Download Respiratory and Metabolic Impacts of Crustacean Immunity: Are

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
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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
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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
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(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
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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.
Acknowledgments
The authors thank Drs Sherry Tamone and Jon
Harrison for the opportunity to contribute to this
symposium and to Dr Julián Hillyer for his critical
review of the manuscript. This is GML Contribution
No. 443.
Funding
This work was supported by the US National Science
Foundation [IOS-0725245, 1147008]. Financial support for this symposium was provided by the U.S.
National Science Foundation [NSF-IOS 1507854]
and the Society for Integrative and Comparative
Biology.
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