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Integrative and Comparative Biology Integrative and Comparative Biology, volume 55, number 5, pp. 843–855 doi:10.1093/icb/icv021 Society for Integrative and Comparative Biology SYMPOSIUM Integrated Immune and Cardiovascular Function in Pancrustacea: Lessons from the Insects Julián F. Hillyer1 Department of Biological Sciences, Vanderbilt University, VU Station B 35-1634, Nashville, TN 37235, USA From the symposium ‘‘Linking Insects with Crustacea: Comparative Physiology of the Pancrustacea’’ presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2015 at West Palm Beach, Florida. 1 E-mail: [email protected] Synopsis When pathogens invade the insect hemocoel (body cavity) they immediately confront two major forces: immune-responses and circulatory currents. The immune response is mediated by circulating and sessile hemocytes, the fat body, the midgut, and the salivary glands. These tissues drive cellular and humoral immune processes that kill pathogens via phagocytosis, melanization, lysis, encapsulation, and nodulation. Moreover, immune-responses take place within a three-dimensional and dynamic space that is governed by the forces of the circulatory system. The circulation of hemolymph (insect blood) is primarily controlled by the wave-like contraction of a dorsal vessel, which is a muscular tube that extends the length of the insect and is divided into a thoracic aorta and an abdominal heart. Distributed along the heart are valves, called ostia, that allow hemolymph to enter the vessel. Once inside the heart, hemolymph is sequentially propelled to the anterior and to the posterior of the body. During an infection, circulatory currents sweep small pathogens to all regions of the body. As they circulate, pathogens encounter immune factors of the insect that range from soluble cytotoxic peptides to phagocytic hemocytes. A prominent location for these encounters is the surface of the heart. Specifically, periostial hemocytes aggregate in the extracardiac regions that flank the heart’s ostia (the periostial regions) and phagocytoze pathogens in areas of high flow of hemolymph. This review summarizes the biology of the immune and circulatory systems of insects, including how these two systems have co-adapted to fight infection. This review also compares the immune and circulatory systems of insects to that of crustaceans, and details how attachment of hemocytes to cardiac tissues and the biology of the lymphoid organ demonstrate that dynamic interactions between the immune and circulatory systems also occur in lineages of crustaceans. Introduction Insects are the most speciose group of animals (Trautwein et al. 2012; Gullan and Cranston 2014; Misof et al. 2014). As a phylogenetic group, the class Insecta diverged from other pancrustaceans between 400 and 500 million years ago (Giribet and Edgecombe 2012; Legg et al. 2013), and its members vary greatly in size, ecology, and lifestyle. Regardless of these differences, all insects share many commonalities. One of these is their constant contact with infectious agents (Vega and Kaya 2012). Viral, bacterial, fungal, protozoan, and metazoan pathogens infect insects through breaches in their exoskeleton and via ingestion. Some insects even infect other insects; for example, parasitoid wasps lay their eggs inside or on the surface of other insects, and the immature stages of these parasitoids subsist on, and eventually kill, their insect hosts (Pennacchio and Strand 2006). Because pathogens pose a menacing threat to their survival, insects have developed multiple barriers that resist infection. As an encompassing physical barrier, the hydrophobic exoskeleton, or cuticle, shields the body from microbes that come into contact with the insect’s outer surface (Siva-Jothy et al. 2005; Lundgren and Jurat-Fuentes 2012). Internally, physical barriers such as the non-cellular peritrophic matrix of the gut and the chitinous trachea of the respiratory system prevent the entry of pathogens into the body cavity, or hemocoel (Siva-Jothy et al. 2005; Kato et al. 2008; Lundgren and Jurat-Fuentes 2012). Additional adaptations such as the cibarial or Advanced Access publication April 21, 2015 ß The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: [email protected] 844 pharyngeal armature of the digestive system physically destroy large pathogens as they are ingested (Omar and Garms 1975; McGreevy et al. 1978). These physical barriers, while effective, are not absolute. To overcome infectious agents that circumvent these physical barriers, insects have developed a powerful immune system. An insect’s immune system, while lacking many aspects of the adaptive immune system of vertebrate animals (e.g., clonal selection), is highly complex in terms of time, space, and mechanism of action (Siva-Jothy et al. 2005; Beckage 2008; Rolff and Reynolds 2009). Pathogens elicit immune-responses as they enter the hemocoel or when they are present in the lumen of the midgut. Inside the hemocoel are cells with immune function as well as cardiovascular pumps that propel hemolymph (insect blood) to all areas of the body. Thus, when pathogens enter the hemocoel they immediately confront at least two major forces: (1) immune-responses with the capacity to destroy pathogens, and (2) circulatory currents that control or affect the movement or migration of pathogens. Although a significant amount of work has been directed toward understanding insects’ immune-responses (Beckage 2008; Rolff and Reynolds 2009), and to a lesser extent the anatomy of the circulatory system (Hertel and Pass 2002; Pass et al. 2006; Wirkner et al. 2013), it has not been until recently that researchers have assessed how circulatory currents affect immune-responses (King and Hillyer 2012). This review summarizes our current understanding of the immune and circulatory systems of insects, and describes how these two systems interact during the course of an infection. Furthermore, this review also compares the immune and circulatory systems of insects to that of crustaceans, and concludes with a description of how the immune and circulatory systems of crustaceans also interact during the course of an infection. J. F. Hillyer cellular and humoral immunity is blurred because many humoral components are produced by hemocytes and participate in cellular immune-responses (Bartholomay et al. 2004; Zou et al. 2008; Baton et al. 2009; Pinto et al. 2009). Following invasion, insects recognize pathogens by the molecular interaction between insect-derived pattern-recognition receptors and pathogen-associated molecular patterns (Fig. 1) (Das et al. 2009; Kurata 2014). In insects, thioester containing proteins, C-type lectins, gram-negative binding proteins, immunoglobulin superfamily proteins, peptidoglycan recognition proteins, and fibrinogen-related proteins may serve as pattern-recognition receptors. Recognition of a microorganism leads to downstream events that kill pathogens via five broadly defined mechanisms: phagocytosis, melanization, encapsulation, nodulation, and lysis (Hillyer 2010; Imler 2014; Satyavathi et al. 2014). Several signaling Immune-response of insects The immune-response of insects is conceptually divided into cellular and humoral components. The cellular response includes phagocytosis, encapsulation, and nodulation by immune cells called hemocytes (Strand 2008; Hillyer and Strand 2014; Honti et al. 2014). The humoral response includes patternrecognition receptors, antimicrobial peptides, phenoloxidase-based melanization, and reactive oxygen and nitrogen intermediates (Christensen et al. 2005; Nappi and Christensen 2005; Das et al. 2009; Hillyer 2010; Jiang et al. 2010; Kurata 2014). Regardless of this conceptual organization, the line between Fig. 1 The immune-response of insects. A pathogen enters an insect’s body through a breach in the cuticle or via ingestion. Once in the hemocoel, host-derived pattern-recognition receptors bind the pathogen, which leads to pathogen-recognition, immune signaling, and the activation of effector mechanisms that kill via phagocytosis, lysis, melanization, encapsulation, or nodulation. The RNA interference pathway (not shown) is also important in the antiviral response. (This figure is available in black and white in print and in color at Integrative and Comparative Biology online.) Insect immune and cardiovascular function pathways modulate these killing mechanisms. The most commonly studied immune signaling pathways in insects are the Toll, IMD, and JAK/STAT pathways, which result in the production of antimicrobial peptides and other effector proteins (Aggarwal and Silverman 2008; Tang 2009; Hwang et al. 2013; Clayton et al. 2014; Imler 2014; Zhong et al. 2012). Other pathways that have received less attention are the CED signaling pathways, which are modulators of the phagocytosis response (Moita et al. 2005; McPhee et al. 2010). These immune signaling pathways encode a certain degree of specificity. In Drosophila, for example, the Toll pathway is primarily activated by fungi and Gram(þ) bacteria whereas the IMD pathway is activated by Gram() bacteria (Aggarwal and Silverman 2008). In mosquitoes, the Toll pathway is primarily activated by fungi, Gram(þ) bacteria, several viruses, and Plasmodium berghei, whereas the IMD pathway is activated by Gram() bacteria and Plasmodium falciparum (Hillyer 2010; Clayton et al. 2014; Severo and Levashina 2014). Likewise, the strength of phagocytosis and melanization is dependent on the nature of the infection (Hillyer et al. 2004). Anatomy of the immune response of insects Pathogens in the hemocoel are contained within an open body cavity that houses all of the insect’s internal organs. Among the tissues that participate in immune-responses are the hemocytes, the fat body, the salivary glands, and the gut (Fig. 2) (Hillyer 2010; Jiang et al. 2010; Mueller et al. 2010; Lemaitre and Miguel-Aliaga 2013). The insect gut extends the length of the body and is divided into functionally discrete regions (Lemaitre and MiguelAliaga 2013). Enteric epithelial cells produce lytic factors that kill pathogens within the lumen of the gut or as they cross from the gut lumen to the 845 hemocoel (Lehane et al. 1997; Osta et al. 2004; Buchon et al. 2009, 2013). The salivary glands are located in the anterior part of the thorax. Salivary acinar cells produce humoral immune factors, some of which affect the viability of the pathogen (Ferrandon et al. 1998; Pinto et al. 2008). The fat body is composed of loosely associated cells that line the insect’s integument (Martins et al. 2011). Cells of the fat body, besides functioning in energy storage and the synthesis of the vitellogenin precursors required for the production of eggs (Arrese and Soulages 2010), synthetize a multitude of humoral factors with lytic activity (Aggarwal and Silverman 2008; Hillyer 2010; Imler 2014). Although the midgut, salivary glands, and fat body are integral components of the immune systems of insects, the primary cells involved in the regulation of immuneresponses are the hemocytes (Strand 2008; Hillyer and Strand 2014; Honti et al. 2014). Hemocytes respond to breaches in the body wall, where, along with transglutaminase and other soluble hemolymph proteins, they coagulate wounds and seal them through the melanization pathway (Lai et al. 2001; Theopold et al. 2004; Babcock et al. 2008). Hemocytes also phagocytoze pathogens within minutes of exposure to them (Hillyer et al. 2003; King and Hillyer 2012), and synthetize pattern-recognition receptors and humoral immune factors that kill pathogens via lytic and melanization pathways (Bartholomay et al. 2004; Zou et al. 2008; Baton et al. 2009; Pinto et al. 2009). Based on their structural and functional characteristics, insects’ hemocytes can be classified using two non-exclusive criteria. The first criterion divides hemocytes by their functional, morphological, enzymatic, and cytochemical characteristics whereas the second criterion divides hemocytes by their anatomical location (Strand 2008; Hillyer and Strand 2014). Studies that have classified hemocytes using the first Fig. 2 Anatomy of the immune-response of insects. Multiple tissues are involved in regulating or executing immune-responses. The primary immune cells are the hemocytes, which mediate the cellular immune-response and participate in the humoral immuneresponse. Hemocytes are found in circulation (circulating hemocytes) and are also found attached to tissues (sessile hemocytes). Some sessile hemocytes are aggregated around the valves (ostia) of the heart, and are called periostial hemocytes. Fat-body cells produce numerous humoral immune factors. The midgut and the salivary glands also participate in the humoral immune-response. (This figure is available in black and white in print and in color at Integrative and Comparative Biology online.) 846 criterion (morphology) have failed to reach a consensus nomenclature that can be used across Insecta. Names used to describe populations of hemocyte cells include plasmatocytes, granulocytes, oenocytoids, crystal cells, prohemocytes, lamellocytes, spherulocytes, adipohemocytes, and thrombocytoids (Hillyer and Christensen 2002; Strand 2008). Not all of these types of cells are present in all insects, and cells that in independent studies have been named differently often overlap (sometimes completely) in structural and functional characteristics (Hillyer and Christensen 2002). Even within a taxon, morphological analyses have often yielded confusing nomenclatures for hemocytes, but functional parallels within and between taxa are often found (Strand 2008; Hillyer and Strand 2014). For example, the granulocytes of mosquitoes (Order: Diptera) and Lepidoptera are phagocytic cells that are functionally analogous to the plasmatocytes of Drosophila melanogaster (Order: Diptera). Likewise, the oenocytoids of mosquitoes and lepidopterans are cells that produce enzymes involved in the melanization pathway and are functionally analogous to the crystal cells of D. melanogaster. Using the second criterion (anatomical location), entomologists have uniformly divided hemocytes into two populations: circulating hemocytes and sessile hemocytes. Specifically, hemocytes that circulate with the hemolymph are called circulating hemocytes whereas those that are attached to tissues are called sessile hemocytes (Babcock et al. 2008; Markus et al. 2009; King and Hillyer 2012, 2013). Sessile hemocytes are distributed throughout the abdominal and thoracic wall, the head, the extremities, and the surface of the internal organs. In adult mosquitoes, approximately 75% of hemocytes occur in circulation whereas approximately 25% occur in a sessile state (King and Hillyer 2013). However, circulating and sessile hemocytes are capable of changing their anatomical location; circulating cells can become sessile and vice versa (Babcock et al. 2008; Markus et al. 2009; Makhijani et al. 2011; King and Hillyer 2012). Presumably, all of the functionally distinct populations of cells (criterion one) can be found both in circulation and in a sessile state (criterion two). The fact that the majority of hemocytes are called circulating hemocytes signifies that these cells are in constant motion. These cells, along with small pathogens that may have entered the hemocoel, are propelled throughout the body by the muscular pumps of the insect’s circulatory system (Hillyer et al. 2007; Babcock et al. 2008; King and Hillyer 2012). J. F. Hillyer Circulation of hemolymph in insects The circulatory system of insects, among other things, functions in the transport of nutrients, waste, hormones, and immune factors to their target sites (Harrison et al. 2012; King and Hillyer 2012; Chapman et al. 2013; Klowden 2013). Anatomically, this cardiovascular system consists of hemolymph (blood), the hemocoel, and a series of muscular pumps (Fig. 3A) (Pass 2000; Hertel and Pass 2002; Pass et al. 2006). The primary pump is the dorsal vessel, which is a tube-like structure that extends along the dorsal midline of the insect and is divided into a thoracic aorta and an abdominal heart (Jones 1977; Wasserthal 2007; Ejaz and Lange 2008; Glenn et al. 2010; League et al. 2015). In most adult insects, the heart periodically alternates between propelling hemolymph toward the head (anterograde direction) and propelling hemolymph toward the posterior of the abdomen (retrograde direction), and this is accomplished by sequentially alternating the direction of wave-like contractions of cardiac muscle (Fig. 3B) (Gerould 1933; Dulcis and Levine 2005; Dulcis et al. 2005; Wasserthal 2007; Glenn et al. 2010; Andereck et al. 2010; League et al. 2015). When the heart contracts anterograde, hemolymph enters the vessel through valves, called ostia (singular: ostium), that are located in each abdominal segment, and exits the vessel through an excurrent opening located near the head (Glenn et al. 2010; League et al. 2015). When the heart contracts retrograde, the abdominal ostia close and hemolymph enters the vessel through one pair of ostia located at the thoraco-abdominal junction and exits through excurrent openings located in the last abdominal segment. Once hemolymph is discharged into the hemocoel, it flows toward the ostia and re-enters the heart, thus completing its circulatory cycle. This description of the physiology of the dorsal vessel reflects what occurs in adult Diptera, and is the basic plan found across Insecta. Variations of this general plan include the presence or absence of (1) segmental blood vessels that originate from the heart, (2) directional reversals of heartbeat (in which case the heart only contracts anterograde), (3) ostia in the aorta, and (4) excurrent (outflowing) ostia in the heart or aorta (Gerould 1933; Hertel and Pass 2002; Pass et al. 2006; Ejaz and Lange 2008; League et al. 2015). The hearts of insects contract myogenically (Jones 1977; Dulcis et al. 2001; Slama and Lukas 2011), but neurohormones and neurotransmitters affect the speed and directionality of the contractions. Among the cardiomodulatory factors are crustacean Insect immune and cardiovascular function 847 Fig. 3 Circulation of hemolymph in insects: focusing on the order Diptera. (A) Structure of the insect circulatory system. The cardiovascular system is composed of hemolymph (blood), a body cavity called the hemocoel (gray), and muscular pumps. The primary hemolymph pump is the dorsal vessel, which is a tube that extends the length of the insect along its dorsal midline. The portion of the dorsal vessel that is in the thorax is called the aorta, and the portion that is in the abdomen is called the heart. Distributed along the heart are paired valves called ostia (singular: ostium) that accept hemolymph from the hemocoel. The dorsal vessel discharges hemolymph back into the hemocoel through excurrent openings located at the ends of the body. Secondary pumps, called accessory pulsatile organs (APOs) or auxiliary hearts, propel hemolymph into narrow areas of the body (e.g., the antennae and wings) or across areas that are distal to the dorsal vessel (the ventral abdomen). (B) Periods of flow of hemolymph in the hemocoel. Wave-like contractions of the heart drive hemolymph through the dorsal vessel. The heart periodically alternates between (1) contracting anterograde (toward the head) while the ventral abdomen is at rest, (2) contracting anterograde while the ventral abdomen contracts retrograde (toward the posterior of the abdomen), and (3) contracting retrograde while the ventral abdomen is at rest. When the heart contracts anterograde, hemolymph enters the dorsal vessel through abdominal ostia, whereas when the heart contracts retrograde hemolymph enters the dorsal vessel through ostia located at the thoraco-abdominal junction. (This figure is available in black and white in print and in color at Integrative and Comparative Biology online.) cardioactive peptide (CCAP), FMRFamide, FMRFamide-like peptides, proctolin, neuropeptide F, serotonin, and glutamate (Dulcis and Levine 2005; Dulcis et al. 2005; Nichols 2006; Ejaz and Lange 2008; Lee et al. 2012; Setzu et al. 2012; Estevez-Lao et al. 2013; Hillyer et al. 2014). Although the action of these neuropeptides and neurotransmitters is rather conserved, there are large, taxon-specific variations in the frequency of contraction of the dorsal vessel. For example, the heart of the stick insect, Baculum extradentatum (order: Phasmatodea), contracts exclusively in the anterograde direction and at a frequency of 0.2 Hz (Ejaz and Lange 2008), whereas the hearts of the adult stages of the fruit fly, D. melanogaster, and the hoverfly, Episyrphus balteatus, contract bidirectionally at 4 and 7 Hz, respectively (Wasserthal 2007; Slama 2012). The heart of the mosquito, Anopheles gambiae, only reverses the direction of contraction during the adult life stage (10 reversals per minute) and contracts at approximately 2 Hz (Glenn et al. 2010; Estevez-Lao et al. 2013; League et al. 2015). Relying on a single vessel to propel hemolymph has limitations. Thus, insects have additional pumps, 848 called accessory pulsatile organs, or auxiliary hearts, that drive hemolymph through areas that would otherwise experience low flow (Fig. 3A) (Pass 2000). For example, to reduce hemolymph stagnation in the ventral abdomen (an area distant from the dorsal vessel), mosquitoes synchronize the contraction of the heart and the contraction of the musculature associated with the ventral diaphragm (Fig. 3B) (Andereck et al. 2010). Additional accessory pulsatile organs found in insects propel hemolymph into the antennae, the wings, and other dead-end structures (Lehmacher et al. 2009; Hertel et al. 2012; Boppana and Hillyer 2014; Hustert et al. 2014). Co-adaptation of the circulatory and immune systems of insects Studies in mosquitoes, fruit flies, and grasshoppers have shown swift flow of hemolymph in the hemocoel (Lee and Socha 2009; Andereck et al. 2010; Glenn et al. 2010; Choma et al. 2011; League et al. 2015). Studies in fruit flies and mosquitoes have also documented the movement of hemocytes in vivo (Babcock et al. 2008; King and Hillyer 2012; Moreira et al. 2013). However, a careful look at the primary scientific literature shows that few studies have assessed whether there is a dynamic interaction between circulatory currents and immune processes. Such an association would be expected to exist, as hemocytes, humoral immune factors, and pathogens exist in intimate association with the circulatory organs of insects and have likely shared such an existence throughout the course of animal evolution (Siva-Jothy et al. 2005; Hartenstein and Mandal 2006). Yet, until recently, whether there was a dynamic interaction between the circulatory and immune systems of insects remained unexplored, in large part due to our poor understanding of hemolymph currents and our poor understanding of the spaciotemporal dynamics of insects’ immuneresponses. In response to injury, circulating hemocytes of D. melanogaster recognize and bind wounds through a process called adhesive capture (Babcock et al. 2008). Hemocytes of other insects also aggregate at the sites of wounds (Rowley and Ratcliffe 1978; Lai et al. 2001; Krautz et al. 2014), and although the mechanics of these occurrences have not been described, it is likely that they occur through a similar, adhesive mechanism. Dynamic changes in the distribution of circulating and sessile hemocytes have also been observed in fruit flies and mosquitoes, and several molecular factors have been implicated in J. F. Hillyer hemocytes’ migratory processes (Markus et al. 2009; King and Hillyer 2013; Moreira et al. 2013). Although the above examples offer a significant glimpse into the behavior of hemocytes in vivo, the best described process that exemplifies the dynamic interaction between insects’ immune and circulatory systems is the hemocyte-mediated sequestration of pathogens on the surface of the heart. In the absence of infection, hemocytes are present in or on the hearts of mosquitoes, stick insects, and presumably other insects (da Silva et al. 2012; King and Hillyer 2012, 2013). Furthermore, in adult mosquitoes (Hillyer et al. 2007; King and Hillyer 2012, 2013) and fruit flies (Elrod-Erickson et al. 2000; Kocks et al. 2005; Cuttell et al. 2008; Akbar et al. 2011; Shiratsuchi et al. 2012; Stone et al. 2012; Horn et al. 2014), infection results in the aggregation of pathogens in distinct regions of the dorsal part of the abdomen. Experimental molecular perturbations in Diptera and Lepidoptera have yielded a similar phenotype. Mainly, RNAi-based knockdown of specific immune genes results in melanization deposits forming in a pattern that is almost identical to that observed after infection (Michel et al. 2005; Schnitger et al. 2007; Bao et al. 2011; Yassine et al. 2014). The pattern of aggregation of pathogens in the dorsal part of the abdomen is intriguing from a circulatory perspective; pathogens aggregate in symmetrical lines that flank the heart within specific regions of each abdominal segment, and these regions of aggregation are immediately lateral of the heart’s ostia (Wasserthal 2007; Glenn et al. 2010; League et al. 2015). The process of the aggregation of pathogens near the heart’s ostia has only been examined at the cellular level in A. gambiae (Fig. 4) (King and Hillyer 2012, 2013). In this mosquito, the extracardiac regions surrounding the ostia, called the periostial regions, contain a resident population of hemocytes that are called periostial hemocytes (King and Hillyer 2012). Within seconds of infection, periostial hemocytes capture and phagocytoze pathogens as they flow with the hemolymph while en route to the lumen of the heart. Then, within minutes of infection, circulating hemocytes move into the periostial regions where they bind the heart-associated musculature and each other, thus increasing the number of periostial hemocytes and amplifying the localized phagocytosis. Periostial aggregation of hemocytes is dependent on time and on the dose of the infection, and once this process is initiated, the number of periostial hemocytes remains elevated for the lifetime of the mosquito (King and Hillyer 2012). Insect immune and cardiovascular function 849 Although the aggregation of hemocytes and pathogens at the periostial regions suggests that a physiological interaction exists between the circulatory and immune systems, this would only hold true if the infection-induced co-aggregation of hemocytes and pathogens that occurs on the surface of the heart is a spatially directed and site-specific event. Further experiments on A. gambiae showed that this is indeed the case. Specifically, infection induces an increase in the number of circulating and sessile hemocytes, but regardless of the status of infection the areas of highest density of hemocytes are the periostial regions, and these are also the only locations where pathogens noticeably aggregate (and are subsequently killed) during the course of an infection (King and Hillyer 2012, 2013). Given that periostial aggregation of hemocytes is induced by infection with multiple bacterial species and malarial parasites, as well as by the injection of inanimate particles and soluble immune elicitors (peptidoglycan and B-1,3-glucan), this immune process on the surface of the heart represents a basal cellular immune-response that relies on the interaction between the circulatory and immune systems (King and Hillyer 2012). The location of this immune response is ideal, as it places immune cells in the areas of the highest flow of hemolymph (Andereck et al. 2010; Glenn et al. 2010; League et al. 2015), thus maximizing the probability that the insect’s primary phagocytes will encounter circulating invaders. Is there a physiological interaction between the immune and circulatory systems of non-insect pancrustaceans? Fig. 4 Co-adaptation of the immune and circulatory systems of insects. In uninfected mosquitoes (top), sessile hemocytes called periostial hemocytes aggregate around the extracardiac regions that surround the heart’s ostia (the periostial regions). In response to infection (bottom), circulating and sessile hemocytes (including periostial hemocytes) phagocytoze pathogens. Infection also induces the migration of circulating hemocytes to the periostial regions, which results in an increase in the number of periostial hemocytes and an amplification of localized phagocytosis. (This figure is available in black and white in print and in color at Integrative and Comparative Biology online.) Insects and their flightless, six-legged relatives form a monophyletic group called the Hexapoda. The Hexapoda, in turn, is nested within a paraphyletic Crustacea (Giribet and Edgecombe 2012; Legg et al. 2013). Comparison of the immune and circulatory systems of insects with their crustacean counterparts shows many similarities (Tables 1 and 2; also see the review in this issue by K.G. Burnett, 2015). For example, the Toll, IMD, and JAK/STAT pathways are all present and active in crustaceans (Hauton 2012; Li and Xiang 2013), and many of the patternrecognition receptor-protein families that are immunologically important in insects have also been identified in crustaceans (Das et al. 2009; Chevalier et al. 2012; Tassanakajon et al. 2013; Wang and Wang 2013; Clark 2014). Furthermore, the phenoloxidase-based melanization cascade is an integral component of the immune response both of insects and crustaceans (Christensen et al. 2005; Amparyup 850 J. F. Hillyer Table 1 Comparison of the immune systems of insects and crustaceans Insects Crustaceans Pattern recognition receptors 3 3 Immune signaling pathways (e.g., Toll, IMD, JAK/STAT) 3 3 Circulating hemocytes 3 Sessile hemocytes 3 3 Periostial hemocytes 3 (at least in Diptera) Unknown Hemocytes in or on the heart 3 3 Lymphoid organ Not present 3 (in Penaeidae) Major humoral immune organ Fat body Hepatopancreas; lymphoid organ Phagocytosis 3 3 Lysis 3 3 Phenoloxidase-based melanization 3 3 Encapsulation 3 (some orders) 3 Nodulation 3 (some orders) 3 (some orders) RNA interference 3 3 3 et al. 2013), and RNA-interference functions in the antiviral response of these two groups of organisms (Bartholomay et al. 2012; Blair and Olson 2014). Finally, the primary immune cells in insects and crustaceans are the hemocytes, and in both groups these circulating and sessile cells mediate both cellular and humoral immune processes (Johnson 1987; Strand 2008; Baton et al. 2009; Pinto et al. 2009; Cerenius et al. 2010; Oliver et al. 2011; Tassanakajon et al. 2013; Hillyer and Strand 2014). Many traits pertaining to the cardiovascular system are also shared between crustaceans and insects (Table 2). The circulatory system of both groups is composed of a hemocoel, hemolymph, and muscular pumps (Wirkner et al. 2013). The primary pump in insects and crustaceans is the heart, and this organ is located along the dorsal midline of the animal in both groups. When compared with insects, however, the crustacean circulatory system shows higher diversity. For example, small cladocerans (e.g., Daphnia pulex) contain a bulb-like heart and a reduced arterial system, whereas large decapods (e.g., shrimp and lobsters) contain a powerful elongated heart that is connected to dense arterial and aortic systems that channel hemolymph to multiple regions of the organism (Wirkner et al. 2013). Regardless of these structural and mechanical Table 2 Comparison of the circulatory systems of insects and crustaceans Insects Crustaceans Hemocoel 3 3 Hemolymph 3 3 Muscular pumps 3 3 Heart is the primary pump 3 3 Heart valves are called ostia 3 3 Accessory pulsatile organs 3 (But not conserved) 3 (But not conserved) Hormonal control of contractions of the heart (e.g., CCAP) 3 3 Vessels emanating from the heart 3 (Segmental blood vessels in some orders) 3 (Arterial and aortic vessels in some orders) Directional reversals of heartbeat 3 (in adults of some orders) No differences, the open nature of the circulatory system is shared between insects and crustaceans, and many of the neuropeptides (e.g., CCAP and FMRFamidelike peptides) and neurotransmitters (e.g., serotonin and octopamine) that influence cardiac physiology in insects also influence cardiac physiology in crustaceans (Grega and Sherman 1975; Stangier et al. 1987; Johnson et al. 1997; Yazawa and Kuwasawa 1992; Walker et al. 2009; Estevez-Lao et al. 2013). To date, the infection-induced aggregation of hemocytes in the periostial regions of the crustacean heart has not been reported. However, direct and indirect data have shown that infection induces the aggregation of hemocytes in various regions of the body. For example, inoculation with bacteria, inanimate particles, or LPS reduces the number of circulating hemocytes in crabs, suggesting that insult to the immune system induces the aggregation of hemocytes in sessile tissues (Johnson et al. 2011). Furthermore, infection with Vibrio campbellii induces the aggregation of hemocytes in the gills of crabs (Johnson 1987; Ikerd et al. 2015), and bacterial infection also induces the aggregation of hemocytes in the gills and hepatopancreas of shrimp (Sahoo et al. 2007). As pertains to circulatory organs, infection induces the aggregation of hemocytes on the wall of the heart and in the arterial vessels of shrimp and crabs (Johnson 1976; Sagrista and Durfort 1990; Sahoo et al. 2007), and fixed phagocytes populate the endothelium of the hepatic arterioles of lobsters (Factor and Beekman 1990; Factor and Naar 1990), but 851 Insect immune and cardiovascular function perhaps the process that most closely exemplifies the co-adaptation of the circulatory and immune systems in crustaceans pertains to the immune activity of the lymphoid organ of penaeid shrimp. The lymphoid organ is a bi-lobed structure that is directly connected to the heart by means of a subgastric artery (Rusaini and Owens 2010). Contractions of the heart propel hemolymph into the subgastric artery, which directly empties into the lumen of each lobe of the lymphoid organ. The lymphoid organ is a major site of pathogen killing: large numbers of hemocytes and hemocyte-derived cells present within this organ contain phagocytozed bacteria and other invasive factors, which indicates that immune cells present in the lymphoid organ phagocytoze pathogens as they exit the heart, or that the lymphoid organ is a location where phagocytic hemocytes aggregate during the course of an infection (or both) (Anggraeni and Owens 2000; van de Braak et al. 2002; Burgents et al. 2005; Burge et al. 2007, 2009; Rusaini and Owens 2010). The lymphoid organ is also a major site where viruses accumulate, and where humoral immune factors involved in lytic and melanization processes are produced (Pongsomboon et al. 2008; Rusaini and Owens 2010). Thus, in a manner analogous to the sequestration of pathogens by periostial hemocytes in mosquitoes, the lymphoid organ of penaeid shrimp is a location where pathogens aggregate and are killed in an area of high flow of hemolymph. Concluding remarks In both insects and crustaceans, circulatory currents affect immune-responses. Specifically, currents of hemolymph drive the constant movement of hemocytes, immune-factors, and pathogens within the hemocoel. In some insects, infection induces the aggregation of hemocytes in the periostial regions of the heart, which places immune cells in areas of high flow of hemolymph, and thus increases the probability that they will encounter circulating pathogens (King and Hillyer 2012). Although this process has not been described in crustaceans, it is unclear whether researchers have investigated its occurrence. Nevertheless, in a manner functionally analogous to what is observed in insects, crustacean hemocytes are present in the wall of the heart and in the arterial system, and in penaeid shrimp the lymphoid organ filters hemolymph immediately after being propelled by the heart (van de Braak et al. 2002; Rusaini and Owens 2010). While it appears clear that the circulatory and immune systems both of insects and crustaceans interact during the course of an infection, many questions remain. For example, periostial aggregation of hemocytes has only been directly or indirectly observed in members of the insect orders Diptera and Lepidoptera (Elrod-Erickson et al. 2000; Kocks et al. 2005; Cuttell et al. 2008; Akbar et al. 2011; Bao et al. 2011; Shiratsuchi et al. 2012; Stone et al. 2012; Horn et al. 2014). Likewise, the presence of hemocytes in the heart and arterial system of crustaceans has mainly been reported within members of the order Decapoda, and the lymphoid organ is only present in members of the family Penaeidae (Factor and Naar 1990; Sagrista and Durfort 1990; Sahoo et al. 2007; Rusaini and Owens 2010). Thus, the evolutionary conservation of these responses within the insect and crustacean lineages remains unknown, as well as whether these interactions occur in other arthropods. Another outstanding question relates to development. Within an insect species, circulatory physiology can change dramatically during development. For example, the heart of an adult mosquito accepts hemolymph through the abdominal ostia and periodically reverses the direction of contraction, whereas the heart of a mosquito larva accepts hemolymph only through an incurrent posterior opening and contracts unidirectionally (League et al. 2015). It remains unclear whether developmentally associated changes in circulatory physiology influence the patterns of aggregation of hemocytes and pathogens during the course of an infection. Finally, various molecular pathways likely drive the processes of migration and phagocytosis by hemocytes. The identities of these pathways remain poorly understood, as well as whether they are conserved between the insect and crustacean lineages. Undoubtedly, future molecular and imaging studies will continue to shed light on the importance of circulatory currents in the antipathogen responses of crustaceans and insects. Acknowledgments I thank Dr Karen G. Burnett, Dr Jonas G. King, Dr Lyric C. Bartholomay, and Ms. Leah T. Sigle for fruitful discussions and for commenting on this manuscript. I am also grateful to Dr Harold Heatwole, Editor-In-Chief of Integrative and Comparative Biology, for editing this manuscript. Funding This work was supported by the U.S. National Science Foundation [IOS-1051636 and IOS1257936]. The funders had no role in the decision to publish or in the preparation of the manuscript. 852 References Aggarwal K, Silverman N. 2008. Positive and negative regulation of the Drosophila immune response. BMB Rep 41:267–77. Akbar MA, Tracy C, Kahr WH, Kramer H. 2011. The full-ofbacteria gene is required for phagosome maturation during immune defense in Drosophila. J Cell Biol 192:383–90. 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