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Transcript
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
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