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Integrative and Comparative Biology Integrative and Comparative Biology, volume 55, number 5, pp. 856–868 doi:10.1093/icb/icv094 Society for Integrative and Comparative Biology SYMPOSIUM Respiratory and Metabolic Impacts of Crustacean Immunity: Are there Implications for the Insects? Karen G. Burnett1 and Louis E. Burnett Grice Marine Laboratory, College of Charleston, 205 Fort Johnson Road and Hollings Marine Laboratory, Charleston, SC 29412, 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 Extensive similarities in the molecular architecture of the crustacean immune system to that of insects give credence to the current view that the Hexapoda, including Insecta, arose within the clade Pancrustacea. The crustacean immune system is mediated largely by hemocytes, relying on suites of pattern recognition receptors, effector functions, and signaling pathways that parallel those of insects. In crustaceans, as in insects, the cardiovascular system facilitates movement of hemocytes and delivery of soluble immune factors, thereby supporting immune surveillance and defense along with other physiological functions such as transport of nutrients, wastes, and hormones. Crustaceans also rely heavily on their cardiovascular systems to mediate gas exchange; insects are less reliant on internal circulation for this function. Among the largest crustaceans, the decapods have developed a condensed heart and a highly arteriolized cardiovascular system that supports the metabolic demands of their often large body size. However, recent studies indicate that mounting an immune response can impair gas exchange and metabolism in their highly developed vascular system. When circulating hemocytes detect the presence of potential pathogens, they aggregate rapidly with each other and with the pathogen. These growing aggregates can become trapped in the microvasculature of the gill where they are melanized and may be eliminated at the next molt. Prior to molting, trapped aggregates of hemocytes also can impair hemolymph flow and oxygenation at the gill. Small shifts to anaerobic metabolism only partially compensate for this decrease in oxygen uptake. The resulting metabolic depression is likely to impact other energy-expensive cellular processes and whole-animal performance. For crustaceans that often live in microbially-rich, but oxygen-poor aquatic environments, there appear to be distinct tradeoffs, based on the gill’s multiple roles in respiration and immunity. Insects have developed a separate tracheal system for the delivery of oxygen to tissues, so this particular tradeoff between oxygen transport and immune function is avoided. Few studies in crustaceans or insects have tested whether mounting an immune response might impact other functions of the cardiovascular system or alter integrity of the gut, respiratory, and reproductive epithelia where processes of the attack on pathogens, defense by the host, and physiological functions play out. Such tradeoffs might be fruitfully addressed by capitalizing on the ease of molecular and genetic manipulation in insects. Given the extensive similarities between the insect and the crustacean immune systems, such models of epithelial infection could benefit our understanding of the physiological consequences of immune defense in all of the Pancrustacea. Introduction Recently published molecular phylogenies and morphologic evidence strongly support the idea that crustaceans, together with insects and other hexapods, form a separate clade, the Pancrustacea, within the arthropod lineage (Regier et al. 2010; Legg et al. 2013). Extensive similarities in the molecular architecture of the crustacean immune system to that of insects certainly support this possibility, with both groups relying on parallel suites of patternrecognition receptors (PRRs), effector functions, and signaling pathways mediated in large part by hemocytes and their soluble products (Chevalier et al. 2012; Hauton 2012; Hillyer 2015). Nonetheless, the physiological contexts in which the immune system operates in crustaceans and insects Advanced Access publication July 29, 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]. Respiratory and metabolic impacts of crustacean immunity are quite different. In both groups the cardiovascular system facilitates the trafficking of hemocytes and the delivery of soluble immune factors, thereby supporting immune surveillance and defense along with other physiological functions such as transport of nutrients, wastes, and hormones. Unlike the insects, crustaceans also rely on their cardiovascular systems to mediate critical functions of gas exchange. In this article, we briefly review our current understanding of the immune and cardiovascular systems in crustaceans, focusing particularly on the widely studied Decapoda. Next, we examine evidence that activating immune defense mechanisms in the highly arteriolized cardiovascular system of the decapod crustaceans can impair respiration and metabolism. Finally, we review some key aspects of the immune and cardiovascular systems of insects, and consider whether there might be metabolic or other physiological costs associated with immune defense in the Insecta. Mechanisms of immunity in crustaceans Despite the absence of an adaptive immune system, crustaceans possess a potent web of innate immune defenses. A tough exoskeleton composed of chitin and reinforced by calcium salts and melanin, along with the peritrophic membrane of the midgut, serves as a first barrier to microbial invasion (Martin et al. 2006; Roer et al. 2015). Molting, the periodic shedding of the exoskeleton, reduces pathogen/parasite load on the external surface and replaces the damaged shell (Moret and Moreau 2012). To counter pathogens that penetrate the exoskeleton, there is a large suite of receptors that recognize potential pathogens and there are multiple effectors that inactivate and eliminate those threats, along with a network of signaling pathways that coordinate and modulate the effector response (Cerenius et al. 2010a; Hauton 2012). PRRs that target certain conserved cell-surface motifs can bind to microbes that penetrate the exoskeleton, leading to downstream activation of immune effectors. PRRs identified in crustaceans include lipopolysaccharide-binding proteins (LGBP) and b-glucan-binding proteins (bGBP), and a recently reported peptidoglycan-binding protein (PGBP) from the tiger prawn Penaeus monodon (Vargas-Albores et al. 1997; Lee et al. 2000; Vargas-Albores and Yepiz-Plascencia 2000; Udompetcharaporn et al. 2014). Other PRRs are lectin-like proteins that bear at least one carbohydrate recognition domain (Luo et al. 2003, 2006; Ma et al. 2007; Yang et al. 2007; Cerenius et al. 2010a). Receptor complexes also play a role in the 857 recognition of pathogens. For example, in the crayfish Pacifastacus leniusculus, serine protease homologs mediate the binding of bacterially-derived peptidoglycans to an LGBP (Liu et al. 2011). Evidence has emerged for PRRs with hypervariable regions that could allow for responses to be tailored to different pathogens and could provide a mechanistic basis for specificity and memory in the invertebrates (Ghosh et al. 2011; Cerenius and Söderhäll 2013; Hauton et al. 2015). At least one of these, the Down syndrome cell adhesion molecule (Dscam), belonging to the immunoglobulin superfamily, can undergo alternative splicing that has the potential to generate unique sets of isoforms in the crayfish after infection with bacteria (Watthanasurorot et al. 2011; Ng et al. 2014; Armitage et al. 2015). When PRRs bind a foreign target, powerful immune defenses can be deployed according to the type and load of pathogen and to the route of entry. These effectors include coagulation, phagocytosis, encapsulation, melanization, antimicrobial peptides, and anti-viral RNA interference. Damage to the exoskeleton triggers coagulation (or clotting) of the hemolymph to limit bleeding and intrusion of water, while maintaining the integrity of the organism and entrapping microorganisms (Theopold et al. 2004). Coagulation is initiated by the release of a hemocyte transglutaminase that cross-links the abundant clotting proteins in hemolymph (Hall et al. 1999; Wang et al. 2001). The ability of hemocytes to engulf, inactivate, and degrade smaller microbes by phagocytosis also constitutes an important component of crustacean immunity. PRRs, peroxynectin, and scavenger receptors are involved in the engulfment while lysosomal enzymes and reactive oxygen species are critical for killing microbes; the activation and regulation of phagocytosis in crustaceans remains largely undefined (Johansson et al. 1995; Johansson 1999; McTaggart et al. 2009; Bi et al. 2015). Encapsulation can be triggered when foreign particles too large to be engulfed by individual hemocytes breach the exoskeleton. Multiple hemocytes form a cellular barrier around the particle, sealing the potential pathogen from the rest of the body and forming a capsular space into which toxic immune products may be released (Kobayashi et al. 1990; Johansson 1999). Coagulation and encapsulation reactions often are closely followed by the deposition of melanin as an end product of the prophenoloxidase cascade (proPO cascade) (Aspán et al. 1995; Cerenius and Söderhäll 2004; Cerenius et al. 2010a; Amparyup et al. 2013). Activation of proPO is often coordinated with the production and release of antimicrobial peptides (AMP) from hemocytes 858 (Cuthbertson 2004; Smith et al. 2008; Rosa et al. 2013; Tassanakajon et al. 2015). Finally, anti-viral RNA interference has also been widely documented in crustaceans. This process confers the ability to recognize, process, and destructively cleave doublestranded RNA and DNA that derive from viral infection (Robalino 2006; Xu et al. 2007; Huang and Zhang 2013; Wang et al. 2014). Evidence for the importance of signaling pathways in activating and coordinating these immune effectors is accumulating rapidly. For example, proPO is activated by proteinases and modulated by melanization-inhibition proteins as well as proteinase inhibitors such as pacifastin, alpha 2-macroglobulins, and serpins (Liang et al. 1997; Söderhäll et al. 2009; Cerenius et al. 2010b; Chaikeeratisak et al. 2012; Wetsaphan et al. 2013; Liu et al. 2015). Notably, in recent years, many components of the Toll, IMD, and JAK/STAT pathways which activate the immune response in a wide variety of other organisms have been identified in crustaceans, and details are emerging of their involvement in the production of AMP and in other responses to bacterial or viral infection (Lan et al. 2013; Wang et al. 2013, 2014; Wen et al. 2014). As the details of its receptors, signaling pathways, and effector functions are revealed, the innate immune system of crustaceans has emerged as a complex, highly regulated, and effective defense against the abundant microbial fauna that populate their habitats. The anatomy of crustacean immune defense Immune surveillance and defense are mediated primarily by circulating hemocytes. Subpopulations of hemocytes can be differentiated morphologically, biochemically, and functionally into three types, hyaline cells that are primarily involved in phagocytosis, semigranular cells that mediate coagulation and encapsulation, and granular cells that produce soluble factors such as AMPs, lectins, prophenoloxidase, peroxynectin, and the transglutaminase that mediates coagulation (Johansson et al. 2000). Circulating hemocytes can be abundant in the hemolymph of apparently healthy decapod crustaceans; for example, 1080 106 hemocytes/mL hemolymph have been reported for species of shrimp, crabs, and lobsters (Lorenzon et al. 2001; Battison et al. 2003; Holman et al. 2004). However, the total number of circulating hemocytes and the relative composition of the subpopulations of these cells can vary widely among individuals within a given species, often as a function of the molt cycle, environmental stress, K. G. Burnett and L. E. Burnett or exposure to microbes (Persson et al. 1987; Le Moullac et al. 1997; Lorenzon et al. 2001; Johnson et al. 2011). In the decapods, hemocytes that are depleted during an immune response or by normal aging of cells are replenished from the hematopoietic tissue, which occurs as a series of ovoid lobules forming a thin sheet on the dorsal part of the foregut (Martin et al. 1987; Johansson et al. 2000). In P. leniusculus, hematopoiesis is under circadian control and new hemocytes are produced throughout the individual’s lifetime (Cerenius et al. 2010a; Lin and Söderhäll 2011). Large and well-vascularized tissues like the gills and the hepatopancreas also play a major role in immune defense by removing particulate materials such as bacteria and carmine particles from the circulating hemolymph of crustaceans (Fontaine and Lightner 1974; Smith and Ratcliffe 1980b; White and Ratcliffe 1982; Martin et al. 1993; Burgents et al. 2005b). Bacteria accumulated in the gill are encapsulated, melanized, and externalized when a new epithelial layer forms and old exoskeleton is shed at the following molt (Martin et al. 2000). The hepatopancreas contains fixed phagocytic cells that engulf, and ultimately degrade, accumulated bacteria (Johnson 1987). The hepatopancreas also produces many proteins that contribute to humoral immunity, including the two most abundant proteins in the hemolymph, clotting protein and hemocyanin, both of which play roles in immune defense (Durstewitz and Terwilliger 1997; Hall et al. 1999). The heart, antennal glands, and Y-organs also accumulate bacteria and viruses, but play only a minor role in removing injected particles from the hemolymph (Johnson 1987; Martin et al. 1993; Alday-Sanz et al. 2002). The lymphoid organ, which is a part of the vascular system in penaeid shrimp, can accumulate injected foreign material and displays characteristic spheroid cells in response to many viral diseases. Whether this tissue plays an active role in immune defense or is a site for accumulation of infiltrating phagocytic hemocytes and spheroid cells is as yet uncertain (van de Braak et al. 2002; Burgents et al. 2005b; Burge et al. 2007, 2009; Rusaini and Owens 2010). The crustacean cardiovascular system The crustacean cardiovascular system is vital for gas exchange, acid-base regulation, immune defense, and many other physiological processes. Not all crustaceans contain hearts, but when a heart is present it is simple and contains a single chamber (see Respiratory and metabolic impacts of crustacean immunity McLaughlin 1983). As an example, the heart of the brine shrimp Artemia franciscana is a single dorsal longitudinal vessel with segmentally arranged ostial valves and alary ligaments dividing the heart into a series of interconnected segmental chambers lying within the pericardial sinus; the chambers serve to separate oxygenated hemolymph about to enter the heart from oxygen-depleted hemolymph in more distal regions of the hemocoel (McMahon 2001). This kind of separation of oxygenated and deoxygenated hemolymph is common in many groups (McMahon 2001). The cladoceran Daphnia magna has a globular heart with no arteries (McLaughlin 1983), but hemolymph is distributed rapidly along specific circuits and oxygenation is highly efficient (Pirow et al. 1999, 2004; McMahon 2001). Simple crustaceans often have body segments equipped with unspecialized phyllopod appendages that are used primarily for filter-feeding and/or locomotion but which also function in gas exchange (McMahon 2001). Vascular systems of these kinds are found among present-day Cephalocarida, which are generally considered basal to the crustacean lineage, as well as in many remipedes and insects (Wilkens 1999; Wirkner et al. 2013). During the evolution of the malacostracans, respiratory structures became localized to specialized regions, where they increased in surface area and complexity. The vascular system was correspondingly modified in ways that maintain efficient respiratory gas transport and separation of oxygenated and deoxygenated hemolymph (McMahon 2001; Wirkner 2009; Wirkner et al. 2013). Among the largest and most complex malacostracans, the decapods have a condensed, single, muscular ventricle suspended in a primer chamber, the pericardial sinus, which pumps hemolymph into seven arteries and subsequently into capillary-like channels that supply metabolically active tissues (McMahon and Burnett 1990; McGaw 2005; Wirkner 2009; Wirkner et al. 2013). Outflow of hemolymph from the heart through cardioarterial valves to particular body regions as well as vascular resistance are under neural and/or neurohormonal control (Kuramoto and Ebara 1984; McGaw et al. 1994, 1995; Wilkens et al. 1996; Wilkens and Taylor 2003). Although classified as an open circulatory system, the decapod cardiovascular system with its highly developed arterial structure has been described as ‘‘incompletely closed’’ (McGaw and Reiber 2002; McGaw 2005). Returning from the tissues, hemolymph drains into intertissue spaces, then through a series of sinuses. These sinuses are distinct structures rather than open spaces, forming precise networks around organs that eventually collect in the 859 infrabranchial sinus (Farrelly and Greenaway 2005; McGaw 2005). In crabs, for example, hemolymph passes from the infrabranchial sinus through the gills along a dense network of narrow lamellar channels that are structured in ways that maximize surface area and the flow of hemolymph while minimizing barriers to gas and ion exchange (McGaw 2005; Towle and Burnett 2007). The delivery of oxygen in decapod crustaceans is maximized further by maintaining high concentrations of the respiratory pigment hemocyanin in circulating hemolymph, which maintains efficient delivery to internal tissues. Although its concentration can vary widely among individuals and as a function of life-history stage, molt stage, and environmental stress, hemocyanin is by far the most abundant protein in crustacean hemolymph (Senkbeil and Wriston 1981; Hagerman 1986; deFur et al. 1990). In addition, hemocyanin can be cleaved to generate antimicrobial and phenoloxidase activities, and likely has a variety of other roles in immune defense (Destoumieux-Garzón et al. 2001; Adachi et al. 2003; Coates and Nairn 2014). Thus, the complex decapod cardiovascular system supports multiple aspects of immune surveillance and defense, in addition to its roles in gas exchange, nutrient and hormone transport, and elimination of waste. Immune responses can impair cardiovascular function and metabolism The high rates of flow in the decapod cardiovascular system that are required to support the metabolic demands of these generally active and sometimes large animals also facilitates the rapid distribution of invading microbes that avoid immobilization at a site of injury (Burgents et al. 2005b). This system also facilitates the rapid distribution of hemocytes, clotting protein, and other soluble immune factors throughout the body (Bachère et al. 2004; Burge et al. 2007, 2009). Nearly immediately after injection of bacteria, carmine, or ink particles, the number of circulating hemocytes rapidly declines (Smith and Ratcliffe 1980a; Martin et al. 1993; van de Braak et al. 2002; Holman et al. 2004; Johnson et al. 2011). Following the same rapid time course, hemocytes become immobilized at the site of injury to tissues (Muñoz et al. 2002; van de Braak et al. 2002; Bachère et al. 2004; Burge et al. 2007, 2009). The fixed hemocytes become more phagocytic and they degranulate to release immune effectors, such as peroxinectin, that may recruit additional hemocytes to the site of injury (Johansson et al. 1995; Martin et al. 1996; Sricharoen et al. 2005). The 860 accumulated hemocytes also encapsulate the area of damage, in order to contain the injected material. For example, the shrimp Litopenaeus vannamei sequestered about 50% of an intramuscular dose of bacteria within 15 min after injection; the immobilized bacteria remained viable and could be cultured from the injection site for more than 4 h (Burgents et al. 2005b). Bacteria that evade containment and hemocytes that remain in circulation form loose aggregates, called nodules. The nodules increase in size, often becoming lodged in narrow hemolymph spaces, such as the circulatory channels of the gill. Once trapped in the gills, the nodules are melanized, and are eventually externalized and eliminated at the subsequent molt (Johnson 1976; Smith 1991; Martin et al. 1998). These and similar observations led White et al. (1985) and Martin et al. (2000) to suggest that aggregates/nodules of hemocytes interfere with other functions of the gills such as respiration and ion regulation, which rely on optimum flow of hemolymph and minimum barriers to the exchange of gas and ions. Indeed, there is good evidence that the presence of nodules in the gills can obstruct the flow of hemolymph, thereby impairing respiratory function. Both the shrimp L. vannamei and the blue crab Callinectes sapidus experience a substantial decline in oxygen uptake (40% and 43%, respectively) for 0.5–24 h following injection of a sublethal dose of Vibrio campbellii (Fig. 1A, B) (Scholnick et al. 2006; Thibodeaux et al. 2009). The observed decrease in aerobic metabolism in C. sapidus is associated with a decrease in the Po2 of postbranchial hemolymph and the disappearance of a change of pH across the gills, indicating reduced oxygenation of the hemolymph and a lower rate of excretion of CO2 at the gill (Fig. 2). At the same time, there is a nearly twofold increase in resistance to the flow of hemolymph through the gill-circuit with no changes in heart rate or in pressure in the branchial chamber (Fig. 3) (Burnett et al. 2006). The same dose of V. campbellii that impairs respiration induces a significant accumulation of hemocytes in the branchial tissues of L. vannamei and increased numbers of hemocyte aggregates (defined as four or more clustered hemocytes) in the gill-lamellae of C. sapidus (Burge et al. 2009; Ikerd et al. 2015). This is accompanied by only slight increases in anaerobic metabolism, as indicated by small but significant changes in lactate and succinate in blue crabs that are injected with bacteria and faced with the energetic demand of walking on a treadmill (Thibodeaux et al. 2009). Taken together, these results substantiate that aggregation of hemocytes in response to the injection of bacteria can occlude K. G. Burnett and L. E. Burnett Fig. 1 The rate of oxygen uptake by (A) the shrimp Litopenaeus vannamei and (B) the blue crab Callinectes sapidus injected with a sublethal dose of Vibrio campbellii (closed circles) or saline (open circles), as measured by flow-through respirometry. Values represent averages over (A) 30-min intervals for 6–9 shrimp SEM or (B) 20-min intervals for 7 crabs SEM. Injection of bacteria leads to a significant decrease in oxygen uptake, which lasts for at least 24 h in shrimp and crabs. Asterisk (*) indicates a significant difference in oxygen uptake between treatments at individual time-points (P50.05) (redrawn from Scholnick et al. 2006; Thibodeaux et al. 2009). the vasculature of the gills, resulting in a loss of diffusive capacity and respiratory function accompanied by a metabolic depression. It is possible that factors other than occlusion of the gills are responsible for, or contribute to, the cardiovascular and metabolic changes elicited by injection of a sublethal dose of bacteria. AMP-activated protein kinase, a central regulator of metabolism, is stimulated by thermal stress and hypoxia; infection might elicit a similar stress response (Frederich et al. 2009). Kim et al. (2004) found that nitric oxide synthase is localized to the epithelium and pillar cells of the gills in the tropical land crab Gecarcinus lateralis, and speculated that production of nitric oxide in response to bacterial challenge elicits alterations in hemolymph pressure. In the shrimp Palaemon elegans and other crustaceans, injections of high doses of bacterial lipopolysaccharide can 861 Respiratory and metabolic impacts of crustacean immunity Fig. 2 Prebranchial and postbranchial oxygen pressures (Po2) and pH in the hemolymph of blue crabs (Callinectes sapidus) 30 min after injection with a sublethal dose of Vibrio campbellii (black bars) or saline control (white bars). Values are means SEM; n ¼ 7 for control and n ¼ 9 for crabs injected with V. campbellii. Injection of bacteria causes a large and significant decline in postbranchial Po2 and the loss of a pH gradient across the gills. Asterisk (**) indicates a significant difference between prebranchial and postbranchial variables (P50.05). * indicates a significant difference of a variable between treatments (P50.05) (redrawn from Burnett et al. 2006). Fig. 3 Drop in pressure across the gills, branchial chamber pressure, and heart rate in the blue crab Callinectes sapidus injected with a sublethal dose of Vibrio campbellii (black bars) or saline (white bars). Data on pressure and heart rate were analyzed using a single-level mixed model with fixed effects of time and treatment (Pinheiro and Bates 2000). Negative pressure in the branchial chambers and heart rate remain unchanged with time. The drop in pressure across the gills increases significantly following injection of V. campbellii. (*; P ¼ 0.0116), indicating an increase in vascular resistance with time. All data are mean SEM; control n ¼ 4, Vibrio-injected n ¼ 5 (from Burnett et al. 2006). trigger an increase in the hemolymph’s concentrations of the neuropeptide, crustacean hyperglycemic hormone (CHH). CHH and various other neuropeptide hormones can regulate vascular resistance and cardiac output, and thus control elements of circulation as well as the distribution of hemolymph (Lorenzon et al. 2004; Christie et al. 2010). It is not clear how such a substantial immunemediated metabolic depression can be sustained. Hardy et al. (2013) found no change in the fractional rate of protein synthesis, a highly energy-expensive cellular process, in shrimp injected with V. campbellii. In the absence of some type of conservation strategy, the metabolic depression associated with the response to invading bacteria has the potential to cause more rapid and severe harm to the host than does a hypoxia-induced depression. This finding is of additional significance to the crustacean immune response itself, in which oxygen-dependent mechanisms play an important role (Mikulski et al. 2000; Burgents et al. 2005a; Tanner et al. 2006) and low levels of environmental oxygen can impair the rate at which bacteria are cleared from the hemolymph (Holman et al. 2004; Burgents et al. 2005a). Energetic tradeoffs between immune defense and other physiological processes have been demonstrated in several other vertebrate and invertebrate species, but their mechanistic bases and functional significance to free-living populations are not well understood (French et al. 2009, 2011). In crustaceans there is now strong evidence to suggest that aggregation of hemocytes in response to a sublethal dose of bacteria can occlude the vasculature of the gills, impairing aerobic respiration and leading to metabolic depression. Given the high density of microorganisms characteristic of the aquatic environment (Schmidt et al. 1998; Fuhrman 1999), the extensive microbial community of the gut and carapace, as well as the high incidence of bacterial infections in crustaceans (Tubiash et al. 1975; Davis and Sizemore 1982; Givens et al. 2013), the metabolic consequences of mounting an immune response in crustaceans could impair energydependent activities such as capturing prey, avoiding predators, migration, and reproduction. Interactions of the immune and cardiovascular systems in insects The insect immune system displays extensive similarity to that of crustaceans, including closely parallel suites of PRRs, signaling pathways, and effector functions (Jiang et al. 2010; Imler 2014; Hillyer 2015). Hemocytes are the primary mediators of immune surveillance and defense in insects, but the functional 862 and morphological classification of subpopulations of hemocytes differs among species and life stages (Hillyer and Strand 2014; Honti et al. 2014; Vlisidou and Wood 2015). Hemocytes can also be divided by anatomical location: those freely circulating in hemolymph and sessile forms that are attached to tissues. Sessile cells are distributed throughout the hemocoel, but in the abdomen a large proportion are attached to the respiratory trachea and the heart (Márkus et al. 2009; King and Hillyer 2013). The lymph gland serves as the primary hematopoietic organ in insect larvae (Lanot et al. 2001). No hematopoietic tissue has been identified in adults and hemocyte counts decline with age in that life stage. However, the number of hemocytes can increase in response to infection, and in the mosquito Anopheles gambiae, circulating and sessile hemocytes have been shown to replicate by mitosis in adults infected with bacteria. Other tissues that contribute to immune defense in insects include the mid-gut, salivary glands, and fat body, all of which synthesize a wide array of AMPs and other humoral immune factors, a process that is largely signaled and orchestrated by hemocytes (Hillyer 2015; Vlisidou and Wood 2015). The strategic response to the damage of tissues and to microbial invasion is similar in crustaceans and insects. When the insect epidermis and cuticle are damaged, the epithelium releases signals that attract hemocytes to the wound where they are bound by adhesive capture. Hemocytes recruited to the site participate in sealing the wound by coagulation and melanization, and repair of tissue, as well as antimicrobial defense that limits the spread of potential pathogens (Babcock et al. 2008; Evans and Wood 2014; Theopold et al. 2014). The process of clot formation in insects is distinct from that of crustaceans, starting with the formation of a soft clot that involves multiple proteins and a transglutaminase, although the primary substrate for the transglutaminase has been difficult to identify. The soft clot is then cross-linked and hardened by phenoloxidase activity, in the process generating toxic reactive oxygen species and depositing melanin directly on larger pathogens, thereby facilitating encapsulation (Scherfer et al. 2004; Volz et al. 2006; Cerenius et al. 2008; Theopold et al. 2014). While clots may eventually be shed by regeneration of the underlying cuticle, aggregates and capsules of hemocytes can persist without any visible harm to the animal (Theopold et al. 2004). Microbes that escape the wounded site and enter the hemocoel are rapidly disseminated within an open circulatory system whose major structural component is a dorsal vessel running through the thorax and abdomen. The abdominal portion of the vessel, K. G. Burnett and L. E. Burnett called the heart, is segmentally arranged into chambers. Peristaltic contraction of cardiac muscle moves hemolymph across the length of the dorsal vessel, where it is released into the hemocoel through excurrent openings located at the anterior and posterior ends of the insect. Once hemolymph is in the hemocoel, its movement is supplemented with secondary pumps called accessory pulsatile organs (Pass 2000; League et al. 2015). Ostia located along the length of the heart control the entry of hemolymph into this portion of the dorsal vessel, supporting rapid, well-defined patterns of flow comparable to the flow of blood cells through vertebrates’ microvessels (Babcock et al. 2008; Glenn et al. 2010; Hillyer 2015; League et al. 2015). In A. gambiae in which this process has been observed and quantified on a cellular level, microbes introduced systemically are rapidly distributed and then engulfed by circulating and sessile hemocytes. Particularly intense aggregations of microbes appear in the extracardiac regions surrounding the ostia of the heart, mediated by a resident population of periostial hemocytes. Minutes later, other circulating hemocytes move preferentially to the periosteal region, further amplifying the phagocytic response and providing a centralized hub for immune defense in which the accumulated hemocytes encounter and trap circulating pathogens before they can be disseminated back to the tissues (King and Hillyer 2012, 2013). From a strategic perspective, the same role of trapping microbes is served by the microvasculature of decapods’ gills, in which venous hemolymph collected into the infrabranchial sinus filters through the gill for oxygenation and elimination of pathogens prior to reentering the circulation. In insects, microbes trapped by the periosteal hemocytes are engulfed by phagocytosis and degraded. In contrast, decapods rely on melanization and molting to externalize pathogens and associated aggregates of hemocytes trapped in the gills. Hemocytes lost in the process are replenished from hematopoietic tissues. In insects then, as in crustaceans, the immune and cardiovascular systems appear to be intimately connected and functionally coordinated, but there are also respiratory and metabolic costs of mounting an immune response in decapod crustaceans. Is there evidence for such physiological trade-offs in the insects? Does mounting an immune response have physiological consequences in the insects? With the invasion of the terrestrial habitat, insects evolved an elaborate tracheal system that allows 863 Respiratory and metabolic impacts of crustacean immunity molecular oxygen to be distributed directly to the cellular level. While pumping of the heart and accessory pulsatile organs facilitates gas exchange by pushing air and hemolymph into the appendages, oxygen transport in insects does not rely on efficient circulation of a respiratory pigment to the tissues. Thus, the cardiovascular systems of insects and crustaceans play quite different roles in respiration, leading to this question. Are there respiratory or metabolic consequences of mounting an immune response in insects, comparable to those seen in decapod crustaceans? While ecological consequences of challenges from pathogens and parasites have been well documented in insects, the physiological consequences of mounting immune responses at the level of the whole organism have received less attention. Most such investigations have focused on the pathologies associated with mortality, and not whether a successful immune response impairs or alters physiological function. Lethal doses of a parasite, bacteria, or virus are administered by injection, or less often by feeding. Physiological responses generally are recorded hours or days after the initial challenge, but prior to the onset of mortalities. Significant metabolic and behavioral changes, such as loss of weight or reduction in feeding, are commonly reported, probably reflecting tissue damage in the gut or increased excretion of urine or feces (Shirasu-Hiza and Schneider 2007). For example, Arnold et al. (2013) noted that 2 days after receiving a lethal dose of Drosophila C virus, Drosophila melanogaster experienced metabolic depression and reduced growth, followed by a loss of locomotor activity on day 3, and mortality on day 5, suggesting that relatively late in this model of infection, energy was being reallocated from growth toward feeding activity and immune defense. Microbial challenges can disrupt patterns of locomotion and physiological function. Increased activity was documented in female Aedes aegypti within 2–6 days after infection with Dengue virus, likely because this virus targets nervous tissues (Lima-Camara et al. 2011). Circadian control of activity was rapidly disrupted in D. melanogaster injected with the grampositive bacteria Streptococcus pneumonia and Listeria monocytogenes (Shirasu-Hiza et al. 2007). In the same study, circadian mutant flies lacking two central clock proteins (timeless or period) had higher mortality rates than did wild-type flies that received a lethal challenge with the same two bacteria, thereby supporting a link between circadian patterns and innate immunity (Shirasu-Hiza et al. 2007). Both fecundity and body mass were significantly altered in malaria-infected A. aegypti (Gray and Bradley 2006). Many reproductive pathologies, as well as perturbations of metabolism and locomotor activity, have also been linked to nonlethal Wolbachia infections in insects (Baldo et al. 2008; Evans et al. 2009). There are even reported benefits such as the pathogen-blocking associated with Wolbachia infection (Hedges et al. 2008; Rancès et al. 2012). The extent to which mounting an immune response might contribute to these and other physiological disruptions, and the mechanisms and metabolic costs associated with mounting a defense are not clear. Recent studies in Drosophila have hinted at intriguing links between immunity and nutrientsensing, hormonal control, and neurodegeneration (Buchon et al. 2014). In one such report, feeding the gram-negative bacterium Erwinia carotovora to D. melanogaster larvae induced an immune response in airway epithelial cells via activation of the IMD pathway, leading to reactivation of tracheal development genes and local thickening and remodeling of the epithelia of the airway (Wagner et al. 2009). The same response was induced with heat-killed bacteria. Impacts on respiration and metabolism were not reported. This study highlights that the idea that interplay among the processes of immune defense, response to pathogens, and homeostasis of the hosts’ epithelia likely play out in the reproductive, intestinal, and respiratory tracts that are responsible for maintaining the integrity of the whole organism. However, with the exception of wound-healing, immune responses in the context of other epithelial surfaces and possible impacts on tissue and wholeorganism physiology have not been widely studied in insects or crustaceans (Roeder et al. 2012; Ferrandon 2013; Buchon et al. 2014). Conclusions The immune systems of crustaceans and insects are equipped with very similar molecular toolboxes that are strategically deployed to provide a primary barrier to the entry of pathogens by coagulation, encapsulation, and melanization. In both groups, hemocytes that are activated by microbial infection redistribute to new sites where they counter the invading microbial challenger. The subsequent fate of the pathogen depends in part on the structure and function of the cardiovascular system in which that immune system operates. In decapods and possibly other crustaceans, aggregates of activated hemocytes and bacteria can occlude hemolymph channels in the gill, impairing respiration and incurring metabolic 864 costs. It is not clear whether activated hemocytes or other components of a successful immune response incur such metabolic or other physiological costs in insects, or in crustaceans with simpler cardiovascular systems. Valuable insights into this question might be gained by the development of new models of epithelial infection in insects for which there are powerful tools for molecular and genetic manipulation. Given the extensive similarities between the insect and the crustacean immune systems, studies in such models of epithelial infection could benefit our understanding of the physiological consequences of immune defense in all of the Pancrustacea. 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