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Structural and functional characterization of the contractile aorta and associated hemocytes of the mosquito Anopheles gambiae
Leah T. Sigle, Julián F. Hillyer
Journal of Experimental Biology 2018 221: jeb181107 doi: 10.1242/jeb.181107 Published 22 June 2018
Leah T. Sigle
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA
Julián F. Hillyer
ORCID record for Julián F. Hillyer
For correspondence: julian.hillyer@vanderbilt.edu
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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.
In insects, the open circulatory system consists of the pulsatile organs, the hemocoel, and the hemolymph (Jones, 1977; Chapman et al., 2013; Klowden, 2013). The main pulsatile organ – or pump – is the dorsal vessel, which extends from the head to the posterior of the abdomen and is positioned along the midline of the organism. The dorsal vessel is divided into an aorta in the head and thorax, and a heart in the dorsal abdomen. In Anopheles gambiae adults, the heart contracts in both anterograde and retrograde directions (Glenn et al., 2010). During anterograde contractions, hemolymph enters the lumen of the heart through six pairs of incurrent ostia that are positioned in abdominal segments 2–7 and exits the dorsal vessel through an excurrent opening located in the head (Glenn et al., 2010; League et al., 2015). During retrograde contractions, hemolymph flowing through a pair of venous channels in the thorax enters the heart through an ostial pair positioned at the thoraco-abdominal junction and exits the heart through an excurrent opening located in the eighth abdominal segment (Glenn et al., 2010). The circulation of hemolymph in areas that are distant from the dorsal vessel is aided by accessory pulsatile organs. These auxiliary hearts are located at the base of the antennae, in the scutellum, and in the ventral abdomen (Clements, 1956; Andereck et al., 2010; Boppana and Hillyer, 2014; Chintapalli and Hillyer, 2016).
Although the structure and function of the adult mosquito heart has been described in detail (Glenn et al., 2010; Leódido et al., 2013; League et al., 2015), the physiology of the aorta is unknown. This is also true for other flies – the members of the order Diptera – even though Drosophila melanogaster is often used as a model for human cardiac disease (Choma et al., 2011; Piazza and Wessells, 2011; Ma, 2016). We speculate that the reason behind this gap in knowledge is that the aorta is not very accessible to researchers. That is, whereas the heart is attached to the dorsal midline of the cuticle as it traverses the abdomen, the aorta is embedded within the indirect flight muscles. Furthermore, the thoracic cuticle is often more sclerotized than the abdominal cuticle, and this prevents the visualization of the aorta through the exoskeleton of intact insects.
In this study, we used a combination of resection, fluorescence staining, and still- and video-imaging to describe the structure and location of the aorta of adult A. gambiae. Furthermore, we characterize the functional mechanics of flow through the aorta and the conical chamber of the heart – including the entry of hemolymph through the thoraco-abdominal ostia – and show that the aorta has contractile activity. Finally, we show that hemocytes are present on the outer surface of the aorta but that they are few in number and do not aggregate in response to infection.
Mosquito rearing and maintenance
Anopheles gambiae Giles sensu stricto (G3 strain) were reared and maintained at 27°C and 75% relative humidity under a 12 h:12 h light:dark photoperiod. Adults were fed a 10% sucrose solution ad libitum and maintained as previously described (Estévez-Lao et al., 2013). Experiments were carried out on adult female mosquitoes at 6 days post-eclosion.
Mosquito dissections
Mosquitoes were anesthetized on ice and dissected under a stereo microscope in order to isolate (i) the entire aorta, (ii) portions of the aorta, or (iii) the aorta and the heart. To initiate the resection procedures, the ventral side of the thorax (including the legs) and a ventral slice of the abdomen were removed by bisecting the mosquito along a coronal plane in alignment with the ventral side of the neck. A slice of the dorsal thorax was then removed along a coronal plane by bisecting the mid-posterior scutum, and the mosquitoes were placed ventral side up on a glass slide in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (Fisher Scientific, Pittsburgh, PA, USA).
To isolate the entire aorta, the alimentary tract was removed, the mosquitoes were bisected along a transverse plane at the thoraco-abdominal junction, and the head and aorta were pulled away from the thorax. The ventral side of the head was then removed in order to expose the pharyngeal plate, and this structure was isolated with the aorta attached. The portion of the aorta containing the anterior aorta and bulbous chamber was isolated as above, except the mosquitoes were bisected along a transverse plane at the junction of the meso- and metathorax. To isolate the portion of the aorta containing the bulbous chamber and the posterior aorta, the head and the alimentary tract were removed, and the aorta was isolated from the thorax. The aorta was separated from the heart by cutting with a fine blade at the anterior junction with the conical chamber. To isolate the aorta with the heart, the head and the alimentary tract were removed, and the heart was detached from the dorsal cuticle by disrupting all alary muscles with an insect pin. The aorta and heart were then removed from the thorax and dorsal cuticle.
Fluorescence labeling
For muscle staining, each mosquito was anesthetized on ice and, using a finely pulled glass needle, 0.15–0.20 µl of 16% formaldehyde was injected into the hemocoel through the thoracic anepisternal cleft. After a 5 min incubation period, the aorta was isolated and immersed in 13 µmol l−1 phalloidin-Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA), 0.16 mmol l−1 Hoechst 33342 (Invitrogen) and 0.1% Triton X-100 (Fisher Scientific) in PBS. The immersed aorta was rocked on a depression slide for 30 min, washed three times in PBS, and mounted in Aqua Poly/Mount (Polysciences, Warrington, PA, USA).
To label hemocytes, Vybrant CM-DiI Cell-Labeling Solution (Invitrogen) – a dye that labels hemocytes in live mosquitoes – was used as previously described (King and Hillyer, 2012). Briefly, mosquitoes were anesthetized and injected with 75 mmol l−1 Vybrant CM-DiI Cell-Labeling Solution and 0.75 mmol l−1 Hoechst 33342 in PBS. Live mosquitoes were incubated at 27°C for 20 min, which allows the dye to be incorporated into the hemocytes. Mosquitoes were anesthetized again and used for either live imaging or aorta resection. To label the muscle of aortas with CM-DiI labeled hemocytes, muscle was stained with phalloidin-Alexa Fluor 488 as described above, with the exception that Triton X-100 was not included in the solution.
Acquisition of still images
Specimens were visualized under brightfield and/or epi-fluorescence illumination using a Nikon 90i compound microscope (Nikon Corporation, Tokyo, Japan) equipped with a Nikon Intensilight C-HGFI fluorescence illumination unit. Image Z-stacks were acquired using a Nikon DS-Qi1Mc CCD camera, a linear encoded Z-motor and Nikon Advanced Research NIS-Elements software. Images were either selected from a layer of the Z-stack or rendered into a two-dimensional image using the Extended Depth of Focus tool of NIS-Elements software.
Video-imaging
For intravital video-imaging of hemolymph flow and hemocytes, intact mosquitoes that had been injected with 1 µm diameter green fluorescent microspheres in PBS (0.001% solids; Invitrogen) or CM-DiI were imaged through the cuticle. For dorsal viewing, mosquitoes were restrained on microscope slides using a non-invasive method previously used to measure circulatory physiology (Boppana and Hillyer, 2014). For lateral or ventral viewing, mosquitoes were positioned on their side or back. A real-time video was acquired under epi-fluorescence illumination on a Nikon SMZ1500 stereomicroscope connected to a Hamamatsu ORCA-Flash 2.8 digital CMOS camera (Hamamatsu Photonics, Hamamatsu, Japan) and NIS-Elements software, or the Nikon 90i compound microscope system described above. Microsphere-injected mosquitoes were immediately viewed or partially dissected to expose the aorta prior to viewing.
For brightfield video-imaging of the contracting aorta, mosquitoes were anesthetized on ice and the head, wings, a slice of the dorsal thorax, and the ventral side of the thorax and abdomen were removed by fine cutting using a blade. After dissection, mosquitoes were immediately placed dorsal side down on a glass slide containing PBS or further dissected to completely isolate the aorta. A real-time video was acquired on the Nikon SMZ1500 stereomicroscope system described above.
Tetracycline-resistant, green fluorescent protein (GFP)-expressing Escherichia coli (DH5 alpha) were grown as previously described (Coggins et al., 2012). Mosquitoes were anesthetized on ice, and E. coli were injected into the hemocoel as described above. Infections were performed with cultures at OD600=5.0, and a plating assay revealed that the infection doses averaged 194,100 E. coli per mosquito. Two other groups of mosquitoes were maintained in parallel: one was injected with sterile LB broth (injury) and the other was left untreated (naïve).
Hemocyte quantification
Hemocytes were labeled with CM-DiI and the bulbous chamber, posterior aorta and conical chamber were isolated. Specimens were immediately visualized at 200–400× magnification, the hemocytes were counted, and still images were acquired using the Nikon 90i compound microscope. For a cell to be considered a hemocyte, it had to be 9–18 µm in diameter, and had to be labeled with both CM-DiI and Hoechst 33342. A minimum of seven mosquitoes were analysed for each treatment group (naïve, injury and infected). Data were analysed by ANOVA using Prism 6 Software (GraphPad, La Jolla, CA, USA).
The location of the aorta
In live A. gambiae, the contracting heart can be observed by intravital video-imaging of the abdomen, as this portion of the dorsal vessel extends along the dorsal midline and is tethered to the fairly translucent tergum by the alary muscles (Glenn et al., 2010; Estévez-Lao et al., 2013; League et al., 2015). At the thoraco-abdominal junction the heart connects to the aorta, which extends to the head. Because the thorax of A. gambiae is highly sclerotized, the aorta cannot be visualized by intravital imaging of intact mosquitoes. Furthermore, in dissected mosquitoes, the dense flight muscles of the thorax make identification of the aorta – which is also composed of muscle – difficult. To overcome these limitations, we examined the aorta using a combination of resection, fluorescence staining techniques, and still- and video-imaging of dissected and intact mosquitoes. Initially, the aorta was observed in dissected thoraces by tracing its trajectory as it extends from its junction with the heart towards the head (Fig. 1A,B). Muscle was stained with Alexa Fluor-conjugated phalloidin and the aorta was visualized by fluorescence microscopy in resected specimens (Fig. 1C).
The aorta and its path through the mosquito Anopheles gambiae. (A) Ventral view of a semi-intact, dissected mosquito with the head, ventral thorax, wings, legs and alimentary tract removed. The aorta (outlined) lies between the indirect flight muscles in the thorax and is anchored by muscle fibers (arrow). A, anterior; P, posterior; L, lateral. The aorta joins the heart at the thoraco-abdominal junction (arrowhead). (B) Outline of the location of the aorta (red) and the heart (green) shown on the exterior of a mosquito (lateral view). (C) Fluorescence image showing the muscle (phalloidin; green) of a resected dorsal vessel (aorta and heart) and associated alary muscles.
The aorta originates in the head and extends posteriorly through the neck along the dorsal midline (Fig. 1B). In the head, the aorta associates with the dorsal plate of the pharyngeal pump (Fig. 2A–C), which is one of two pumps that facilitate feeding (Kim et al., 2011; Ha et al., 2015). Upon entering the prothorax, the aorta bends dorsally and then straightens such that it travels near the lateral midline with a slight, ventral slant as it crosses the mesothorax and metathorax (Fig. 1B). At a point near the thoraco-abdominal junction, the aorta bends sharply until it reaches the dorsal cuticle, and at the thoraco-abdominal junction it joins a region of the heart called the conical chamber.
The location of the aorta in the head, neck and prothorax. (A) A mosquito head with the ventral side removed, exposing the pharyngeal plate (pp). (B) Resected pp with the aorta and alimentary tract attached. (C) A resected aorta (phalloidin; green) attached to the pp. (D) A resected aorta showing its position relative to the proboscis and alimentary tract, and its attachment to the pp; gc, gastric caeca; mp, maxillary palp. (E–G) In the thorax, the aorta lies just dorsal of the midgut, the crop and the location where the gc diverge from the midgut. The specimen in E is magnified in F (lateral view) and G (dorsal view). (H) The corpora allata lie alongside the aorta. Specimens were imaged under brightfield (monochrome) and/or fluorescence illumination (phalloidin, green; Hoechst 33342, blue). A, anterior; P, posterior; L, lateral; D, dorsal; V, ventral. Scale bars: A–G, 100 µm; H, 50 µm.
In relation to other tissues, the anterior portion of the aorta is dorsal to the pharyngeal plate (Fig. 2A–C), which is also where the recurrent nerve passes between the lobes of the brain (Christophers, 1960). Along its path, the aorta lies just dorsal to the alimentary tract: in the head and neck it is dorsal to the foregut (pharynx and esophagus), and in the thorax it is dorsal to the midgut, the crop and the junction between the gastric caeca and the midgut (Fig. 2D–G). The gastric caeca are diverticula located at the junction of the foregut and midgut (Jones and Zeve, 1968; Okech et al., 2008), and although in the prothorax they diverge ventral of the aorta, they extend dorsally such that the pair straddle the aorta laterally (Fig. 2D–G). The corpora allata, which are a pair of neuroendocrine glands located in the prothorax, lie alongside the aorta (Fig. 2H), and the salivary glands are distal, as they lie ventral to the alimentary tract (Fig. 2F).
Like the heart, the aorta is a muscular tube formed by striated muscle arranged in a helical twist (Fig. 3A–F). The diameter of the aorta varies along its length but on average is narrower than the heart (Figs 1C and 3A). Based on both the diameter of the vessel and the arrangement of muscle fibers, the aorta is anatomically formed of three contiguous but distinct regions: the anterior aorta, the bulbous chamber and the posterior aorta (Fig. 3). At the anteriormost portion of the aorta – in the head – is an excurrent opening from which hemolymph exits the aorta and enters the hemocoel (Fig. 3B).
The aorta is composed of three regions: the anterior aorta, the bulbous chamber and the posterior aorta. (A) The entire aorta showing the arrangement of muscle fibers (phalloidin, green) and cardiomyocyte nuclei (Hoechst 33342, blue). Four high-resolution images were stitched to assemble this image, and portions (dotted squares) are magnified in B–F. am, alary muscle. (B,C) The excurrent opening (B) and musculature (C) of the anterior aorta (AA). (D,E) The anterior (D) and posterior (E) regions of the bulbous chamber (BC), including the cluster of nuclei at the posterior. (F) The posterior aorta (PA). Specimens were imaged under fluorescence illumination (phalloidin, green; Hoechst 33342, blue).
The anterior aorta and its excurrent opening
The anterior portion of the aorta originates in the head and extends into the neck. It makes up a little less than a quarter of the total length of the aorta and begins with an excurrent opening located above the dorsal pharyngeal plate of the head (Fig. 3B). The area of the excurrent opening resembles a prolate spheroid with an opening in the anterior end. Posterior to this structure, the aorta narrows significantly and consists of muscle fibers arranged in both right- and left-handed helical twists, with discontinuous longitudinal muscles (Fig. 4A). The cardiomyocyte nuclei are paired along the length of the anterior aorta (Fig. 4B,C). Compared with the heart, the density of muscle fibers is higher in the anterior aorta. The anterior aorta ends where the aorta bends dorsally in the prothorax, and the transition between the anterior aorta and the bulbous chamber typically contains a cluster of cardiomyocyte nuclei (Fig. 4C).
The anterior aorta. (A) Fluorescence image showing a portion of the anterior aorta, including the muscle fibers (phalloidin, green) arranged in both right- and left-handed helical twists (arrows), and the discontinuous longitudinal muscles (arrowheads). (B) DIC and fluorescence overlay of a portion of the anterior aorta showing the paired cardiomyocyte nuclei (Hoechst 33342, blue). (C) Surface intensity plot showing the distribution of nuclei (Hoechst 33342 staining) across the entire aorta and the conical chamber. To the right of the dashed vertical line the nuclei of the alary muscles overlap with those of the posterior aorta and conical chamber. AA, anterior aorta; BC, bulbous chamber; PA, posterior aorta; CC, conical chamber.
The bulbous chamber
Immediately posterior of the anterior aorta is the bulbous chamber. It makes up just over a quarter of the aorta. The bulbous chamber begins at the dorsal bend in the prothorax and extends into the mesothorax (Figs 3A and 5A). The bulbous chamber is the region of the aorta with the densest muscle arrangement, and relative to the anterior aorta the cardiomyocytes are closer together (Figs 3A and 4C). As in the anterior aorta, the muscle arrangement in the bulbous chamber is in both right- and left-handed helical twists (Fig. 5B), but the diameter of the aorta gradually widens as it transitions from the anterior aorta into the bulbous chamber. Thin muscle fibers attach to the bulbous chamber and these fibers extend through the flight muscles and presumably tether the aorta to the cuticle (Figs 1A and 5C). Fat body is often adhered to the bulbous chamber, with most of it associated with the sites where the thin muscle fibers adhere to the aorta (Fig. 5C–E). The end of the bulbous chamber is formed of a cluster of cardiomyocyte nuclei, and the posterior aorta begins where the aorta narrows and the density of muscle decreases (Fig. 3E).
The bulbous chamber. (A,B) Fluorescence images (phalloidin, green) showing the anterior (A) and central (B) portions of the bulbous chamber, including the bend in the prothorax (A), and the double helical orientation of muscle (B, arrows). (C–E) Fluorescence (phalloidin, green; Hoechst 33342, blue) and DIC overlays showing thin muscle fibers (arrowheads) and fat body (arrows) attached to the posterior region of the bulbous chamber. Scale bars: A and B, 50 µm; C–E, 100 µm.
The posterior aorta
The posterior aorta constitutes approximately half of the aorta and extends from the middle of the mesothorax to the thoraco-abdominal junction, where it joins the heart. At the transition between the bulbous chamber and the posterior aorta the muscle changes from the densest arrangement to the sparsest arrangement, and this is evident by both the intensity of muscle staining and the increased spacing between the cardiomyocyte nuclei (Figs 4C and 6A). The muscle fibers in the posterior aorta are arranged in a left-handed helical twist, which is different from the overlapping left- and right-handed twists present in the anterior aorta and the bulbous chamber (Fig. 6B). As the posterior aorta approaches the heart, the muscle density modestly increases and the cardiomyocyte spacing decreases to levels similar to the heart (Fig. 4C). The posterior aorta widens at its end where the conical chamber of the heart begins (Fig. 6C).
The posterior aorta. (A,B) Fluorescence image (phalloidin, green; Hoechst 33342, blue) of the posterior aorta showing the decrease in muscle density as the bulbous chamber transitions into the posterior aorta (A, arrowheads), the narrowing of the posterior aorta, and the single helical arrangement of muscle (B, arrows). (C) DIC and fluorescence overlay showing the widening of the posterior aorta as it joins the conical chamber (arrowheads), the alary muscles (arrows), and the nuclei of pericardial cells (pc).
The conical chamber at the thoraco-abdominal junction, and associated ostia
The aorta terminates at the thoraco-abdominal junction where it joins a diamond-shaped region of the heart called the 'conical chamber' (Fig. 7A–C). The conical chamber begins where the musculature transitions into overlapping left- and right-handed twists, as in the anterior aorta and bulbous chamber (Fig. 7B). The shape of the conical chamber is present in both intact (Fig. 7C) and resected specimens (Figs 6C and 7A), indicating that it is not solely maintained by the tension of the alary muscles. The posterior aorta and the conical chamber are both anchored to the cuticle by an incomplete pair of alary muscles that join the dorsal vessel near where the anteriormost portion of the first abdominal tergite meets the postnotum. About a quarter of the alary muscle bundle associates with the posterior aorta, whereas the other three-quarters attaches to the conical chamber (Fig. 7C,D). The posterior myofibers of these alary muscles extend along the dorsal vessel such that they meet the alary muscles from the second abdominal segment (Fig. 7C). On abdominal segments 2–7, pericardial cells associate with the alary muscles of the heart (Glenn et al., 2010; Martins et al., 2011; King and Hillyer, 2012). We discovered that pericardial cells also associate with the alary muscles that attach to the posterior aorta and conical chamber (Fig. 7D,E). Compared with the pericardial cells present in abdominal segments 2–7, these cells are similar in terms of size, their binucleated nature, and their flanking of the dorsal vessel.
The aorta joins the heart at the conical chamber. (A,B) A resected aorta showing that the conical chamber is composed of muscle in overlapping left- and right-handed twists (arrowheads); panel B is an individual slice of the region outlined in A, and shows the junction of the posterior aorta and the conical chamber (arrows). (C,D) Ventral view of the thoraco-abdominal junction, showing the aorta, the heart, and the venous channels (vc). The aorta joins the heart at the diamond-shaped conical chamber (cc), which associates with an incomplete pair of alary muscles (C, arrows). Panel D shows an individual slice of the region outlined in C, and shows the pericardial cells (arrowheads, inset shows resected pericardial cells) and alary muscles (arrows) attaching to the posterior aorta. (E,F) Ventral view showing the nuclei of the thoraco-abdominal ostia in the posterior half of the conical chamber (E, dotted white circles), and the angling of the ostia towards the posterior of the body (F, arrows). Muscle is shown in green (phalloidin) and nuclei are blue (Hoechst 33342). A, anterior; P, posterior; L, lateral. Scale bars: A,B,D–F, 50 µm; C, 100 µm.
In the conical chamber is the heart's first ostial pair, called the thoraco-abdominal ostia (Fig. 7F). Like the ostia in abdominal segments 2–7, this ostial pair is located slightly posterior of the anterior suture of the first abdominal segment, but with this being the shortest segment, this ostial pair is only slightly anterior of the transverse midline instead of in the anterior portion of the segment. Thus, these ostia are positioned in the posterior half of the conical chamber – near where the posterior two bundles of the alary muscle attach to the heart – and the funnel-shaped lips of the ostia are angled towards the posterior of the body (Fig. 7E,F).
Hemolymph flow in the conical chamber and the venous channels
To determine the functional mechanics of hemolymph flow at the thoraco-abdominal junction, we conducted intravital video-imaging of live mosquitoes and observed the movement of CM-DiI-labeled hemocytes and density-neutral fluorescent microspheres in the dorsal portion of the posterior thorax and the first abdominal segment. These regions contain the venous channels, the conical chamber and the thoraco-abdominal ostia. Intravital imaging revealed that during retrograde heart contractions, hemocytes and fluorescent microspheres flowing through the venous channels as well as the dorsal portion of the first abdominal segment enter the heart through the thoraco-abdominal ostia and are propelled towards the posterior of the body (Movie 1). When the heart switches from contracting retrograde to contracting anterograde, hemolymph flow through the venous channels continues momentarily and then pauses or slows (Movie 1). During the period of pause or slowing, this hemolymph oscillates between moving anterograde and retrograde, but there is a small, net displacement towards the thoraco-abdominal ostia (Movie 1). Also, during anterograde heart contractions the hemolymph in the dorsal portion of the first abdominal segment flows away from the heart, whereas during retrograde heart contractions it flows towards the thoraco-abdominal ostia.
Based on these observations we infer that during retrograde heart contractions the thoraco-abdominal ostia receive hemolymph from the venous channels and the abdominal hemocoel of the first segment, and once in the lumen of the dorsal vessel this hemolymph is propelled towards the posterior of the body. During anterograde heart contractions, the thoraco-abdominal ostia close, and thus hemolymph does not enter the lumen of the dorsal vessel. Instead, the retrograde movement of hemolymph travelling through the venous channels significantly slows, and this hemolymph flows past the thoraco-abdominal ostia and into the first abdominal segment, causing hemolymph in this segment to flow slowly away from the heart.
Contractile activity of the aorta
To observe the aorta in live specimens, mosquitoes were anesthetized, the ventral side of the thorax was removed in a manner that exposed the aorta, and the tissues were visualized by intravital video-imaging. Visualization of the aorta while in situ revealed that it pulsates: the diameter expands and retracts (Movie 2). Because of challenges associated with this procedure we were only able to measure the aortic contraction rate in a limited number of mosquitoes. In those mosquitoes, where the aorta remained within the indirect flight muscles, the aortic contraction rate was 1.5–2.0 Hz, similar to the 1.3–2.2 Hz contraction rate we typically observe for the heart (Glenn et al., 2010; Estévez-Lao et al., 2013; League et al., 2015). These contractions appear different from the contractions of the heart. Whereas the heart contracts in a smooth, wave-like manner and alternates between contracting in the anterograde and retrograde directions (Glenn et al., 2010; League et al., 2015), the aorta contracts in two phases that result in the shortening of distinct regions of the vessel (Movie 2). Specifically, in the first phase the posterior aorta shortens, and this is followed by a second phase where the bulbous chamber shortens. The contraction of the bulbous chamber is significantly more vigorous than the contraction of the posterior aorta, and the sequence of these contractions is suggestive of net displacement in the anterograde direction (Movie 2). On occasions, one of the pulsations occurred without the other, but we hypothesize that this was because of the disruption associated with the dissection of the mosquito.
To determine whether aortic contractions are dependent on the heart, we isolated the aorta and examined its contraction dynamics. When the aorta was attached to the anterior half of the heart, the heart did not contract, yet the aorta contracted (Movie 2). The majority of contractions originated from the bulbous chamber. When the aorta was isolated from the heart, contractions still occurred (Movie 2). Together, these data strongly suggest that contractions of the bulbous chamber are not dependent on the wave-like contractions of the heart.
Hemolymph flow dynamics through the aorta
Although the aorta cannot be directly visualized in the thorax or head of intact mosquitoes, hemolymph released into the head via the anterior excurrent opening of the aorta has to flow in the retrograde direction through the hemocoel of the neck as it returns to the thorax and abdomen. To determine whether hemolymph flow through the aorta is continuous, we used intravital video-imaging to examine flow within the neck of intact mosquitoes. In the neck, hemolymph only flows in the retrograde direction, but this flow periodically pauses for about 5 s and does so approximately six times per minute (Movie 3). These pauses roughly correspond to the number of heartbeat directional reversals, and the time the heart spends contracting in the retrograde direction (Glenn et al., 2010; Estévez-Lao et al., 2013; League et al., 2015). During periods of pause, hemolymph in the neck oscillates back and forth but there is no net displacement. This indicates that hemolymph flows through the aorta only in the anterograde direction, and does so only during periods of anterograde heart contractions. This also indicates that the pulsations of the aorta – which are continuous – do not propel hemolymph during periods of retrograde heart contractions.
Hemocytes and the aorta
Hemocytes are always present at the periostial regions of the heart, which are the locations that flank the incurrent ostia of abdominal segments 2–7 (King and Hillyer, 2012; Sigle and Hillyer, 2016). Upon infection, additional hemocytes aggregate in these regions of high hemolymph flow, where they phagocytose and kill pathogens (King and Hillyer, 2012; Sigle and Hillyer, 2016). To determine whether hemocytes are also attached to the aorta, the hemocytes of live mosquitoes were labeled with CM-DiI, the aorta was isolated, and fluorescence microscopy was used to visualize these cells. In some but not all mosquitoes, hemocytes were indeed present on the surface of the aorta. When present, hemocytes were very rarely seen in the anterior aorta. Instead, they were predominantly in the bulbous chamber and occasionally on the posterior aorta, but their number was always very low. For example, uninfected mosquitoes usually had fewer than two hemocytes (Fig. 8A–C).
Hemocytes are present on the aorta. (A) Number of hemocytes on the aorta of naïve, injured and E. coli-infected mosquitoes at 24 h post-treatment (columns are the means and whiskers represent the s.e.m.; ANOVA, P=0.3756). (B–E) Fluorescence images (B,C: CM-DiI, red; phalloidin, green; Hoechst 33342, blue) and DIC overlays (D,E: E. coli, green) of hemocytes on the bulbous chamber (B,D,E) and posterior aorta (C). (F) Dark melanin pigments associating with the attachment muscles and fat body on the bulbous chamber.
To determine whether infection induces the aggregation of hemocytes on the surface of the aorta, mosquitoes were injured, infected with E. coli or left unmanipulated, and the number of hemocytes present on the bulbous chamber and posterior aorta counted 24 h later. Overall, injured or E. coli-infected mosquitoes had two to three hemocytes on the aorta, which is similar to that observed in naïve mosquitoes (Fig. 8A–E; ANOVA, P=0.3756). Some mosquitoes, regardless of treatment, lacked hemocytes on the aorta. Upon infection, hemocytes on the aorta phagocytosed GFP-expressing E. coli, indicating that they are immunologically active (Fig. 8D,E). Occasionally, melanization was observed on the surface of the bulbous chamber, but melanin deposits were associated with fat body and not the hemocytes (Fig. 8F).
In this study we used both ex vivo and in vivo imaging to characterize the structure and function of the aorta and conical chamber of adult A. gambiae. Furthermore, we describe how alary muscles, pericardial cells and hemocytes attach to these tissues, and we illustrate how hemolymph flows through the aorta and the conical chamber, including how it flows through the thoraco-abdominal ostia (Fig. 9).
Diagrammatic representation of the heart and aorta of mosquitoes. (A) Anatomical location of the dorsal vessel, including the location of the aorta, heart, ostia, excurrent openings and venous channels. (B) Hemolymph flow during anterograde heart contractions, including the entry of hemolymph into the heart through the abdominal ostia. (C) Hemolymph flow during retrograde heart contractions, including hemolymph into the heart through the thoraco-abdominal ostia of the conical chamber. Diagrams show lateral view on the left and dorsal view on the right.
Holometabolous insects undergo drastic morphological and physiological changes during their development, and in some the structure of the dorsal vessel varies greatly between life stages whereas in others it stays largely the same (Jones, 1977). In mosquitoes, the heart of larvae and adults is structurally similar (Leódido et al., 2013; League et al., 2015). However, in A. gambiae the adult heart contracts in both anterograde and retrograde directions whereas the larval heart contracts only in an anterograde direction. Furthermore, hemolymph enters the adult heart through ostia located in abdominal segments 2–7, but in larvae the abdominal ostia are always closed (Glenn et al., 2010; League et al., 2015). Instead, hemolymph enters the larval heart through a pair of incurrent openings located at the posterior of the body – structures that in adults only have excurrent function (League et al., 2015). Comparison of the aorta of larvae and adults reveals that they are structurally similar, but that there are minor differences in the path the aorta takes. In A. gambiae larvae and adults, the aorta extends from the head to the thorax in a similar fashion, with the anterior portion of the bulbous chamber slanting dorsally as it expands in diameter (League et al., 2015). In larvae, hemolymph flowing in an anterograde direction through the aorta exits into the hemocoel in the posterior region of the head (League et al., 2015), which is the same location where hemolymph exits the aorta of adults. However, the path of the posterior aorta differs between larvae and adults; in larvae it extends along a linear plane, whereas in adults it bends dorsally to reach the heart. These differences reflect differences in the adult and larval body plans: the larval thorax and abdomen are more streamlined when compared with the adult.
The aorta has been cursorily described in other mosquito species (Jones, 1952, 1954; Christophers, 1960). These studies did not publish micrographs – making it difficult or impossible to assess and compare structures – and did not visualize the flow of hemolymph. Comparing the structure of the aorta of A. gambiae to what has been described of the aorta of larvae, pupa and adults of Aedes aegypti and Anopheles quadrimaculatus reveals similarities in structure and general location (Jones, 1952, 1954; Christophers, 1960). These include a smaller lumen compared with the lumen of the heart, a bulbous-like sinus in the prothorax, an excurrent opening in the head, and a lack of ostia. The excurrent opening of the aorta of A. quadrimaculatus and A. aegypti larvae and adults has been described as being located between the brain and the pharynx (Jones, 1954; Christophers, 1960). Furthermore, in A. aegypti adults the excurrent opening is described as 'semicircular in section with a flat floor where it lies upon the narrow anterior portion of the dorsal plate of the oesophageal pump' (Christophers, 1960), similar to our observations in resected specimens. Altogether these data suggest that the aorta terminates above the dorsal pharyngeal plate. However, earlier studies provided conflicting statements on whether the aorta extends beyond the anterior of the pharynx (Clements, 1956; Christophers, 1960). In the present study, we observed the aorta terminating as a prolate spheroid immediately above the dorsal pharyngeal plate. Although we did not observe additional musculature extending anterior of this region, if such muscle were to be present it would serve as attachment muscle to the anterior of the head and as a means of supporting the antennal hearts (Clements, 1956; Sun and Schmidt, 1997; Boppana and Hillyer, 2014). In summary, comparison of our data with earlier cursory descriptions of the aorta of other mosquito species suggests that the function of this region of the dorsal vessel is conserved across the mosquito lineage (Jones, 1952, 1954). Comparison to the aorta of adult Drosophila is difficult because the only description does not include micrographs (Miller, 1950); however, the general location of the aorta appears to be conserved.
Although the aorta has often been described as a vessel that does not contract and merely transports hemolymph propelled by the heart (Klowden, 2013), contraction of the aorta has been reported in some insects, including the adult blowfly Phormia regina, the mealworm beetle Tenebrio molitor, the Vietnamese stick insect Baculum extradentatum, the larval fruit fly Drosophila melanogaster, the locust Locusta migratoria and multiple Lepidopteran species (Hessel, 1969; Angioy and Pietra, 1995; Markou and Theophilidis, 2000; Hertel and Pass, 2002; Dasari and Cooper, 2006; Ejaz and Lange, 2008; da Silva et al., 2011). In some of these insects the contractions are intrinsic, as contractions continue after the aorta has been separated from the heart (Dasari and Cooper, 2006; Ejaz and Lange, 2008; da Silva et al., 2011). This is similar to what we observed in A. gambiae. Furthermore, the insect heart contracts myogenically, but the contraction rate and the proportional directionality is influenced by neurotransmitters and neurohormones (Hillyer, 2015). Although nerve processes have been described along the wall of the aorta in Baculum extradentatum and Drosophila larvae (Johnstone and Cooper, 2006; Ejaz and Lange, 2008), innervation of the mosquito heart has only been cursorily described (Estévez-Lao et al., 2013). We hypothesize that, like the heart, aortic contractions are myogenic. Although the aorta contracts continuously, these contractions appear to play a minor role in propelling hemolymph to the head during anterograde heart contractions. Instead, these contractions, together with the narrow nature of the aorta, may maintain pressure during periods of anterograde heart flow and prevent backflow during periods of retrograde heart flow.
A lack of alary muscles is often used as a defining feature differentiating the aorta in the thorax from the heart in the abdomen (Clements, 1992; Nation, 2008); however, we show that there is an incomplete alary muscle pair that originates in the thorax and supports the posterior of the aorta as it bends sharply as it approaches the thoraco-abdominal junction and joins the conical chamber. Also, thin muscle fibers anchor the bulbous chamber in the prothorax, which could be an additional, delicate alary muscle pair. This, and previous reports of alary muscle associating with the aorta of an insect (Ejaz and Lange, 2008), suggests that varying body plans require different types of aortic muscle support, and these muscles may be necessary for the maintenance of shape during contractions. Broadly, the aorta and heart differ significantly in muscle structure and contractility, and while variations in the presence or absence of muscles supporting the aorta exist, when present these muscles are less robust in comparison with those of the heart. As with the alary muscles of the heart, the alary muscles of the posterior aorta contain pericardial cells. This is similar to Culex fatigans, where pericardial cells are present at the posterior border of the metathorax (Pal, 1944).
The heart of adult mosquitoes periodically alternates between contracting in the anterograde and retrograde directions (Glenn et al., 2010; League et al., 2015). Early in adulthood the majority of heart contractions propagate in the anterograde direction, whereas later in life the proportion of contractions is evenly split between anterograde and retrograde (Doran et al., 2017). In this study, we report that during retrograde heart contractions, hemolymph in the venous channels and first abdominal segment enters the conical chamber through the thoraco-abdominal ostia, and flows in a retrograde direction via the lumen of the heart. During anterograde heart contractions, however, the thoraco-abdominal ostia close and do not allow the passage of hemolymph. This is opposite from the incurrent ostia of abdominal segments 2–7, which in mosquitoes are only open during anterograde heart contractions. This is also different from Drosophila species, where the thoraco-abdominal ostia have been described as either accepting hemolymph into the dorsal vessel during both anterograde and retrograde heart contractions (Wasserthal, 2007), or allowing inflow of hemolymph during retrograde heart contractions and outflow during the latter periods of anterograde heart contractions (Sláma, 2010). Furthermore, in adult Drosophila species and Calliphora vicina, two ostial pairs are present within the conical chamber: one in the first abdominal segment and one in the second (Wasserthal, 1999, 2007). In comparison, the conical chamber in A. gambiae does not extend into the second abdominal segment and only contains one pair of ostia: the thoraco-abdominal ostia. Finally, we did not detect any ostia on the aorta. In Drosophila larvae, cells expressing seven up (svp) give rise to the ostia of the heart; however, svp-expressing cells in the aorta do not differentiate into functional ostia (Ponzielli et al., 2002). The muscle formations that associate with these svp-expressing cells is reminiscent of what we observe in the bulbous chamber and anterior aorta of adult A. gambiae.
Hemocytes are immune cells that circulate with the hemocoel or are attached to tissues (sessile). In adult mosquitoes, an infection induces the aggregation of hemocytes at the periostial regions of the heart (King and Hillyer, 2012, 2013). This is advantageous because it places immune cells in the areas of the hemocoel with the highest hemolymph flow, thus allowing for the sequestration and killing of pathogens (Glenn et al., 2010; King and Hillyer, 2012; Sigle and Hillyer, 2016). In mosquito larvae, however, hemocytes do not aggregate at the periostial regions of the heart and instead aggregate in the tracheal tufts (League and Hillyer, 2016). This occurs because of differences in hemolymph flow dynamics between larvae and adults; in larvae the abdominal ostia are always closed, and thus the periostial regions are not locations of high hemolymph flow (League et al., 2015). Instead, hemolymph enters the heart via a posterior excurrent opening that is enveloped by the tracheal tufts, and thus in larvae there is also a high density of hemocytes in the areas of the heart that experience the most flow. Here we assessed whether hemocytes attach to the exterior of the aorta. We observed that although hemocytes are present on the aorta of adult mosquitoes, there are usually fewer than five hemocytes even in an infected mosquito, which is significantly fewer than the >100 hemocytes that adhere to the periostial regions of the heart (King and Hillyer, 2012, 2013; Sigle and Hillyer, 2016). The lack of hemocyte aggregation on the aorta following an infection is not surprising. Ostia are lacking in this portion of the dorsal vessel. Thus, the external regions of the aorta do not experience high hemolymph flow.
Across Insecta there are significant differences in the structure of the aorta. This includes the presence or absence of chambers, diverticula, loops, folds and ostia (Jones, 1977). In the present study we characterize the structural mechanics and functional role of the aorta of adult A. gambiae, a mosquito of epidemiological relevance because of its ability to transmit malaria. This research expands our understanding of how hemolymph is circulated throughout the body, and how the circulatory and immune systems interact during the course of an infection.
We thank Tania Estévez-Lao for mosquito husbandry, discussions and critically commenting on this manuscript. We also thank Dr Garrett League for enlightening conversations.
The authors declare no competing or financial interests.
Conceptualization: L.T.S., J.F.H.; Methodology: L.T.S., J.F.H.; Validation: L.T.S., J.F.H.; Formal analysis: L.T.S., J.F.H.; Investigation: L.T.S., J.F.H.; Resources: J.F.H.; Data curation: L.T.S., J.F.H.; Writing - original draft: L.T.S., J.F.H.; Writing - review & editing: L.T.S., J.F.H.; Visualization: L.T.S., J.F.H.; Supervision: J.F.H.; Project administration: J.F.H.; Funding acquisition: J.F.H.
This research was funded by the US National Science Foundation (NSF) grant IOS-1456844 and a Vanderbilt University Chancellor Faculty Fellowship to J.F.H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of this manuscript.
Supplementary information available online at http://jeb.biologists.org/lookup/doi/10.1242/jeb.181107.supplemental
Accepted April 22, 2018.
© 2018. Published by The Company of Biologists Ltd
http://www.biologists.com/user-licence-1-1/
Andereck, J. W.,
King, J. G. and
Hillyer, J. F.
(2010). Contraction of the ventral abdomen potentiates extracardiac retrograde hemolymph propulsion in the mosquito hemocoel. PLoS ONE 5, e12943. doi:10.1371/journal.pone.0012943
Angioy, A. M. and
Pietra, P.
(1995). Mechanism of beat reversal in semi-intact heart preparations of the blowfly Phormia regina (Meigen). J. Comp. Physiol. B 165, 165-170. doi:10.1007/BF00260807
Boppana, S. and
(2014). Hemolymph circulation in insect sensory appendages: functional mechanics of antennal accessory pulsatile organs (auxiliary hearts) in the mosquito Anopheles gambiae. J. Exp. Biol. 217, 3006-3014. doi:10.1242/jeb.106708
Chapman, R. F.,
Douglas, A. E. and
Siva-Jothy, M. T.
(2013). Circulatory system, blood and the immune sytem. In The Insects: Structure and Function (ed. S. J. Simpson and A. E. Douglas), pp. 107-131. Cambridge: Cambridge University Press.
Chintapalli, R. T. V. and
(2016). Hemolymph circulation in insect flight appendages: physiology of the wing heart and circulatory flow in the wings of the mosquito Anopheles gambiae. J. Exp. Biol. 219, 3945-3951. doi:10.1242/jeb.148254
Choma, M. A.,
Suter, M. J.,
Vakoc, B. J.,
Bouma, B. E. and
Tearney, G. J.
(2011). Physiological homology between Drosophila melanogaster and vertebrate cardiovascular systems. Dis. Model Mech. 4, 411-420. doi:10.1242/dmm.005231
Christophers, S. R.
(1960). Aedes aegypti: the Yellow Fever Mosquito. New York: Cambridge University Press.
Clements, A. N.
(1956). The antennal pulsating organs of mosquitoes and other Diptera. Q. J. Microsc. Sci. 97, 429-433.
(1992). The circulatory system. In The Biology of Mosquitoes, vol. 1, pp. 195-205. London: Chapman and Hall.
Coggins, S. A.,
Estévez-Lao, T. Y. and
(2012). Increased survivorship following bacterial infection by the mosquito Aedes aegypti as compared to Anopheles gambiae correlates with increased transcriptional induction of antimicrobial peptides. Dev. Comp. Immunol. 37, 390-401. doi:10.1016/j.dci.2012.01.005
Dasari, S. and
Cooper, R. L.
(2006). Direct influence of serotonin on the larval heart of Drosophila melanogaster. J. Comp. Physiol. B 176, 349-357. doi:10.1007/s00360-005-0058-3
da Silva, S. R.,
da Silva, R. and
Lange, A. B.
(2011). Effects of crustacean cardioactive peptide on the hearts of two Orthopteran insects, and the demonstration of a Frank-Starling-like effect. Gen. Comp. Endocrinol. 171, 218-224. doi:10.1016/j.ygcen.2011.01.015
Doran, C. R.,
(2017). Mosquito aging modulates the heart rate and the proportional directionality of heart contractions. J. Insect Physiol. 101, 47-56. doi:10.1016/j.jinsphys.2017.06.013
Ejaz, A. and
(2008). Peptidergic control of the heart of the stick insect, Baculum extradentatum. Peptides 29, 214-225. doi:10.1016/j.peptides.2007.07.036
Estévez-Lao, T. Y.,
Boyce, D. S.,
Honegger, H.-W. and
(2013). Cardioacceleratory function of the neurohormone CCAP in the mosquito Anopheles gambiae. J. Exp. Biol. 216, 601-613. doi:10.1242/jeb.077164
Glenn, J. D.,
(2010). Structural mechanics of the mosquito heart and its function in bidirectional hemolymph transport. J. Exp. Biol. 213, 541-550. doi:10.1242/jeb.035014
Ha, Y.-R.,
Lee, S.-C.,
Seo, S.-J.,
Ryu, J.,
Lee, D.-K. and
Lee, S.-J.
(2015). Comparison of the functional features of the pump organs of Anopheles sinensis and Aedes togoi. Sci. Rep. 5, 15148. doi:10.1038/srep15148
Hertel, W. and
Pass, G.
(2002). An evolutionary treatment of the morphology and physiology of circulatory organs in insects. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 133, 555-575. doi:10.1016/S1095-6433(02)00251-9
Hessel, J. H.
(1969). The comparative morphology of the dorsal vessel and accessory structures of the Lepidoptera and its phylogenetic implications. Ann. Entomol. Soc. Am. 62, 353-370. doi:10.1093/aesa/62.2.353
(2015). Integrated immune and cardiovascular function in Pancrustacea: lessons from the insects. Integr. Comp. Biol. 55, 843-855. doi:10.1093/icb/icv021
Johnstone, A. F. M. and
(2006). Direct innervation of the Drosophila melanogaster larval aorta. Brain Res. 1083, 159-163. doi:10.1016/j.brainres.2006.02.007
Jones, J. C.
(1952). Prothoracic aortic sinuses in Anopheles, Culex, and Aedes. Proc. Ent. Soc. Wash 54, 244-246.
(1954). The heart and associated tissues of Anopheles quadrimaculatus Say (Diptera: Culicidae). J. Morphol. 94, 71-123. doi:10.1002/jmor.1050940104
(1977). Circulatory System of Insects. Springfield, Illinois: Charles C. Thomas.
Jones, J. C. and
Zeve, V. H.
(1968). The fine structure of the gastric caeca of Aedes aegypti larvae. J. Insect Physiol. 14, 1567-1575. doi:10.1016/0022-1910(68)90092-9
Kim, B. H.,
Kim, H. K. and
Lee, S. J.
(2011). Experimental analysis of the blood-sucking mechanism of female mosquitoes. J. Exp. Biol. 214, 1163-1169. doi:10.1242/jeb.048793
(2012). Infection-induced interaction between the mosquito circulatory and immune systems. PLoS Pathog. 8, e1003058. doi:10.1371/journal.ppat.1003058
(2013). Spatial and temporal in vivo analysis of circulating and sessile immune cells in mosquitoes: hemocyte mitosis following infection. BMC Biol. 11, 55. doi:10.1186/1741-7007-11-55
Klowden, M. J.
(2013). Circulatory systems. In Physiological Systems in Insects, pp. 365-413. San Diego: Academic Press.
League, G. P. and
(2016). Functional integration of the circulatory, immune, and respiratory systems in mosquito larvae: pathogen killing in the hemocyte-rich tracheal tufts. BMC Biol. 14, 78. doi:10.1186/s12915-016-0305-y
League, G. P.,
Onuh, O. C. and
(2015). Comparative structural and functional analysis of the larval and adult dorsal vessel and its role in hemolymph circulation in the mosquito Anopheles gambiae. J. Exp. Biol. 218, 370-380. doi:10.1242/jeb.114942
Leódido, A. C. M.,
Ramalho-Ortigão, M. and
Martins, G. F.
(2013). The ultrastructure of the Aedes aegypti heart. Arthropod. Struct. Dev. 42, 539-550. doi:10.1016/j.asd.2013.09.005
Ma, L.
(2016). Can the Drosophila model help in paving the way for translational medicine in heart failure? Biochem. Soc. Trans. 44, 1549-1560. doi:10.1042/BST20160017C
Markou, T. and
Theophilidis, G.
(2000). The pacemaker activity generating the intrinsic myogenic contraction of the dorsal vessel of Tenebrio molitor (Coleoptera). J. Exp. Biol. 203, 3471-3483.
Martins, G. F.,
Ramalho-Ortigão, J. M. and
Pimenta, P. F. P.
(2011). Morphological features of the heart of six mosquito species as revealed by scanning electron microscopy. Int. J. Trop. Insect. Sci. 31, 98-102. doi:10.1017/S1742758411000178
Miller, A.
(1950). The internal anatomy and histology of the imago. In Biology of Drosophila (ed. M. Demerec), pp. 420-534. New York: John Wiley & Sons, Inc.
Nation, J. L.
(2008). Dorsal vessel: heart and aorta. In Encyclopedia of Entomology (ed. J. L. Capinera), pp. 1236-1238. Dordrecht: Springer Netherlands.
Okech, B. A.,
Boudko, D. Y.,
Linser, P. J. and
Harvey, W. R.
(2008). Cationic pathway of pH regulation in larvae of Anopheles gambiae. J. Exp. Biol. 211, 957-968. doi:10.1242/jeb.012021
Pal, R.
(1944). Nephrocytes in some Culicidae-Diptera. Indian J. Entomol. 6, 143-148.
Piazza, N. and
Wessells, R.
(2011). Drosophila models of cardiac disease. Prog. Mol. Biol. Transl. Sci. 100, 155-210. doi:10.1016/B978-0-12-384878-9.00005-4
Ponzielli, R.,
Astier, M.,
Chartier, A.,
Gallet, A.,
Therond, P. and
Semeriva, M.
(2002). Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the Bithorax Complex genes and hedgehog signaling. Development 129, 4509-4521.
Sigle, L. T. and
(2016). Mosquito hemocytes preferentially aggregate and phagocytose pathogens in the periostial regions of the heart that experience the most hemolymph flow. Dev. Comp. Immunol. 55, 90-101. doi:10.1016/j.dci.2015.10.018
Sláma, K.
(2010). Physiology of heartbeat reversal in adult Drosophila melanogaster (Diptera: Drosophilidae). Eur. J. Entomol. 107, 13-31. doi:10.14411/eje.2010.002
Sun, B. D. and
Schmidt, J. M.
(1997). The structure of the antennal heart of Aedes aegypti (Linnaeus). Can. J. Zool. 75, 444-458. doi:10.1139/z97-055
Wasserthal, L. T.
(1999). Functional morphology of the heart and of a new cephalic pulsatile organ in the blowfly Calliphora vicina (Diptera: Calliphoridae) and their roles in hemolymph transport and tracheal ventilation. Int. J. Insect Morphol. Embryol. 28, 111-129. doi:10.1016/S0020-7322(99)00012-4
(2007). Drosophila flies combine periodic heartbeat reversal with a circulation in the anterior body mediated by a newly discovered anterior pair of ostial valves and 'venous' channels. J. Exp. Biol. 210, 3707-3719. doi:10.1242/jeb.007864
Dorsal vessel
Hemolymph
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text:
Ketchikan Cruise Port Guide and Information
Known as the "gateway to Southeast Alaska," Ketchikan is one of the southernmost cities on the Inside Passage—a network of waterways that snake through the lush Tongass National Forest and alongside the snowy peaks of the Alaska Coast Range.
Downtown Ketchikan, Alaska.
The Ketchikan area was initially used as a fish camp by Tlingit Natives, who named the site "Kitschk-hin," meaning "thundering wings of an eagle."
Today, the city still showcases Native culture with attractions such as Chief Johnson's Totem Pole, which can be found in the heart of town. Ketchikan actually has one of the largest collections of totem poles to be found in Alaska, some of which date back more than a century.
Westerners first arrived at Ketchikan in the 1880s to establish a saltery (in which fish are salted for market) and cannery. By the 1890s, Ketchikan was serving as a supply center for gold miners hoping to strike it rich. However, fish canning, logging and tourism have historically been the town's most valuable economic ventures.
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Cruise Ship Dock
A view from the pier in Ketchikan.
Cruise ships dock in the heart of Ketchikan. The port can accommodate up to four ships at a time, which means that, depending on the size of the vessels in port, up to 21,00o passengers and crew might be visiting on any given day. Occasionally the port will receive a fifth cruise ship that has to anchor and ferry passengers ashore via tender boats. This means that on a peak day, there could be twice as many visitors to the town as there are residents!
Ketchikan Weather
Temperatures range from around 30 degrees in the winter to the mid-70s in the summer season. The city receives a fair amount of rain and fog. The fog usually burns off by midday but the rain can stay with you for a while. June, July, and August are the months that receive the least amount of rainfall.
Local Tastes
With its coastal setting on Revillagigedo Island, Ketchikan offers some of the freshest seafood found anywhere in the country.
Staples include richly flavored Chinook salmon (also called King salmon), buttery Pacific halibut and sweet King crab. Try restaurants such as Annabelle's Keg & Chowder House or the Alaska King Crab Company, both of which receive high marks from visitors and locals alike.
For Mediterranean and American cuisine featuring a variety of vegetarian options, visit New York Café. New China Restaurant offers excellent Thai and Chinese menu items, as well as traditional beverages such as coconut juice and milk tea.
Whether you want to buy a hard hat or gold, you can easily find it in Ketchikan's historic downtown, lined with everything from galleries to candy stores.
Ketchikan Dry Goods
Soho Coho Gallery
Tongass Trading Company
Simply Bella
Starboard Frames & Gifts
Ketchicandies
Ketchikan is rich with cultural gems. Art lovers might begin at the Scanlon Gallery, which has been showcasing items including paintings, ceramics, sculptures, woodwork, photography and jewelry since 1972. The Tongass Historical Museum features exhibits on Native fish camps and the importance of salmon to people in the region.
Historical photographs and artifacts help bring displays to life. The Arctic Spirit Gallery showcases traditional Native arts and crafts. The Totem Heritage Center houses Native objects such as beaded regalia and cedar bark baskets, and it also offers classes in Native arts.
Popular Cruise Excursions
Explore Ketchikan's gorgeous seaside and mountainous landscape by foot or by boat. Make sure you take a stroll down historic Creek Street to see the salmon ladders, stores and the famous Married Man's Trail, which offers a short hike to a gorgeous view.
Popular hiking routes include the 2.5-mile Deer Mountain Trail to the roughly 3,000-foot summit, and the 2.6-mile out-and-back Rainbird Trail, providing stunning coastal views.
Allen Marine Tours, Inc. hosts a 4.5-hour narrated boat tour to the beautiful Misty Fjords National Monument.
Snorkel Alaska, an eight-time winner of Princess Cruises Shore Excursion of the Year Award, takes intrepid travelers beneath the surface to find colorful sea life as vibrant as what can be seen in tropical coral reefs.
George Inlet Lodge Crab Feast. The lodge is a cannery location in a remote area. Guests board a crab boat and pull up crab pots and get a wildlife tour, followed by an all-you-can-eat dungeons crab feast. This is accessible by floatplane or a 25-minute motorcoach ride.
READ MORE: Juneau Cruise Port and Information Guide
Visiting Denali National Park and Preserve in Alaska
What is the Alaska Railroad?

redpajama_set_name: RedPajamaCommonCrawl


text:
package main

import (
	"encoding/json"
	"fmt"
	"net"
	"sync"
	"time"

	"github.com/alienth/go-fastly"
	"github.com/juju/ratelimit"
)

type rateBucket struct {
	ratelimit.Bucket
	lastUsed time.Time
}

type ipRate struct {
	ip      *net.IP
	buckets map[Dimension]*rateBucket
	// Used to signal
	limited          bool
	shouldLimit      bool
	list             *IPList
	limitedOnService map[string]bool

	FirstHit    time.Time `json:"first_hit,omitempty"`
	LastHit     time.Time `json:"last_hit,omitempty"`
	LastLimit   time.Time `json:"last_limit,omitempty"`
	Hits        int       `json:"hits,omitempty"`
	Strikes     int       `json:"strikes,omitempty"`
	Expire      time.Time `json:"-"`
	LimitExpire time.Time `json:"limit_expire,omitempty"`

	rwMutex
}

type ipRates []*ipRate
type hitsSortableIPRates struct{ ipRates }

func (r ipRates) Len() int                       { return len(r) }
func (r ipRates) Swap(i, j int)                  { r[i], r[j] = r[j], r[i] }
func (r hitsSortableIPRates) Less(i, j int) bool { return r.ipRates[i].Hits < r.ipRates[j].Hits }

type limitMessage struct {
	service   *fastly.Service
	ipRate    *ipRate
	operation fastly.BatchOperation
}

var limitCh = make(chan *limitMessage, 200)

func (ipr *ipRate) getAllBuckets() []rateBucket {
	ipr.RLock()
	defer ipr.RUnlock()

	buckets := make([]rateBucket, 0)

	if ipr.list.DimensionShared {
		sharedBuckets := ipr.list.sharedBuckets
		sharedBuckets.RLock()
		for _, bucket := range sharedBuckets.m {
			buckets = append(buckets, *bucket)
		}
		sharedBuckets.RUnlock()
	} else {
		for _, bucket := range ipr.buckets {
			buckets = append(buckets, *bucket)
		}
	}

	return buckets
}

func (ipr *ipRate) overAnyLimit() bool {
	buckets := ipr.getAllBuckets()

	for _, bucket := range buckets {
		if bucket.Available() < 0 {
			return true
		}
	}

	return false
}

// Initializes bucket if one doesn't already exist.
func (ipr *ipRate) getBucketByDimension(dimension *Dimension) *rateBucket {
	ipr.Lock()
	defer ipr.Unlock()

	var found bool
	// If DimensionValues were specified in our IPList, check to see if the
	// dimension passed matches that value. If it doesn't, zero out the
	// Dimension so that we just track by IP address.
	if len(ipr.list.DimensionValues) > 0 {
		for _, value := range ipr.list.DimensionValues {
			if value == dimension.Value {
				found = true
				break
			}
		}
		if found != true {
			dimension = &Dimension{}
		}
	}
	var bucket *rateBucket
	if *dimension != (Dimension{}) && ipr.list.DimensionShared {
		sharedBuckets := ipr.list.sharedBuckets
		sharedBuckets.Lock()
		if bucket, found = sharedBuckets.m[*dimension]; !found {
			bucket = &rateBucket{Bucket: *ratelimit.NewBucketWithQuantum(ipr.list.Time.Duration, ipr.list.Requests, ipr.list.Requests)}
			sharedBuckets.m[*dimension] = bucket
		}
		sharedBuckets.Unlock()
		ipr.buckets[*dimension] = bucket
	}
	if bucket, found = ipr.buckets[*dimension]; !found {
		bucket = &rateBucket{Bucket: *ratelimit.NewBucketWithQuantum(ipr.list.Time.Duration, ipr.list.Requests, ipr.list.Requests)}
		ipr.buckets[*dimension] = bucket
		if ipr.list.Verbose {
			logger.Printf("Initializing token bucket for IP %s\n", ipr.ip.String())

		}
	}

	return bucket
}

// Records a hit and returns true if it is over limit.
func (ipr *ipRate) Hit(ts time.Time, dimension *Dimension) bool {
	bucket := ipr.getBucketByDimension(dimension)

	ipr.Lock()
	defer ipr.Unlock()

	var overlimit bool
	if ipr.list.Verbose {
		logger.Printf("Hit by %s. %d tokens remaining.", ipr.ip.String(), bucket.Available())
	}

	// Don't track overlimits on IPs that are already limited.  This is a
	// bit of a hack to prevent IPs which are already limited from
	// inflating their strikes and LimitExpire up to huge values.
	//
	// This is necessary in an environment where blocked IPs will continue
	// to generate logged hits. The original mechanism assumed that once an
	// IP is blocked, no new hits will arrive.
	//
	// TODO: Address this more sanely.
	if !ipr.limited {
		waitTime := bucket.Take(1)
		bucket.lastUsed = ts
		if waitTime != 0 {
			overlimit = true
		}
	}

	if ipr.FirstHit.IsZero() {
		ipr.FirstHit = time.Now()
	}
	ipr.LastHit = ts
	ipr.Hits++
	ipr.Expire = time.Now().Add(ipr.list.Expire.Duration)
	return overlimit
}

// Limit adds an IP to a fastly edge ACL
func (ipr *ipRate) Limit(service *fastly.Service) error {
	ipr.Lock()
	defer ipr.Unlock()

	if !noop && ipr.shouldLimit {
		if ipr.list.Verbose {
			logger.Printf("Ratelimit exceeded. Queuing block for %s.\n", ipr.ip.String())
		}
		msg := limitMessage{service: service, ipRate: ipr, operation: fastly.BatchOperationCreate}
		limitCh <- &msg
	}

	if noop && !ipr.limited {
		// Pretend we limited here. Duplicates some of pushACLUpdates.
		ipr.Strikes++
		limitDuration := ipr.list.LimitDuration.multiply(float64(ipr.Strikes))
		ipr.LimitExpire = time.Now().Add(limitDuration.Duration)
		logger.Printf("NOOP: Would issue limit on IP %s on service %s, for duration of %v.\n", ipr.ip.String(), service.Name, limitDuration.Duration)
		// Pretend we're limited in noop mode so that messages don't spam.
		ipr.limited = true
	}

	return nil
}

const APIBulkLimit = 1000

// Processes the limitCh queue and fans out to separate service-specific
// channels, which are handled by individual processServiceQueue() goroutines.
// Also calls hook service, if applicable.
func queueFanout() {
	channelByService := make(map[*fastly.Service]chan *limitMessage)
	var ok bool

	for msg := range limitCh {
		var channel chan *limitMessage
		if channel, ok = channelByService[msg.service]; !ok {
			channelByService[msg.service] = make(chan *limitMessage, 200)
			channel = channelByService[msg.service]
			go processServiceQueue(msg.service, channel)
		}
		// Enqueue async to prevent downstream blocking from causing limitCh to pile up.
		go func(channel chan *limitMessage, msg *limitMessage) { channel <- msg }(channel, msg)
		// Call our webhooks, if they've been set.
		switch op := msg.operation; op {
		case fastly.BatchOperationCreate:
			go func(ipr ipRate) {
				err := hook.Add(ipr)
				if err != nil {
					logger.Printf("Error calling webhook on IP addition for %s: %s\n", ipr.ip.String(), err)
				}
			}(*msg.ipRate)
		case fastly.BatchOperationDelete:
			go func(ipr ipRate) {
				err := hook.Remove(ipr)
				if err != nil {
					logger.Printf("Error calling webhook on IP removal for %s: %s\n", ipr.ip.String(), err)
				}
			}(*msg.ipRate)
		}
	}
}

type ipOp struct {
	ip        string
	operation fastly.BatchOperation
}

type timestamp struct {
	mu sync.Mutex
	time.Time
}

var lastPush timestamp

// sync with pushACLUpdates to ensure we don't process a single service concurrenly.
func processServiceQueue(service *fastly.Service, channel chan *limitMessage) {
	interval := time.Duration(15 * time.Second)
	ticker := time.NewTicker(interval)

	batch := make([]*limitMessage, 0)

	ratesQueued := make(map[ipOp]bool)

	for {
		select {
		case msg := <-channel:
			key := ipOp{ip: msg.ipRate.ip.String(), operation: msg.operation}
			if !ratesQueued[key] {
				batch = append(batch, msg)
				ratesQueued[key] = true
				if len(batch)+20 >= APIBulkLimit || (len(channel) <= 20 && sendImmediately(interval)) {
					pushACLUpdates(service, batch)
					batch = make([]*limitMessage, 0)
					ratesQueued = make(map[ipOp]bool)
				}
			}

		case _ = <-ticker.C:
			if len(batch) > 0 {
				pushACLUpdates(service, batch)
				batch = make([]*limitMessage, 0)
				ratesQueued = make(map[ipOp]bool)
			}
		}
	}

}

// pushACLUpdates takes a slice of *limitMessage and builds a slice of
// fastly.ACLEntryUpdate, and then calls fastly to execute those updates. It
// If a failure occurs along the way, it will requeue the batch messages into
// limitCh.
func pushACLUpdates(service *fastly.Service, batch []*limitMessage) {
	lastPush.mu.Lock()
	lastPush.Time = time.Now()
	lastPush.mu.Unlock()
	updates := make([]fastly.ACLEntryUpdate, 0)
	acl := aclByService[service]
	// Holds the ipRates which we need to update the entries for after
	// creating ACL entries.
	ratesToUpdate := make(ipRates, 0)

	// Used to track what iprates we're going to be creating, deleting, or
	// updating so that we don't try to double create or double delete.
	creating := make(map[*ipRate]bool)
	deleting := make(map[*ipRate]bool)
	updating := make(map[*ipRate]bool)

	entries, _, err := client.ACLEntry.List(service.ID, acl.ID)
	if err != nil {
		logger.Printf("Error fetching ACL Entry list for %s. Requeuing pending changes. Error: %s\n", service.Name, err)
		go requeueBatch(batch)
		return
	}

	for _, message := range batch {
		ipr := message.ipRate
		var existingEntry *fastly.ACLEntry
		for _, entry := range entries {
			// TODO handle cases where we are in the ACL multiple times?
			if entry.IP == ipr.ip.String() {
				existingEntry = entry
				break
			}
		}

		var update fastly.ACLEntryUpdate
		update.Operation = message.operation

		switch op := update.Operation; op {
		case fastly.BatchOperationDelete:
			if existingEntry == nil || deleting[ipr] {
				continue
			}
			logger.Printf("Unlimiting IP %s on service %s\n", ipr.ip.String(), service.Name)
			deleting[ipr] = true
			update.ID = existingEntry.ID
		case fastly.BatchOperationCreate:
			if existingEntry != nil || creating[ipr] {
				// someone else already updated it
				continue
			}
			creating[ipr] = true
			update.IP = ipr.ip.String()
		case fastly.BatchOperationUpdate:
			if existingEntry == nil {
				// someone else deleted it
				continue
			}
			updating[ipr] = true
			update.IP = ipr.ip.String()
			update.ID = existingEntry.ID
		}

		ipr.Lock()

		if update.Operation == fastly.BatchOperationCreate {
			if !ipr.limited {
				// Only increase the duration time if we're not already
				// limited.  This is because we might just be applying a limit
				// to a new service that we saw a hit on.
				// TODO change this - doesn't work well if we want to update a ban.
				//
				// Strikes should increment in the event that
				// they were banned, and then unbanned, and
				// then caused trouble again.
				ipr.Strikes++
			}

			limitDuration := ipr.list.LimitDuration.multiply(float64(ipr.Strikes))
			ipr.LimitExpire = time.Now().Add(limitDuration.Duration)
			durationText := fmt.Sprintf("%d minutes", int(limitDuration.Minutes()))
			if int(limitDuration.Seconds())%60 != 0 {
				durationText += fmt.Sprintf(" %d seconds", int(limitDuration.Seconds())%60)
			}
			logger.Printf("Limiting IP %s for %s on service %s\n", ipr.ip.String(), durationText, service.Name)
			ipr.LastLimit = time.Now()
			ipr.Expire = time.Now().Add(time.Duration(24) * time.Hour)
		}

		if update.Operation != fastly.BatchOperationDelete {
			comment, err := json.Marshal(ipr)
			// This will probably never happen
			if err != nil {
				logger.Printf("Unable to prepare update for %s on %s: %s", ipr.ip.String(), service.Name, err)
				continue
			}
			update.Comment = string(comment)
		}
		updates = append(updates, update)
		ratesToUpdate = append(ratesToUpdate, ipr)
		ipr.Unlock()
	}

	// This will happen if, for example, all of the iprates in the batch
	// were already present in the ACL. That can happen because the
	// mainloop might see more requests which push us over limit after the
	// IP has already been added to the acl, as the addition to the ACL has
	// some lag time.
	if len(updates) == 0 {
		return
	}

	if _, err := client.ACLEntry.BatchUpdate(service.ID, acl.ID, updates); err != nil {
		logger.Printf("Error updating ACL for %s. Requeuing pending changes. Error: %s\n", service.Name, err)
		go requeueBatch(batch)
	}

	// If this fails, then a RemoveLimit() call might assume an entry is
	// still in the ACL when it isn't. Not disasterous.
	if err = ratesToUpdate.syncWithACLEntries(service); err != nil {
		if err != nil {
			logger.Printf("Error syncing ip rates with ACL entries for service %s: %s\n", service.Name, err)
		}
	}

}

// Takes a batch of messages and sends em back to the limitCh
func requeueBatch(batch []*limitMessage) {
	for _, msg := range batch {
		limitCh <- msg
	}
}

// syncWithACLEntries operates on a set of ipRates. It takes a fastly service,
// lists the ACLEntries on that service, and then updates the ipRate's
// limitedOnService field based on the results from Fastly.
func (rates ipRates) syncWithACLEntries(service *fastly.Service) error {
	entries, _, err := client.ACLEntry.List(service.ID, aclByService[service].ID)
	if err != nil {
		return err
	}

	for _, ipr := range rates {
		ipr.Lock()
		found := false
		for _, entry := range entries {
			if entry.IP == ipr.ip.String() {
				found = true
				break
			}
		}
		if found {
			ipr.limitedOnService[service.ID] = true
			ipr.limited = true
		} else {
			ipr.limitedOnService[service.ID] = false
		}

		// See if we're limited by any services, and if not, signal
		// that this ip is no longer limited.
		if ipr.limited {
			found := false
			for _, limited := range ipr.limitedOnService {
				if limited {
					found = true
					break
				}
			}
			if !found {
				ipr.limited = false
			}
		}
		ipr.Unlock()
	}

	return nil
}

// sendImmediately checks our clients remaining API ratelimit. Takes a
// time.Duration as an indicator of the maximum amount of time we'd like to
// wait before sending. If the remaining time in the ratelimit divided by our
// maximum allowed duration is less than the remaining amount of actions in the
// ratelimit, return false as we lack the number of requests necessary to meet
// our guarantee.
// Also returns false if we already performed an API push within the last second.
func sendImmediately(guarantee time.Duration) bool {
	lastPush.mu.Lock()
	defer lastPush.mu.Unlock()
	if time.Now().Sub(lastPush.Time) < time.Duration(1)*time.Second {
		return false
	}
	rate := client.RateLimit()
	if rate == nil {
		// We don't know the current ratelimit.
		return false
	}

	if int(rate.Reset.Sub(time.Now())/guarantee)+20 > rate.Remaining {
		return false
	}

	return true
}

// Removes an IP from ratelimits
func (ipr *ipRate) RemoveLimit() {
	if !noop {
		ipr.RLock()
		for service, _ := range aclByService {
			if ipr.limitedOnService[service.ID] {
				msg := limitMessage{service: service, ipRate: ipr, operation: fastly.BatchOperationDelete}
				limitCh <- &msg
			}
		}
		ipr.RUnlock()
	} else {
		ipr.Lock()
		logger.Printf("NOOP: Would remove all limits for IP %s.\n", ipr.ip.String())
		ipr.limited = false
		ipr.Unlock()
	}

}

// clean will free any shared bucket from our IPList if this ipRate was the last
// to utilize that shared bucket.
func (ipr *ipRate) cleanSharedBuckets() {
	ipr.Lock()
	defer ipr.Unlock()
	sharedBuckets := ipr.list.sharedBuckets
	sharedBuckets.Lock()
	defer sharedBuckets.Unlock()
	for dimension, bucket := range ipr.buckets {
		if bucket.lastUsed == ipr.LastHit {
			delete(sharedBuckets.m, dimension)
		}
	}
}

redpajama_set_name: RedPajamaGithub


text:
The Lemon Orchard
i am thrilled to give you a first look at the cover of my 2013 novel--THE LEMON ORCHARD. it will come out on July 2, but you can pre-order now if you like. it's never too soon to dream about summer reading!
Very thankful to my librarian friends for this lovely mention of THE LEMON ORCHARD in Library Journal: House-sitting for her aunt and uncle in Malibu, with only her dog for company, Julia seeks solitude so that she can quietly mourn her daughter's death. Then she befriends Roberto,who tends the nearby lemon orchard and has sorrows of his own: his daughter has disappeared, but he has yet to give up hope. Classic Rice and doubtless another best seller.
In Books News, Luanne's Blog, Notes Tags Library Journal, Luanne Rice, The Lemon Orchard
starred reviews for LITTLE NIGHT
we're excited to share with you these early reviews, both starred, for LITTLE NIGHT: From Publisher's Weekly:
* Little Night Luanne Rice. Viking/Pam Dorman, $26.95 (336p) ISBN 978-0-670-02356-1 After bludgeoning her sister's abusive husband with a burnt log, Clare Burke is whisked away to jail in the dramatic opening of Rice's 30th novel (after Secrets of Paris). Based on Anne's false testimony in defense of her husband, Clare serves two years for assault, the sisters become estranged, and the story picks up 18 years later in 2011 in New York City, where Clare is a blogger and birdwatcher. Though she's never fully recovered from the trauma of her sister's betrayal, Clare desperately wants to reconnect with Anne, who has since cut all ties with her family at the behest of her manipulative husband. But when Anne's 21-year-old daughter, Grit, shows up on Clare's doorstep seeking a family that loves her, Clare and her niece bond, though the subject of their common tie—Anne—is never far from either of their minds. The two support one another as they attempt to create a relationship and reconnect with the woman who hurt them. Poetic and stirring, Rice's latest beautifully combines her love of nature and the power of family. Agent: Andrea Cirillo, the Jane Rotrosen Agency. (June 5) Reviewed on: 04/16/2012
Other Formats Compact Disc - 978-0-307-70494-8 Hardcover - 491 pages - 978-1-4104-4886-6
From Library Journal:
*Rice, Luanne. Little Night. Pamela Dorman: Viking. Jun. 2012. c.336p. ISBN 9780670023561. $26.95. F In 1993, Clare Burke attacked her sister Anne's abusive husband, Frederik, and went to prison for assault. Once close, the sisters grew estranged after Anne lied in court about what precipitated the attack. Clare, however, never stopped missing and worrying about Anne and her two children, Gillis and Margarita (Grit), who continued to live with a man they often feared. Now working as a birder and blogger in New York City, Clare is stunned to receive a letter from Grit asking to stay with her for a few days. Days turn into months as the aunt and niece get to know each other and try to live in the present while understanding the past. VERDICT Best-selling author Rice's 30th book is an outstanding read that both chills and warms the soul. Her descriptions of abuse are startling and unnerving, while her vibrant verbal paintings of birds and nature are calming and uplifting. This hard-to-put-down story about how family ties can be undone and sometimes retied is compelling and will undoubtedly resonate with fans of contemporary women's fiction. Highly recommended. [See Prepub Alert, 12/5/11.]—Samantha J. Gust, Niagara Univ. Lib., NY
Source: http://luannerice.net/wp-content/uploads/2...
In Luanne's Blog, News, Notes Tags Library Journal, Little Night, Luanne Rice, Publishers Weekly, starred review


text:
Stanley Spencer (ur. 30 czerwca 1891 w Cookham, zm. 14 grudnia 1959 w Cliveden (Buckginghamshire)) – angielski malarz.

Życie i twórczość
Artysta był silnie związany z rodzinnymi stronami i przez całe życie pozostawał ważną osobistością swojego miasteczka. Już podczas nauki w Slade School of Fine Art Stanley Spencer zdobył przydomek Cookham. Atrakcyjne położenie na brzegu Tamizy zapewniały mu nie tylko inspirację, ale także plenery, które odpowiadały bardzo osobistemu podejściu do sztuki. Oryginalne, charakterystyczne obrazy ukazujące miasteczko jako raj na ziemi łączyły w sobie duchowość z ziemską swojskością. Do najsłynniejszych z tych prac należy Rezurekcja w Cookham (1924-27), radosna, przejmująca wizja dnia sądu ostatecznego odbywającego się na lokalnym przykościelnym cmentarzu.

Podczas I wojny światowej Spencer został wcielony do wojska. Początkowo służył jako szpitalny sanitariusz, potem jako zwykły żołnierz w Macedonii. Doświadczenia te stały się fundamentem murali wykonanych dla Sandham Memorial Chape w Burghclere (Hapshire). W czasie II wojny światowej otrzymał zlecenie namalowania stoczni w Port Glasgow w Szkocji. Dopiero później powrócił do tematu zmartwychwstania.

Elementy autobiograficzne przewijają się w całej twórczości Spencera, ich wyboru autor dokonywał według swej osobistej skali ważności. Zaabsorbowanie miłością religijną i cielesną zaowocowało niedokończonym (chociaż ambitnym) projektem zwanym Chrch-House lub Chapel of Me. Ok. 1937-1938 powstało Piękno miłości, odważna erotycznie seria obrazów, które przedstawiają ekscentryczne, niedobrane fizycznie pary. Spencer malował także intymne, naturalistyczne portrety swojej rodziny i własne, jak Double Nude Portrait: The Artist and His Second Wife (1937), mimo że ludzkie postacie w większości jego prac są naiwnie stylizowane.

Główne dzieła
 Travoys Arriving with Wounded Soldiers at a Dressing Station at Smol, Macedonia, 1919 (Imperial War Museum, Londyn)
 Rezurekcja w Cookham, 1924-27 (Tate Collection, Londyn)
 Double Nude Portrait: The Artist and His Second Wife, 1937 (Tate Collection, Londyn)
 Piękno miłości, ok. 1937-38 (kilka kolekcji, w tym m.in. Stanley Spencer Gallery, Cookham)
 Wioska w niebie, 1937 (Manchester City Art Gallery, Manchester)
 Shipbuilding on the Clyde, 1940-46 (Imperial War Museum, Londyn)

Przypisy

Brytyjscy malarze
Uczestnicy I wojny światowej (Imperium brytyjskie)
Urodzeni w 1891
Zmarli w 1959

redpajama_set_name: RedPajamaWikipedia


that I filled with setscrews. As I'm now shooting my more recently (April 2017) aquired FWB LP100, the LP80 has been sitting 9in the pistol case untouched. I decided to mount an optic and perhaps use it for NRA Bullseye practice. The problem was finding a set of rings that would engage the recoil lug recess at the rear of the mount. Most rings I tried didn't work, as the recess is so far to the rear of the rail that there isn't enough rail behind it to support a ring. But way back in the rear of my scope mount box was a long discontinued B-Square mount I bought back in the 1980s. If you look at the photo at the top of this article, you'll see that it hangs way over the rear of the scope rail- but it's still solidly mounted.
The recoil forces acting on the moving part of this recoilless pistol are actually pretty violent- the receiver only moves a short distance, but it's very light, and the movement needed to counterbalance the forces exerted by the spring and piston is sharp and rapid. When I tried to mount a scope using rings without a recoil lug, three shots was all it took to dislodge the rings from the rail. I mounted a Millet SP1 for my test, as it's a pretty rugged sight given that it sell for around $50.


text:
{ "item_title" : "Case Closed, Vol. 73, 73", "item_author" : [" Gosho Aoyama "], "item_description" : "Can Detective Conan crack the case...while trapped in a kid's body? Jimmy Kudo, the son of a world-renowned mystery writer, is a high school detective who has cracked the most baffling of cases. One day while on a date with his childhood friend Rachel Moore, Jimmy observes a pair of men in black involved in some shady business. The men capture Jimmy and give him a poisonous substance to rub out their witness. But instead of killing him, it turns him into a little kidJimmy takes on the pseudonym Conan Edogawa and continues to solve all the difficult cases that come his way. All the while, he's looking for the men in black and the mysterious organization they're with in order to find a cure for his miniature malady. A wealthy woman with a clock obsession is murdered after receiving a threat from the Guardian of Time. What is the secret of the old clock? Can Conan make a timely arrest? Then Conan takes on a dangerously tasty case at a noodle shop where the specialty of the house is Ramen to Die For. And at the scene of a murder, Conan and Rachel meet a teen detective who matches them at sleuthing and martial arts ", "item_img_path" : "https://covers3.booksamillion.com/covers/bam/1/97/470/961/1974709612_b.jpg", "price_data" : { "retail_price" : "9.99", "online_price" : "9.99", "our_price" : "9.99", "club_price" : "9.99", "savings_pct" : "0", "savings_amt" : "0.00", "club_savings_pct" : "0", "club_savings_amt" : "0.00", "discount_pct" : "10", "store_price" : "9.99" } }
Case Closed, Vol. 73, 73
by Gosho Aoyama
On Order. Usually ships in 2-4 weeks
Can Detective Conan crack the case...while trapped in a kid's body?
Jimmy Kudo, the son of a world-renowned mystery writer, is a high school detective who has cracked the most baffling of cases. One day while on a date with his childhood friend Rachel Moore, Jimmy observes a pair of men in black involved in some shady business. The men capture Jimmy and give him a poisonous substance to rub out their witness. But instead of killing him, it turns him into a little kid Jimmy takes on the pseudonym Conan Edogawa and continues to solve all the difficult cases that come his way. All the while, he's looking for the men in black and the mysterious organization they're with in order to find a cure for his miniature malady.
A wealthy woman with a clock obsession is murdered after receiving a threat from the Guardian of Time. What is the secret of the old clock? Can Conan make a timely arrest?
Then Conan takes on a dangerously tasty case at a noodle shop where the specialty of the house is Ramen to Die For. And at the scene of a murder, Conan and Rachel meet a teen detective who matches them at sleuthing and martial arts
Publish Date: January 2020
Gosho Aoyama
$9.99 Paperback
Case Closed, Vol. 78


Forests are essential for global development and preventing dangerous climate change but currently, markets fail to realise this.
We support partnerships that deliver on commitments for deforestation-free commodities, reduce the pressure on forests, and improve livelihoods by 2020.
We provide grant finance and technical assistance to propose alternatives to business as usual in the land use sector. We support the private sector in partnerships with the public sector and people – the communities that depend on forests – that can deliver on deforestation-free commitments and improve livelihoods by 2020.
Our projects work across all forest landscape types and entire market systems because there is no one-size-fits all solution to deforestation and forest degradation. Our portfolio primarily consists of Forest Partnerships which span four hotspot regions for deforestation and forest degradation – West and Central Africa, East Africa, Southeast Asia and Latin America.
We are always looking for new ideas for partnerships that can catalyse investment in forests and sustainable land use. If you have an idea for a Forest Partnership, enabling conditions project or a demand-side project, you can send it to us through our online application portal.
Partnerships for Forests and TechnoServe are working together to protect Ethiopia's ecologically unique and biodiverse habitats while increasing the livelihoods of forest coffee farmers. "We were handed the forest by our parents, and we must preserve it," says Amanuel Trefe (pictured above, right), before setting off into the Gabrebecho Forest in western Ethiopia's Kaffa zone.


Infinergy Solar Hot Water is the New Zealand representative (importer, distributor and reseller) via a network of professionals of world leading brand CHROMAGEN. Trusted quality since 1962. Proven in NZ conditions for over ten years.
to get the name of your nearest representative / installer.


Each month JustOT like to recognise one of our locums for their personal achievements and commitment to the Occupational Therapy Service and this month we have the pleasure of recognising Brooke, our wonderful OT from Adelaide.
Brooke started working for JustOT earlier this year and has since received excellent feedback from both her clients and her recruitment consultant, Julie.
Brooke was nominated by Julie for her continuous hard work, who also added 'Brooke is very deserving of the Locum of the Month title for August and she is a pleasure to work with'.
What do you enjoy about being a locum? I enjoy being able to travel to different locations in the UK. I love meeting lots of new people and all the different role opportunities.
Most memorable moment within your career? Doing a home visit to a house boat as the owner kept falling over in the boat and was worried she would fall from the gang plank getting onto the house boat!


Kansagra said it's possible that parents under-reported kids using devices at night, but more likely, the technology is simply interfering with sleep hygiene. "For example, children who are allowed to keep devices in their room may be more likely to avoid a good sleep routine, which we know is helpful for sleep," he said.
Other recommendations for good sleep hygiene include not exercising (physically or mentally) too close to bedtime; establishing a regular sleep schedule; limiting exposure to light prior to sleep; avoiding stimulants such as alcohol, caffeine, and nicotine in the hours before bedtime; and creating a dark, comfortable and peaceful sleep environment.


In the last few years, Gyumri has seen a lot of movement. The Gyumri Technology center, or "Techno park", opened its doors in 2014, and more recently, the Friends of Gyumri foundation, Gyumri Project Hope, and dozens of international conferences and events have taken root. You may remember last year's Luys i Luso installation in October 2015, or this year's workshop by John Stanmeyer of National Geographic. This summer a documentary screenwriting residency brought together Armenian, Georgian and Turkish screenwriters and directors to promote regional exchange. Slowly but surely, things are starting to bubble.
Anna, for one, is convinced that Gyumri has the potential to become "the next Tbilisi" (understand: a hotspot for European travelers, architecture lovers, and party enthusiasts). But until it achieves this kind of international visibility, Gyumri remains a core national artistic hub, and that's the reason why we're so drawn to it.
We went there on Saturday to partake in one of our favourite activities: location scouting. Finding a good location is one of the key success factors for any pop up gallery, and it comes with its own set of rules: we need a space that's empty, full of character, in line with the exhibit's theme, equipped with amenities such as heating and running water (a bathroom is always a plus), and that's not too far from the city center….
We don't want to disclose too much for now, but let us say this: we're impressed. Very impressed.
But HAYP prep-work is not only about taking Instagram pictures in beautiful, dusty houses (although if you're into that… you should definitely follow us on Instagram). This weekend, we also reconnected with Susanna from KASA foundation (where we gave a talk back in February) and discussed potential collaboration for this upcoming project.
We also had the chance to catch up with Michel Davoudian, CEO of APAGA Technologies. If the name sounds familiar to you, that's because APAGA sponsored HAYP4.0: IN MOTION – when we took over the Yerevan-Gyumri train with art and performance for 4 days.
Last but not least, on Sunday, after hearing about it for months, we finally got to check out Rob's Home, a new artsy bar that we quickly dubbed the "Calumet of Gyumri".
We'll go back there very soon, and in the meantime, expect to hear from us! We'll soon publish a call to events and performers for our HAYP 8.0 Event Week, from November 18 to 28.


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Today, January 6, is not just the 12th Day of Christmas, otherwise known as Epiphany, when the Wise Men slapped their foreheads and said "Doh! So that's who this kid is! By the way, who was supposed to buy the card?"
If everyone out there bought one Smith a drink today, think what a better world this would be. And if we could just keep the spirit of National Smith Day alive in our hearts year 'round, there'd be no more wars, hurricanes, traffic accidents, or ferret bites. Sigh...I'm a dreamer, it's true.
Anyway, check out this goofy holiday site and try to see past the vertigo-inducing design to its underlying truth: nearly every day is a holiday. Every day, someone, somewhere, is observing a special time, taking a few moments to mark the year's passing, wiping away a wistful tear at the memory of Leap-Second Time Adjustment Days gone by.
Thanks to Thomas Roy of Lebanon, PA, we can now celebrate National Dimpled Chad Day (you missed it this past Tuesday), or better yet, Northern Hemisphere Hoodie-Hoo Day, February 20, in which you wave a beach blanket at the sun at high noon and shout "Hoodie-Hoo!" twice, in hopes of warmer weather. To me, shouting "Hoodie-Hoo!" seems like a durn good idea just about any day or time, except maybe during a board meeting or a funeral.
See, Mr. Roy invents holidays. He is, in a sense, a holidaysmith.
On the holiday site I discovered that my birthday takes place on National Town Criers Day and during National Farriers Week, which seems fitting for a Smith.
Check out your own birthday. Sorry if it's July 15.


Can I customize my signup form with CSS?
I am trying to put a sign up form on my home page but it does not look good with the rest of my site. 1. How do you remove the white background that is by default? The HTML in the sign up form builder isn't really allowing me to make any real adjustments. 2. Is there a way to edit the CSS? For example, I don't want a thin grey border around the form. 3. I want to move my submit tag up next to the email field, how do I edit that table? I don't see anything in the HTML for that either.
If you decide to do it, you'll go to your account settings and click the API key tab to generate the API key. You can check out the calls and help documentation here: http://api.myemma.com. You'll want to carefully look over the sample documentation to make sure it's within your skill-set because support is not provided for this feature.


The young architect was typical of a generation that grew up in an early twentieth century China recently defeated in the Opium wars, and one that was seeking to carve its own identity away from Western influence. As such, he led the way in creating a modern look with a distinctly Chinese flavour, despite having received an education in L'Ecole Nationale des Beaux Arts in Paris. After graduating and returning to China he became a professor at the University of Nanjing; later on receiving commissions to design residential buildings, including the Carlton building on Huanghe Lu. There was a bit of a professional blip, however, when he fled with his family to the USA after the Japanese invasion, to run first a Laundromat then a chicken farm, before returning to teaching and architecture in the '60s. He died in his nineties, and is remembered as the first Chinese Art Deco architect.
The Chinese American architect – born in New York – had a design style that reflected his mixed background, often combining Art Deco elements with Chinese-style features such as pagoda roofs and latticework. Before sailing to China, Lee worked for architect Henry K Murphy who, according to Historic Shanghai, 'was a firm advocate of adapting traditional Chinese styles to modern uses'. Lee designed YMCAs and YWCAs throughout China, in eleven cities in total, with four in Shanghai – including one on Yuanmingyuan Lu (Rockbund), with a strikingly carved entrance. In September of this year, Lee was honoured with a retrospective of his work at the Museum of Chinese in America.
One of several Chinese architects who studied at the University of Pennsylvania under the tutelage of Paul Philippe Cret, Fan – also known as Fan Wenzhao – Robert Fan toured extensively in Europe and America resulting in a preference for modern, Western architecture – he is responsible for the 'pure Art Deco' Majestic Theatre on Jiangning Lu in Jingan, built in 1941 and the Shanghai Concert Hall beside Yanan Dong Lu, a neoclassical design that looks wholly European.


Now for the hardest question of all: who's your favorite pokemon?
Difficult, right? There's only hundreds to choose from. And what about the different types? Do you like lightning or dragon? Fairy or water? Do you prefer a specific generation? What about a specific evolution? When all the factors are considered, there's practically too many to choose from. Picking a favorite is near impossible.
Even though everyone will have slightly different tastes on pokemon, we can all probably agree on one particular pokemon being fabulous: Pikachu. We all fell in love with Ash's adorable yellow pokemon when those red cheeks started crackling for the first time. Even if Pikachu's not your be-all-end-all of favorite pokemon, every pokemon fan has a soft spot for this little lightning zapper. So feel free to show off your adoration of Pikachu with this Pokemon Pikachu Smile Juniors Hooded Raglan Shirt. This white shirt has a rounded hemline and contrasting black sleeves. On the front is a picture of that oh-so-happy Pikachu with his name underneath. There's even a hood! This shirt is perfect for pokemon hunting, or simply showing off your love of Pikachu.


I'm ambivalent about this Mel Gibson-as-antihero movie. On the one hand, Gibson's character really has no redeeming values. He commits murder for revenge or to prove a point. He is someone I hope my neighbors don't admire. On the other hand, he is also smart, and watching him outwit his opponents is often satisfying. This is one of the most entertaining action movies since last summer. The energy was great, and the 60s cop-funk soundtrack was the perfect complement. If you are morally stable and unsqueamish, give it a try.
As producer, Gibson had parts of the film re-shot and re-edited to make his character not such a bad guy, even though that was the point of the picture. And he still came across as morally impoverished.
Out of Sight, Steven Soderbergh, 1998, a film that just had the same feel. The music, the moral ambiguity, and the anti-hero hero make these films flow together in my mind.