the heart, a process that occurs because of the phagocytic activity of periostial hemocytes King and Hillyer, A total of 39 mosquitoes was assayed, with paired data obtained for the contractions of both the wing heart and the dorsal vessel.
Mosquitoes were anesthetized, injected with microspheres and restrained as detailed above, except that only one wing was held in the extended position while the other wing was allowed to remain resting on the mosquito's dorsum.
Initially, videos were used to qualitatively determine the directional flow of hemolymph across each wing vein, and later, they were used to quantitatively monitor the trajectory, distance, velocity and maximum acceleration of microspheres as they traveled in the wing space Fig.
Quantitative measurements were performed using the Object Tracker module of NIS-Elements, with hemolymph entry into the wing being measured within the radius and hemolymph exit out of the wing being measured within the ambient costa. For quantitative analyses, 20 mosquitoes were imaged, and for each mosquito, five microspheres were tracked while they flowed into the wing and five microspheres were tracked while they flowed out of the wing microspheres in each vein.
Data comparing the contraction rates of the wing heart and the dorsal vessel were tested for normality and analyzed using a paired, two-tailed t -test. Correlation analyses were performed by plotting the contraction rate of the wing heart of a mosquito against the contraction rate of the dorsal vessel of the same mosquito, and then calculating the correlation coefficient R as well as the Pearson correlation P -value of the entire dataset.
Data on hemolymph velocity and maximum acceleration were tested for normality. Then, data on velocity were compared using an unpaired, two-tailed t -test, and data on maximum acceleration were compared using the Mann—Whitney test.
To identify the presence and location of the wing heart of A. Injection of fluorescent microspheres into the hemocoel resulted in their dissemination throughout the body, and their aggregation in specific sites.
One of the locations where the microspheres aggregated was within the scutellum, and fluorescence imaging of this area revealed a pulsatile structure and the vigorous movement of hemolymph Movie 1 , Fig. Then, examination of sectioned thoraces by light microscopy uncovered an unpaired wing heart whose diaphragm forms a posterior-facing semicircle that anchors in two points of the anterior of the scutellar hemocoel Fig. Further examinations of intravital video recordings, together with inferences from light and scanning electron microscopy, showed that when the diaphragm contracts it assumes a slightly more flattened form.
This expands the scutellar sinus and aspirates hemolymph out of both wings Figs 1 , 2 , Movies 1 , 2. When the pulsatile diaphragm relaxes, the volume of the scutellar sinus is reduced, which results in the expulsion of hemolymph into the thoracic hemocoel. The pulsatile diaphragm of the unpaired wing heart of mosquitoes. A Fluorescence microscopy image through the thorax of a mosquito showing the outline of the scutellum solid lines and the outline of the single pulsatile diaphragm of the wing heart dashed lines.
The unpaired pulsatile diaphragm can be observed because of the aggregation of fluorescent microspheres around its periphery. B Bright-field microscopy image of a sectioned scutellum solid lines showing the pulsatile diaphragm of the wing heart dashed lines.
Relationship between the wing heart, the scutellar arms and the wings of mosquitoes. A,B Scanning electron microscopy images showing a scutellar arm extending from the scutellum and hence, the wing heart to the posterior margin of the left wing. Also noted are the location of the costa an anterior wing vein with afferent flow; inflow and the ambient costa a posterior wing vein with efferent flow; outflow. The area in the square in A is magnified in B.
Hemolymph being aspirated by the wing heart exits the wings via veins located at the posterior of each appendage. This hemolymph moves from the wing to the wing heart via channels called axillary cords that connect to scutellar arms diagrammed in a general sense in Pass et al.
Visualization of hemolymph flow within the wings and scutellum revealed that, unlike what is seen in the heart of adult mosquitoes Andereck et al. Analysis of the spatial shift of the pulsatile diaphragm of the wing heart by intravital video imaging showed that it contracts at an average rate of 3. Analysis of the dorsal vessel of the same mosquitoes showed that this organ contracts at an average rate of 2.
Finally, for each video, the contraction of the wing heart was measured at both the left and the right regions of the pulsating diaphragm.
S2 , further confirming that the wing heart is unpaired a single pulsatile diaphragm. Contraction rates of the wing heart and dorsal vessel of mosquitoes. For the dorsal vessel, rates were determined by visualizing the abdominal portion of the vessel, also known as the heart.
To map hemolymph flow within the wing, we intrathoracically injected fluorescent microspheres and tracked their movement across the network of wing veins. For the purpose of this exercise, we used the wing vein nomenclature described by Knight and Laffoon Light and scanning electron microscopy images of a wing of A. Hemolymph flow across the wing. A Scanning electron microscopy image and B bright-field microscopy image of a mosquito wing.
C Nomenclature of the veins of a mosquito wing. D Map of hemolymph flow across the veins of a mosquito wing. Acronyms are defined at the bottom of the figure, and the direction of the arrows indicates the direction of flow afferent, toward the distal end of the wing; efferent, toward the thorax.
Initial experiments aimed to determine the flow diameter of the circulatory circuit of the wing. When 0.
Having established 0. Hemolymph enters each wing and proceeds distally afferent flow via veins that emanate from the thorax at a location that is anterior of the scutellum. Specifically, hemolymph enters each wing via the costa, sub-costa and radius, and travels toward the distal end of the appendage.
Partial flow of hemolymph from the sub-costa to the costa occurs via a humeral crossvein that is located near the proximal end of the appendage, and the sub-costa joins the costa at a location that is approximately halfway into the anterior margin of the appendage. Hemolymph entering each wing via the radius also proceeds distally until this vein divides into the radius 1 and the radial sector.
The radius 1 is a longitudinal extension of the radius, and hemolymph flows in an afferent direction until it joins the costa at the distal margin of the appendage.
The junction of the costa and the radius 1 marks the beginning of the ambient costa, where hemolymph flows in a posterior and then proximal direction efferent flow along the periphery of the appendage. The other vein that bifurcates from the radius, the radial sector, briefly extends toward the posterior before proceeding distally. Hemolymph within the radial sector flows distally until it reaches the radiomedial crossvein, at which point the hemolymph flows toward the posterior via this crossvein.
Hemolymph in the media flows proximally until it merges with the cubitus anterior at a location that is near the proximal end of the appendage, and exits the wing. The mediocubital crossvein deposits hemolymph into the cubitus anterior, and this hemolymph flows to the proximal end of the wing and exits the appendage. In addition to receiving hemolymph from the mediocubital crossvein, the cubitus anterior also receives hemolymph from the ambient costa, and this hemolymph flows in the proximal direction until it exits the wing.
Although Knight and Laffoon mention the presence of a cubitus posterior, flow was not observed at that location. Also intersecting with the ambient costa, at a location that is at the posterior of the wing and proximal to the junction between the ambient costa and the cubitus anterior, is the anal vein.
The anal vein accepts hemolymph from the ambient costa and delivers it to the cubitus anterior at a location that is near the proximal end of the appendage. Hemolymph in the ambient costa that does not enter the anal vein continues its efferent flow along the posterior margin of the wing until it exits the appendage. Hemolymph exiting each wing enters a scutellar arm Fig. Visual examination of flowing microspheres illustrated that a major afferent vein is the radius and a major efferent vein is the ambient costa.
To determine whether hemolymph velocity changes within the wing space, we quantitatively measured the flow of 0. Using microspheres as a proxy for hemolymph flow, we found that the velocity of hemolymph increases as it exits the wing. It is important to note that 0. Taken altogether, these data show that hemolymph flow throughout the wing is swift, and that the flow of hemolymph is faster and more forceful as it exits the wing.
Quantitative analysis of hemolymph flow in the radius and the ambient costa of the mosquito wing. A Image of a wing, where the rectangles indicate the locations where hemolymph flow was measured within the radius for hemolymph entry into the wing; afferent flow and the ambient costa for hemolymph exit out of the wing; efferent flow.
The structure of the wing accessory pulsatile organ varies amongst taxa. It can be a modification of the dorsal vessel, as is seen in most hemimetabolous Exopterygota and some holometabolous Endopterygota insects, or it can be separate from the dorsal vessel, as is seen in Hemiptera and many holometabolous insects Krenn and Pass, a , b. Furthermore, the wing hearts that are separate from the dorsal vessel can either be paired, in which case there is one wing heart for each wing, or unpaired, in which case one wing heart aspirates hemolymph from both wings.
A phylogenetic reconstruction of the evolution of the wing circulatory organ revealed that dorsal vessel modifications are the ancestral state, and that the evolution of paired and unpaired diaphragms occurred multiple times during the course of insect evolution Pass et al. Furthermore, the secondary loss of wings, as has occurred in Siphonaptera, coincides with the loss of the scutellum and the scutellar arms, supporting the hypothesis that these cuticular structures evolved to house the machinery required for moving hemolymph across the wings Krenn and Pass, a , b.
Melanogenesis and associated cytotoxic reactions: applications to insect innate immunity. Insect Biochem Mol Biol. Lasley EL, et al. Tribolium information bulletin. In: William HM, editor. Agricultural research institute. New York: Chazy; The SignalP 4. Accessed 29 Jun Accessed 15 Jun Characterization of tyrosine hydroxylase from Manduca sexta. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Subunit composition of pro-phenol oxidase from Manduca sexta : molecular cloning of subunit ProPO-P1. Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning. Characterization and properties of a 1,3-beta- d -glucan pattern recognition protein of Tenebrio molitor larvae that is specifically degraded by serine protease during prophenoloxidase activation.
J Biol Chem. Involvement of phenoloxidase in browning during grinding of Tenebrio molitor larvae. Download references. Performed the experiments HT. All authors ensure that this is the case. All authors read and approved the final manuscript.
We thank Dr. Karl Kramer for advice about hemolymph plasma proteins in T. Lisa Brummett for helping with the Western blotting experiment. All experimental data in this study were included in this article. Additional figures and tables are available in this article. The funders did not have a role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Hiroko Tabunoki, Neal T. Dittmer, Maureen J. You can also search for this author in PubMed Google Scholar. Correspondence to Hiroko Tabunoki. Identifying the sex of pupal and adult T. The structure of the genital papillae is markedly different between female and male pupae.
The pupae were sexed by examining the structure of the genital papillae on the last abdominal segment , which are markedly different between female and male pupae indicated with black arrow.
Figure S2. Observation of the reaction curve in experimental condition 1. The absorbance A was monitored, and calculated as mO. A; larva control, B; larva M.
Figure S3. Observation of the reaction curve in Experimental condition 2. Figure S4. Observation of the reaction curve in Experimental condition 3.
Figure S5. Observation of the reaction curve in Experimental condition 4. Figure S6. Observation of the reaction curve in Experimental condition 5. Figure S7. Observation of the reaction curve in Experimental condition 6. Table S1. Table S2. Assay buffer combinations tested for measuring phenoloxidase PO activity. Table S3. Phenoloxidase activity in experimental combination 1 to 5. Reprints and Permissions. Tabunoki, H.
Development of a new method for collecting hemolymph and measuring phenoloxidase activity in Tribolium castaneum. BMC Res Notes 12, 7 Download citation. Received : 29 August Accepted : 31 December Published : 07 January Anyone you share the following link with will be able to read this content:.
Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Download PDF. Dittmer 2 , Maureen J. Rob DeSalle, Curator in the Division of Invertebrate Zoology at the American Museum of Natural History in New York City, offers this explanation: The major difference between insect blood and the blood of vertebrates, including humans, is that vertebrate blood contains red blood cells.
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