Insect immune responses to nematode parasites

Insect immune responses to nematodeparasitesJulio Cesar Castillo1, Stuart E. Reynolds2 and Ioannis Eleftherianos1

1 Insect Infection and Immunity Lab, Department of Biological Sciences, The George Washington University, 336 Lisner Hall, 2023 G

Street NW, Washington, DC 20052, USA2 Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK

Host innate immunity plays a central role in detectingand eliminating microbial pathogenic infections in bothvertebrate and invertebrate animals. Entomopathogenicor insect pathogenic nematodes are of particular impor-tance for the control of insect pests and vectors ofpathogens, while insect-borne nematodes cause seriousdiseases in humans. Recent work has begun to use thepower of insect models to investigate host–nematodeinteractions and uncover host antiparasitic immunereactions. This review describes recent findings on in-nate immune evasion strategies of parasitic nematodesand host cellular and humoral responses to the infection.Such information can be used to model diseases causedby human parasitic nematodes and provide clues indi-cating directions for research into the interplay betweenvector insects and their invading tropical parasites.

Parasitic nematodesNematode parasites constitute one of the major threats tohuman health, causing diseases of major socioeconomicimportance worldwide. Recent estimates indicate thataround 3 billion people are infected with nematodes [1].More than a hundred species of nematodes are parasites ofhumans, and other species have a substantial effect on theglobal economy by directly or indirectly causing severedamage to agricultural crops, leading to a significant re-duction in food availability [2]. Parasitic nematodes alsohave a major long-term impact on livestock and domesti-cated animals [3]. In addition, certain nematode parasitespecies harbor bacterial pathogens that are virulent to theinsect host [4]. Importantly, the manipulation of the im-mune system of the host by mammalian parasitic nema-todes is a key determinant for their survival within theinsect vector.

Entomopathogenic nematodes are parasitic worms thatinfect and kill insects [5]. They are also excellent animalmodels to investigate interspecies associations such as mu-tualism/symbiosis, parasitism, pathogenicity and vector-borne diseases [6]. Several insect species act as hosts ofnematode parasites that survive, develop, multiply andcomplete their complex life cycles within an insect(Table 1). A common morphological feature in the biologyof most entomopathogenic nematodes is the infective juve-

Review

Glossary

Axenic nematodes: nematodes that lack bacterial symbionts. They are useful to

investigate the relative roles of the worms and the bacteria in pathogenicity.

Cell spreading: certain insect hemocyte types have the ability to strongly

adhere to foreign surfaces and spread to phagocytose a small particle or

encapsulate a larger foreign object.

Cellular capsule: a multicellular aggregate of hemocytes that isolates a foreign

object. In some insects the capsule is composed of melanin.

Compatible vector: the ability of an insect vector to support the development

of a pathogen and transmit the disease.

Diapause: an anticipated, typically long-term, cyclical interruption in growth or

development of an organism due to one or more environmental factors that

occur well before adverse environmental conditions are manifest.

Eicosanoids: derivatives of polyunsaturated fatty acids that are important in the

oviposition and reproduction of insects. There is also some evidence that

eicosanoids mediate the immune response of insects to bacteria in the

hemolymph.

Encapsulation: a cellular immune response of insects in which moderate-sized

foreign material, microorganisms, parasites or parasitoids are surrounded or

enveloped by hemocytes. Encapsulation occurs around objects too large for

hemocyte phagocytosis. It involves cooperation between hemocytes and is

mediated by cytokines and adhesion molecules.

Entomopathogenic: an organism that infects insects and causes a disease.

Fat body: a large organ and major immune-responsive tissue that is located in

the insect open circulatory body cavity and is functionally equivalent to the

mammalian liver.

Filarial nematodes: thread-like roundworms that cause the infectious tropical

disease filariasis. They have a simple life cycle, involving a sexual stage giving

birth to microfilarial larvae in the mammalian host, and an insect vector in

which the larval stages develop.

Hemocytes: insect blood cells suspended in the hemolymph. Certain hemocyte

types are professional phagocytes that patrol the hemolymph and engulf

invaders.

Hemocyte aggregation: the process of forming adhesions between hemocytes

that surround and trap invading microorganisms.

Hemolymph: the insect blood that circulates around the body, bathing the

tissues directly. It consists of fluid plasma in which hemocytes are suspended.

The plasma, because of its function of maintaining the tissues throughout the

body, contains many chemicals.

IkB: protein that inhibits the NF-kB (nuclear factor kappa-light-chain-

enhancer of activated B cells) transcription factor by preventing it from

entering the nucleus. It is involved in propagating the cellular response to

inflammation.

Imd pathway: in Drosophila, the immune deficiency pathway mainly responds

to Gram-negative bacterial infection and controls antibacterial peptide genes

(e.g. Diptericin). The Imd pathway shares some similarities with the vertebrate

tumor necrosis factor receptor pathway.

Lectins: proteins that have binding sites for specific mono- or oligosaccharides.

They were originally named for the ability of some to selectively agglutinate

human red blood cells of particular blood groups.

Melanization reaction: a branch of insect immunity that involves the rapid

synthesis and deposition of a brown–black pigment (called melanin) at the site

of infection and injury. Melanin possesses cytotoxic activity towards micro-

organisms, restrains the invasion of microbial pathogens into the body cavity

of the host and assists in wound healing. Melanization is triggered after the

recognition of microbial compounds, such as b-1,3-glucans, lipopolysacchar-

ides and peptidoglycans.

Microfilariae: the larval filarial nematodes are known as microfilariae and are

found in the blood or in the tissue beneath the skin. This facilitates

nile (IJ) or dauer juvenile (Box 1). The bacteriaPhotorhabdus and Xenorhabdus colonize the nematode

transmission by the insect when it comes for its next feed.

Corresponding author: Eleftherianos, I. ([email protected]).

1471-4922/$ – see front matter . Published by Elsevier Ltd. doi:10.1016/j.pt.2011.09.001 Trends in Parasitology, December 2011, Vol. 27, No. 12 537

mailto:[email protected]

http://dx.doi.org/10.1016/j.pt.2011.09.001

Review Trends in Parasitology December 2011, Vol. 27, No. 12

Mimicking molecule: compounds produced by the parasite and exposed on its

body surface. These compounds enable the parasite to become antigenically

closely related to the host and thus avoid inducing host immune responses.

Nematode cuticle: it is the nematode exoskeleton that provides structural

integrity, environmental protection and plays a role in hydrostatic skeleton

function. It mainly consists of collagens, lipids, carbohydrates and glycopro-

teins. It is produced by the hypodermis, an ectodermal cell layer that surrounds

the body of the animal. The nematode cuticle is permeable and can grow

between molts (unlike arthropod exoskeleton).

Nodulation: the binding of multiple hemocytes to aggregations of bacteria.

Opsonin: substance that binds to the surface of a particle and enhances the

uptake of the particle by a phagocyte.

Opsonization: process of coating foreign objects with an opsonin.

Phagocytosis: the engulfing of microorganisms or other cells and foreign

particles by phagocytes.

Plasmatocytes: a class of insect hemocytes important in the cellular immune

response.

Prophenoloxidase activating system: melanization is controlled by phenolox-

idase, a key enzyme that catalyzes the oxidation of phenols to quinones leading

to the formation of melanin. Phenoloxidase exists as an inactive precursor

called prophenoloxidase and, following either physical injury or microbial

challenge, it is released by hemocytes and is activated via limited proteolysis

through the action of a serine protease cascade.

Serine proteases: a group of endoproteases from both animal and bacterial

sources that share a common reaction mechanism based on formation of an

acyl-enzyme intermediate on a specific active serine residue. Serine proteases

are irreversibly inactivated by naturally occurring inhibitors (serpins).

Toll pathway: an evolutionary conserved signaling cascade that plays a key

role in several developmental and immune-related processes. In Drosophila,

the Toll pathway is triggered by the proteolytic cleavage of the Toll ligand,

Spaetzle, and leads to the activation of the Rel proteins DIF and Dorsal. The Toll

pathway is mainly activated by Gram-positive bacteria and fungi and largely

controls the expression of antimicrobial peptides active against fungi (e.g.

Drosomycin).

Table 1. Examples of interactions between parasitic nematodes a

Nematode Symbionta Insect hosts

Heterorhabditis sp. Photorhabdus sp. Manduca sexta,

Musca domestica

Heterorhabditis

bacteriophora

Photorhabdus

luminescens

Drosophila melanogaster,

Galleria mellonella

Lepidoptera, Coleoptera,

Orthoptera

Heterorhabditis

marelatus

Photorhabdus

luminescens

Leptinotarsa decemlineata

Heterorhabditis

megidis

Photorhabdus

luminescens

Locusta migratoria

Steinernema sp Xenorhabdus sp. Musca domestica,

Lepidoptera, Coleoptera,

Orthoptera

Steinernema

carpocapsae

Xenorhabdus

nematolphila

Mythimna unipuncta

Steinernema feltiae Xenorhabdus

bovienii

Galleria mellonella

Steinernema glaseri Xenorhabdus

poinarii

Popillia japonica

Brugia pahangi Wolbachia spp. Aedes aegypti,

Simulium ornatum,

Anopheles quadrimaculatu

Brugia malayi Wolbachia

pipientis

Armigeres subalbatus,

Aedes aegypti

Dirofilaria immitis Wolbachia spp. Armigeres subalbatus,

Aedes aegypti

Wuchereria bancrofti Wolbachia spp. Culex quinquefasciatus

Onchocerca lienalis Wolbachia spp. Simulium vittatum

aBacteria symbiotic to the nematodes.

bThe interaction between the nematode and insect host is either pathogenic, killing the

within the insect host without killing it.

cImmune responses of the insect host against the nematodes.

538

Heterorhabditis and Steinernema, respectively, in a mutu-alistic association, and they are also pathogens of insects.The mutualistic relationship between nematodes and bac-teria is not obligate, as nematodes are viable in the absenceof their mutualistic bacteria [7,8]. Nematode infection fol-lows a specific series of events until the parasites establishthemselves in the insect body cavity (Figure 1) [7,9]. Tosurvive within the insect and complete their life cycle,nematodes employ tactics to evade or suppress the hostimmune response (Box 2). Insects have developed a rangeof morphological, behavioral, physiological and moleculardefense reactions to combat infection and eliminate theintruding parasites (Table 1).

Filarial parasitic nematodes (see Glossary) cause seri-ous diseases in humans (lymphatic filariasis, onchocercia-sis/river blindness) and animals (heart filariasis in dogs,verminous hemorrhagic dermatitis in cattle) [10]. Thefilarial nematodes that cause these diseases are transmit-ted by different mosquito species of the genera Aedes,Anopheles and Culex, and black flies of the genus Simu-lium, which act as intermediate hosts or compatible vectors[11]. Filarial nematode parasites have complex life cyclesthat share certain features (Box 1). A general characteris-tic of their biology is that they undergo cyclodevelopmentaltransmission during which the parasites change morpho-logically and develop within the insect vector, but do not

nd their insect hosts

Interactionb Immune responsesc Refs.

Pathogen Humoral, cellular, melanization [34,94]

Pathogen Humoral, hemolymph

coagulation, cellular

[48,50,82–84]

Pathogen Cellular [67]


Pathogen Cellular [82,94]


Pathogen Prophenoloxidase cascade,

humoral, cellular, melanization

[68,69,80,81]

Pathogen Melanotic encapsulation [66]

s

Pathogen/

vector

Humoral?, cellular [39,47,61,95]

Vector Melanization, melanotic

encapsulation, humoral?

[60,88]

Vector Melanotic encapsulation,

cellular

[33,59,96]

Vector – [54]

Vector Cellular [63]

insect, or the insect is a vector, whereby the nematode completes lifecycle stages

Box 1. Life cycles of insect nematode parasites

Heterorhabditis and Steinernema nematodes

Heterorhabditis and Steinernema are entomopathogenic (insect

pathogenic) nematodes belonging to the families Heterorhabditidae

and Steinernematidae, respectively [9,92]. They are obligate parasites

completing their entire life cycle in insect hosts, and they are mass-

produced and marketed worldwide as biocontrol agents for the control

of a wide range of insect species of agricultural or medical importance.

The general characteristics of the life cycles of these nematodes are

relatively similar. Symbiotic bacteria of the genus Photorhabdus and

Xenorhabdus are found colonizing the gut (Table 1) of a specialized

form of the nematodes called the infective juvenile (IJ), which is a

nonfeeding stage equivalent to the dauer juvenile stage of the model

nematode Caenorhabditis elegans and is the only stage that is able to

survive outside of the host [7,9]. The IJ is an obligate part of the life

cycle of the nematodes and is required for successful infection of insect

hosts, which starts with the rapid detection of the host, followed by the

attachment of the parasite to the cuticle, penetration through natural

openings (mouth, anus, spiracles, tracheae), invasion into the insect

body cavity and establishment in the hemolymph (insect blood)

(Figure 1). Once inside the insect, the nematodes release their symbiotic

bacteria which rapidly proliferate and kill the host by secretion of

several toxins and virulence factors that cause septicemia in the midgut

and fat body tissues. The symbiotic bacteria of the nematodes replicate

within the insect, and the IJ exits diapause and enters the IJ recovery

stage that involves development into adult hermaphrodite worms. The

nematodes reproduce while feeding on the bacteria within the dead

insect; the nematodes then lay eggs that hatch and develop through

four larval stages into adult nematodes. The cycle continues, reprodu-

cing two to three generations before entering diapause, thus forming IJ

due to a lack of nutrients within the insect cadaver. The nematode–

bacterium complex finally emerges from the insect carcass into the soil

in search of new suitable hosts. Under optimal laboratory conditions,

approximately 100 000 IJ nematodes can be produced from a single IJ

infecting an individual insect. Owing to their complex life cycles and

close association with the virulent bacterial pathogens Photorhabdus

and Xenorhabdus, Heterorhabditis and Steinernema nematodes are

used in combination with genetically amenable insects in basic

research; they provide a fascinating model for studying insect

immunity and disease progression, bacterial pathogenicity/symbiosis

and nematode parasitism and the interaction between these important

biological processes [70].

Filarial nematodesThe major life cycle stages of filarial nematodes are: (i) infective

larvae, which are transmitted to humans by insect bites; (ii) adult

worms, which develop from larvae and reside in the lymphatic

vessels; and (iii) microfilariae, which are produced by adult female

worms, are found circulating in the host bloodstream.

Insect vectors ingest microfilariae from the blood of mammalian

hosts, and the ingested parasites penetrate the insect midgut

epithelium to gain access to the hemolymph (Figure 2). From there,

they travel to the thoracic musculature and invade the flight muscles

where microfilariae go through two molts to become third-stage

infective larvae. These migrate to the head and reach the proboscis

from where they escape when the insect feeds on the host. Following

an insect blood meal, the infective larvae enter the mammalian host,

resume development and move to the lymphatic system. The third

juvenile stage (or infective larval stage) is the transitional life stage

between the insect vector and the mammalian host, and thus it is the

crucial stage for infection [10,12]. Adult male and female parasites

mature in lymphatic vessels and, following mating, female worms

generate microfilariae, which migrate to the peripheral blood.

Microfilariae can then be transferred to a vertebrate host through

ingestion by a blood-feeding insect vector.


proliferate [12]. However, the successful development andtransmission of filarial nematodes depends on the ability ofthe insect vector to encounter internal damage and surviveinfection by employing robust immune defenses during the

Entomopathogenic bacteria released into thgut and hemocoel.

Direct ent

Ingested worms

Gut wall

Oral infection

Hemocytes[Encapsulation]

Figure 1. Diagram showing the most common routes used by entomopathogenic (or ins

the insect body cavity through the mouth, anus or spiracles. Once nematodes gain ac

damage various insect tissues and organs, such as the gut and fat body. In the case o

symbiotic bacteria (Photorhabdus and Xenorhabdus, respectively) into the insect host le

key cellular immune mechanisms (e.g. phagocytosis). In turn, this leads to a pathologic

shows that nematodes are potentially able to cross the disrupted midgut epithelium. T

complete their life cycles before they emerge as a complex from the insect carcass in s

sessile hemocytes and fat body) are shown. Abbreviation: AMP, antimicrobial peptide.

development of the parasites (Figure 2). Understandingthe molecular mechanisms of antifilarial worm response ininsect vectors will potentially lead to the development ofnovel means to control disease transmission. Interestingly,

Penetration through spiracles

Destruction of gut integrity?

Fat body [AMP production]

Sessile hemocytes [resting state]

e

ry through cuticle

Anus

Inhibition of immune responses- Apoptosis- Tissue lysis- AMP degradation?- Impaired phagocytosis

TRENDS in Parasitology

ect pathogenic) nematodes to infect their insect hosts. Infective juveniles (IJs) enter

cess into the hemocoel (the insect open circulatory system), they may physically

f the insect parasitic nematodes Heterorhabditis and Steinernema, the release of

ads to suppression of the insect immune response as the bacteria are able to inhibit

al state within the insect (septicemia) that results in rapid insect death. Inset figure

he nematodes and their symbiotic bacteria replicate within the insect where they

earch of new suitable hosts. The main insect immune-related tissues (circulating,

539

Box 2. Nematode strategies to evade the host immune response

Once inside the insect, entomopathogenic nematodes face a highly

potent immune response by which the host attempts to eliminate or

reduce an infection. However, the immune response of a host to

nematode parasites may not always be effective because of the ability

of the latter to survive the immune defenses. Insect parasitic

nematodes employ immune evasion strategies by interfering with,

disrupting or manipulating immune defenses at the stage of the

innate immune response or the early-induced response [93].

Immune evasion tactics of parasites can be classified into three main

groups:

i. Anatomical seclusion: involves the migration of the parasites to

host tissues where they can avoid immune reactions, such as

encapsulation by hemocytes.

ii. Camouflage strategy: involves the production of mimicking

molecules or the sequestration of immune factors from the host

hemolymph. These molecules are deposited onto the body

surface of the parasite and help to avoid recognition and induction

of host immune responses.

iii. Interference strategy: involves the suppression, disruption or

modulation of host immune defenses (humoral or cellular) by the

parasite.

In recent years, studies on host–parasite interactions and in

particular nematode evasion of the insect immune system have

benefited from genomic approaches in certain parasitic nematode

species and model insect hosts. Infection of Aedes aegypti with the

filarial parasite Brugia malayi affected the transcription of only a few

genes, possibly suggesting an immune evasion and/or suppression

strategy by the parasite [28]. Similarly, whole genome microarray

analysis of different mosquito genera transmitting filarial worms

showed that the parasites modulate only a few immunity genes in

these vectors [30–32,54], which is consistent with the idea that

mosquito-borne pathogens have evolved sophisticated mechanisms

to evade innate immune responses to spread the disease. More

recently, transcriptomic analysis of the induced Steinernema carpo-

capsae parasitic phase revealed clusters of genes that were predicted

to encode putative secreted proteins that may possibly interfere with

host immune mechanisms [74]. Similar studies will provide important

information related to the molecular basis of the series of actions that

take place during the parasitic process including nematode invasion,

adaptation to the insect immune responses and successful coloniza-

tion of the host.


most filarial nematodes, including the three major causa-tive agents of filarial disease in humans: Brugia spp.,Onchocerca volvulus and Wuchereria bancrofti, also carrythe Gram-negative intracellular bacteria Wolbachia thatare common endosymbionts of arthropods (Table 1) [13].These endosymbionts have evolved a mutualistic associa-

Mosquito takes a blood mealand ingests microfilariae

Microfilariae move thrthe esophagus into th

Microfilariae develop in thethoracic muscles

Third stage larvae migrate to thehead and proboscis

Larvae are injected to thehost during blood feeding

Figure 2. Interactions of filarial nematode parasites with its mosquito vector. When m

esophagus into the gut, penetrate the midgut epithelium, gain access to the hemolymph

and turn into third-stage infective larvae. These migrate through the hemocoel to the h

the host during blood feeding. The immune response of mosquito to filarial nematode p

fat body and hemocyte-mediated melanotic encapsulation response. In the latter cas

associated with blackening of the capsule due to melanization.

540

tion with their nematode host [14], and because they arerequired for worm survival and fertility, Wolbachia bacte-ria are an important target for antifilarial therapy [10].Although Wolbachia can be released from worms andinduce inflammatory responses due to death of nematodeswithin tissues of the mammalian host [15], it is currently

ough e gut

Microfilariae gain access to hemolymph and migrate to the throracic muscles

Fat body

Hemocytes

Melanization and Encapsulation responses

Production of AMPs


osquitoes ingest filarial parasites in a blood meal, nematodes move through the

and migrate to the thoracic muscles. In this tissue, microfilariae undergo two molts

ead, eventually reaching the mosquito proboscis from which they are injected into

arasites involves synthesis and secretion of antimicrobial peptides (AMPs) from the

e, hemocytes form a multicellular capsule around the parasite, a process that is


unknown whether there is an interaction between theinsect vector immune system and the nematode symbionts.

Insect immune defense mechanismsInnate immunity is present in all multicellular forms oflife. Metazoan animals possess mechanisms that allowthem to detect, process and trigger specific responsesagainst infection or injury. Microbial or parasitic infectionslead to the activation of recognition and effector-codinggenes in the host, which enables a general and rapidimmune response against the invading pathogen [16].Several innate immune reactions in insects and otherinvertebrates are shared with innate immune responsesin vertebrates. Only vertebrates, however, possess theadaptive or acquired antibody-based arm of the immunesystem that generates antigen-specific mechanisms for thedestruction of infectious agents [17]. The conservation ofcertain components of innate immunity between insectsand mammals demonstrates that insects are compellingmodels for elucidating innate immune functions in animals[18]. The insect immune system is controlled by a complexand diverse array of signaling pathways that involve adedicated cluster of defense mechanisms usually dividedinto humoral and cellular reactions. These act locally andsystemically, and together contribute to protection againstattacks by foreign microorganisms [19].

Humoral immune responses involve the induced tran-scriptional activation of a large number of effector genesthat leads to the synthesis of antimicrobial peptides(AMPs) predominantly in the fat body and their secretioninto the hemolymph (Figure 1). Insect AMPs are small,cationic, membrane-active molecules that accumulate inthe hemolymph, reaching high concentrations in responseto infection; they exhibit a broad range of activitiesagainst different classes of pathogens and can still bedetected in the hemolymph up to several weeks afterchallenge [20]. Cellular immune responses involve theaction of circulating hemocytes, which differ among insectspecies and are divided into certain classes based onmorphological characteristics, antigenic properties andfunctional features. Hemocytes derive from the lymphgland (a specialized hematopoietic organ) or embryoniccells during the larval stages of the insect and can persistinto the adult stage. Their number rapidly increasesduring an infection, and they are responsible for severalcellular defenses including cell spreading, formation ofcell aggregates, nodulation, phagocytosis and encapsula-tion; they can also take part in humoral reactions [21]. Inaddition, insects can activate complex proteolytic cas-cades that regulate coagulation and melanization of he-molymph, defenses associated with production of reactiveoxygen and nitrogen species and epithelial responses inthe gut that also play important roles in fighting microbialinfections [22,23].

Immune recognition of nematodesThe insect immune system is equipped with sensitivemechanisms for the detection of microbial attack. Previousand recent evidence has pointed to the perception that theresponse to invading microorganisms is initiated by recog-nition of certain microbial structures by host receptors of

the innate immune system and pathogen-induced processesthat contribute to the progression of disease. In particular,recognition of microbial infection is controlled by patternrecognition proteins (PRPs) that bind to conserved microbe-associated molecular pattern (MAMP) molecules producedby microbes such as bacteria and fungi [24,25]. Microbial cellmembrane components, such as lipopolysaccharide (LPS),and various types of peptidoglycans and glucans, are themajor immune-eliciting microbial ligands that have so farbeen characterized in insects [26]. Despite impressiveadvances in the recognition of bacterial and fungal patho-gens by the insect immune system [27], our understandingof the molecular mechanisms involved in nematode recog-nition remains incomplete.

Transcriptomics analysis has recently been employed toidentify recognition genes in mosquitoes infected withfilarial worms. Examination of the mosquito transcrip-tomic profile following infection of Aedes aegypti withthe filarial parasite Brugia malayi, which causes humanlymphatic filariasis, resulted in a small number of changesthat included genes encoding putative PRPs and signalmodulator genes [28]. However, a distinct transcriptionalprofile was obtained in Armigeres subalbatus mosquitoesinfected with Brugia pahangi or B. malayi [29,30]. Inthe latter case, which represents a compatible mosquito-filarial worm association, a peptidoglycan recognition pro-tein (PGRP), C-type lectins and calreticulin were detectedin the antifilarial worm immune response [31]. This sug-gests that lectins may play a role in the detection of filarialworms upon nematode infection in mosquitoes. To gaininsight into the detection of filarial worms in mosquitoes,researchers previously constructed six cDNA librariesfrom tissues of adult female A. subalbatus infected withvarious pathogens including the nematodes B. malayi andDirofilaria immitis. Expressed sequence tags (ESTs) fromthese libraries were generated, annotated and subjected toanalysis. The results identified Aslectin, a lectin with afibrinogen-like domain previously shown to be involved inmosquito immune responses against bacteria [32]. In ad-dition, induction of a hemocyte-produced b1,3-glucan rec-ognition protein from A. subalbatus in response to thefilarial worm D. immitis has been observed previously.Functional characterization of this molecule suggested apotential role in the initiation of the melanization andencapsulation immune reactions (see below) [33]. In thelepidopteran model insect Manduca sexta, symbiotic Het-erorhabditis nematodes (worms carrying virulent bacteria)and Photorhabdus bacteria alone were shown to upregu-late the mRNA of specific PRPs at similar levels, whereasaxenic nematodes (worms lacking Photorhabdus) inducedlower immune gene transcription in the fat body tissue.These results imply that Heterorhabditid nematodes arenot recognized by the insect immune system or are muchless efficiently detected than their symbiotic bacteria, andthat the induced expression of PRPs is an importantcomponent of the immune defense of the insect againstthis pathogen [34]. Activation of recognition genes in Dro-sophila following nematode (symbiotic or axenic) infectionhas not yet been tested [35]. Finally, it is of particularinterest that certain nematode parasites are able to inter-fere with the insect immune system by evading immune

541

Antimicrobial Peptides

Key:

OpsoninsBacterial toxinsEntomopathogenic bacteria

]

Phenoloxidase activityDead hemocyte

Activated hemocyteCuticular layerMAMPs

Opsonizationand hemocyte recruitment

Capsule formation

(a) (b)

Hemocytes secrete melanin

Opsonins

Sequestration of host’s hemolymph proteins

Bacterial toxins

Dissarming of the immune system by symbiotic bacteria

Apoptosis of plasmatocytes

Circulating hemocytessecrete AMPs

Resistant nematodeSusceptible nematode

Circulating hemocytessecrete AMPs

Parasite cuticular lipids

Fat body

AMPsAMPs

Fat body

Phagocytosis of bacteriaby plasmatocytes

Apoptosis of lamellocytes

No opsonization

Inactivation of the PO cascade

Live nematode

Crystal cells

Hemocytes secrete melanin

Lamellocytes

Lamellocytes


Dead nematode

Cuticle barrier

Figure 3. Interaction of nematode cuticle with insect immune mechanisms. (a) Susceptible nematodes are potentially recognized by the immune system either via the

interaction of parasite surface proteins or carbohydrate motifs with certain insect opsonins (molecules that render foreign microorganisms susceptible to phagocytosis or

encapsulation) or by direct contact with insect hemocytes. This leads to the local activation of the phenoloxidase (PO) cascade, production of melanin and encapsulation

response by hemocytes through capsule formation. Immune recognition also triggers the production and secretion of antimicrobial peptides (AMPs) and other immune

proteins that result in slowing down or eliminating the infection. (b) By contrast, resistant nematodes are able to use their cuticular lipids to recruit host hemolymph

proteins. They use these hemolymph proteins to coat themselves which allows them to avoid opsonization and encapsulation by insect hemocytes. Certain insect

pathogenic nematodes, such as Heterorhabditis and Steinernema, are able to employ their symbiotic bacteria (Photorhabdus and Steinernema, respectively) that interfere

with host immune defenses. These bacterial pathogens possess several toxin genes and virulence factors that are expressed upon infection and are capable of disarming

insect humoral (degradation of AMPs), cellular (apoptosis of hemocytes) and melanization (inactivation of the PO cascade) responses. Abbreviations: AMPs, antimicrobial

peptides; FB, fat body; H, hemocytes; MAMPs, microbe-associated molecular patterns; PO, phenoloxidase.


recognition, thus preventing the activation of immunedefenses (Box 2, Figure 3).

Humoral immune responses to nematodesActivation of signaling pathways dependent on microbialrecognition results in the restriction or elimination of theinvading pathogen by antimicrobial effectors [27]. Thebest-known antimicrobial factors of the insect immunesystem are the AMPs that are secreted into the hemo-lymph. The first of these to be identified were the Cecro-pins, which are best characterized in Drosophila andcertain Lepidoptera [36]. In flies, AMP-encoding genesare regulated by two distinct and well-studied NF-kBintracellular cascades: the Toll pathway, which is requiredfor countering some Gram-positive bacteria and fungi, andthe immune deficiency pathway that is activated in re-sponse to Gram-negative bacteria [37]. Mutations in cer-tain genes of this pathway cause an immune-deficientphenotype characterized by the lack of expression of sev-eral AMP genes and a marked sensitivity to microbialinfections [38].

There is little information in the literature that relatesthe effects of AMP to the development of filarial nematodeparasites in vivo (Figure 2). Inoculation of A. aegyptiwith microfilariae of B. pahangi has led to purification,

542

identification and functional characterization of the AMPDefensin [39]. This peptide has also been detected in thehemolymph of mosquitoes inoculated with microfilariae ofB. malayi or D. immitis [40]. It has further been shown thatCecropins are active against filarial worms in vitro [41]. Inaddition, although W. bancrofti filarial nematodes havebeen shown to upregulate the mRNA levels of defensin,cecropin and transferrin in infected A. aegypti mosquitoes[42], no defensin transcription was found in the fat body of A.subalbatus transmitting B. malayi parasites [43].

Wolbachia bacterial symbionts are also harbored bymost insect species [44]. A strain of Wolbachia, calledwMelPop or ‘popcorn’, has been identified to over-replicatein somatic tissues, which leads to reduced lifespan of itsDrosophila melanogaster host under laboratory conditions[45]. Stable transfection of this Wolbachia strain from D.melanogaster into A. aegypti also results in reduced lon-gevity of mosquitoes in the lab [46]. Interestingly, wMel-Pop infection leads to the upregulation of a variety ofimmune-response related genes (immune preactivation)in A. aegypti and decreases the development of Brugiafilarial nematodes in mosquitoes [47].

The crucial role of the inducible antimicrobial defenseagainst nematode infection is demonstrated by the immu-nosuppressive properties of the nematode parasite cuticle


and its interaction with hemolymph humoral components.In the greater wax moth Galleria mellonella, Heterorhab-ditis bacteriophora and its symbiotic bacterium Photorhab-dus luminescens were found to produce a specificextracellular proteinase that is secreted into the insectand inhibits the AMP Cecropin [48]. This suppressionprotects the nematodes, allows the release of symbioticbacteria and enables the nematode–bacterium complex toestablish infection. A similar study involving Galleria andthe entomopathogenic nematode Steinernema feltiaeshowed that the parasite employs cuticle-surface factorsagainst the activation of immune pathways and productionof antimicrobial peptides in Galleria larvae [49]. The in-hibitory properties of the nematode cuticle were attributedto the binding specificity of parasite–body surface factorsthat are able to remove hemolymph immune-related pro-teins, and thus rendering nematodes resistant to the im-mune response (Figure 3).

To investigate the impact of the nematode parasiteitself, separately from its symbiotic bacteria, it is impor-tant to generate and work with axenic nematodes. It wasrecently reported that mRNA levels of cecropin were un-detectable in the fat body of Manduca infected with onlyaxenic Heterorhabditis, whereas infection with symbioticworms (containing Photorhabdus bacteria) was associatedwith elevated cecropin mRNA levels [34]. Similarly, in theDrosophila model, natural infection of transgenic larvaewith symbiotic Heterorhabditis resulted in the transcrip-tional activation of four AMPs, as shown by reporter con-structs based on green fluorescent protein. By contrast,axenic Heterorhabditis failed to induce significant tran-scription of AMP genes, which implies that the immuneresponse is to the presence of the nematode with itssymbiont Photorhabdus [50]. However, it is interestingto note that although certain genes encoding differentAMP genes are overexpressed in Manduca and Drosophilain response to Photorhabdus injection, which implies thatthe antibacterial responses of the insect host are deployedagainst infection by these pathogens [51,52], Xenorhabdusbacteria are able to inhibit the expression of Cecropin inthe beet armyworm, Spodoptera exigua [53].

At the genomic level, it has been reported that AMPgenes are transcribed in response to filarial worm infectionin A. subalbatus [31], although low transcription levels ofcertain AMPs were detected following the analysis of cDNAlibraries that were generated from whole body A. subalba-tus exposed to filarial worm parasites [32]. However, otherrecent studies suggested that identification of a low num-ber of immune-responsive genes activated against filarialworm infection could reflect strategies for tolerating tissuedamage caused by the nematodes and not antifilarial wormreactions [28,54]. Finally, given that the regulation of AMPtranscripts in response to pathogens is a highly complexprocess in mosquitoes [55], the interpretation that nema-tode parasites are able to impede host humoral mecha-nisms and decrease transcriptional abundance thereforeremains speculative.

Cellular immune responses to nematodesAlthough insect humoral immune defenses are still betterunderstood than cellular responses, the gap is rapidly

closing as significant progress has been made recently withthe aid of genetic techniques in model insects that haveallowed the identification of molecules required for regu-lating cellular defenses. In Drosophila, it has been shownthat the cellular and humoral responses act in concert tocombat infection [56,57]. The Toll pathway in flies has beenfound to control hemocyte proliferation/density and plays asignificant role in the cellular immune response, whichincludes the encapsulation and killing of parasites [58].Mutations in the gene cactus (the Drosophila homolog ofIkB) or the constitutive expression of the gene dorsal (NF-kB transcription factor) can induce differentiation of lamel-locytes, a specialized group of hemocytes that participatein the encapsulation of foreign particles [20].

The interaction of mosquito hemocytes with microfilar-iae of the filarial worms D. immitis and B. malayi has longbeen reported in A. aegypti and A. subalbatus, respectively[59,60]. It was later described that trapping and destruc-tion of microfilariae in the body cavity of mosquitoesinvolves both humoral and cellular reactions: melanization(see below) and encapsulation (Figure 2). In particular,cellular encapsulation of B. pahangi microfilariae in themosquito Anopheles quadrimaculatus involves the activa-tion of hemocytes (plasmatocytes) and their attachment tothe already melanized microfilariae. The encapsulationprocess starts with the synthesis of a melanin layer andterminates with the formation of a membrane-like struc-ture on the external surface of the cellular capsule [61].Similar results reporting the combined action of melani-zation and encapsulation functions for restraining filarialparasites were also obtained in mosquito vectors inoculat-ed with D. immitis microfilariae [62]. The importance of theinsect cellular response in fighting nematode parasites hasalso been shown in Simulium black flies that vector micro-filariae of Onchocerca lienalis, which represents one of fourfilarial nematodes that cause onchocerciasis in humans.The intrathoracic injection of O. lienalis microfilariae intoadult black flies significantly increased total numbers offreely circulating hemocytes in infected flies comparedwith control flies [63].

In the entomopathogenic nematode Steinernema, carry-ing symbiotic Xenorhabdus bacteria, different moleculesconferring cytotoxic activity and unbinding effects in vitrotowards specific lepidopteran hemocyte types have beenidentified previously, functionally characterized and sepa-rated using biochemical techniques [64]. Heterorhabditisnematodes carrying symbiotic Photorhabdus orally infectorthopteran insect pests; the nematodes have been shownto interfere with the cellular immune defense by prevent-ing the phagocytic capacity of hemocytes at early timepoints of the infection, resulting in hemocyte killing later[65]. However, the factors responsible for these effects werenot identified. Nematode cuticular surface lipids have beenfound to interact with the cellular immune response ofdifferent insect species. Steinernema glaseri nematodeparasites avoid encapsulation in larvae of the Japanesebeetle, Popillia japonica, by using the surface coat proteinSCP3a that not only destroys host hemocytes but alsoprotects unrelated nematode species from being detectedand eliminated [66]. Although the structure of this proteinhas not been solved, it is clear that it acts at the early

543


stages of infection and plays a major role in the establish-ment and survival of the nematodes. In search of hostfactors that limit Heterorhabditis survival and reproduc-tion in the Colorado potato beetle, an unidentified hemo-lymph factor was shown to affect nematode encapsulationby hemocytes both in vivo and in vitro, but did not directlyinfluence the development of the worms [67]. In addition,previous results suggest that the nematode S. feltiae isable to attract hemolymph components onto its cuticularsurface and thus avoid encapsulation by preventing hemo-cytes from attaching to its cuticle (Figure 3) [68]. The samenematode has also been shown to protect its associatedbacteria by sequestering opsonization factors from theinsect hemolymph that resulted in reduced phagocytosisby host hemocytes in Galleria [69]. Recently, symbioticHeterorhabditis nematodes, but not axenic worms, werefound to decrease certain cellular immune reactions, suchas numbers of viable hemocytes and hemocyte aggregationfollowing infection of Manduca larvae [34]. However, bothsymbiotic and axenic Heterorhabditis were able to reducethe phagocytotic ability of hemocytes, as estimated by thenumber of nonpathogenic Escherichia coli cells that wereengulfed in phagocytic cells. Although the factor respon-sible for these effects was not determined, these resultsagain highlight the dynamic interference between para-sitic nematodes with the insect cellular defense mecha-nism. Notably, Photorhabdus and Xenorhabdus alsointerfere with insect cellular immunity. Photorhabdusbacteria have been found to evade the cellular immuneresponse by killing hemocytes and by employing mecha-nisms that suppress key cellular responses in insects [70],whereas Xenorhabdus bacteria use distinct effectors orsecretion mechanisms to overcome host cellular functions[71].

Whole genome transcriptomic analysis of A. subalbatushemocytes and whole body tissues from A. subalbatus fedon blood meal containing infective filarial worms generat-ed dissimilar results with only four transcripts beingshared between RNA from the two sources [31]. Thisfinding implies the high tissue specificity of the antifilarialimmune response in this mosquito species. In the samestudy, a transcript that was significantly upregulated in B.malayi infected mosquitoes was calreticulin, which is in-volved in encapsulation responses in Galleria and is also amarker for phagocytosis of apoptotic cells in Drosophila[72,73]. Other microarray studies also reported that thetranscriptional response of mosquitoes to filarial worminfection mainly comprises upregulation of genes encodingcellular components or molecules that are involved incellular signaling and repair [28,32].

Experimental evidence in the entomopathogenic nema-tode Steinernema using EST analysis recently identifiedgenes which are expressed in the parasitic phase of thenematode and are predicted to encode putative secretedproteins that could act as virulence factors against insecthemocytes [74]. Another study has provided further infor-mation on differential expressed genes in Heterorhabditisupon treatment of the nematodes with hemolymph con-taining insect hemocytes [75]. Overall, these results indi-cate that nematode parasites are able to interfere with thecellular host immune response, to target and inactivate

544

immune cells that in turn will facilitate rapid suppressionand efficient evasion of the insect immune system.

Interaction of nematode parasites with thephenoloxidase and coagulation responsesThe phenoloxidase (PO) immune response is an immediatereaction against invading microbes in insects. It involvesthe formation of short-lived toxic substances (e.g. chemi-cally reactive quinones) and long-lived products such as themelanin (a brown–black pigment) that is deposited atwound sites, participating in encapsulation and killingof microbial pathogens [76]. The PO response is initiatedthrough PRPs, and the enzyme initially exists as theinactive precursor prophenoloxidase (proPO). This is syn-thesized and released by specialized circulating cells intothe hemolymph after microbial challenge and is promptlyactivated by plasma serine proteases [77]. Recent evidenceimplies that the melanization cascade (or proPO activatingsystem) overlaps the humoral and cellular insect immunedefenses. It is strongly associated with the coagulationresponse (or hemolymph clotting) that acts to seal woundsand prevent microorganisms from entering and spreadingthroughout the insect body cavity and with factors thatstimulate phagocytosis [78,79].

There is evidence that insect parasitic nematodes arecapable of interacting with the PO system, to successfullyinfect their hosts. S. feltiae nematodes have been shown touse body surface molecular components that interfere withthe LPS-mediated proPO activation pathway in Gallerialarvae. This leads to a quick reduction in hemolymph POactivity and suppression of the host encapsulation re-sponse [80]. Incubation of S. feltiae cuticles alone withGalleria hemolymph results in a significant decrease inPO activity. This PO inhibitory effect was attributed to anunknown lipidic component of the surface of the parasitethat sequesters host-interacting proteins from the hemo-lymph, thus inactivating the PO response (Figure 3). Al-though this feature of S. feltiae has not been observed forother insect parasitic nematodes, it was proposed that thiscould form a disguise strategy that would help the parasiteto evade host immune mechanisms (Box 2) [81]. In anotherstudy showing that host immune responses against nema-todes differ greatly among insect species, certain parasiticnematodes exhibited different abilities to suppress mela-nization and escape from encapsulation. It was suggestedthat differences in successful insect infection or host sus-ceptibility might reflect variation in body surface proteincomposition in different entomopathogenic nematode spe-cies [82]. More recently, it was demonstrated that knock-down of Drosophila transglutaminase, a conservedcomponent of clotting cascades in insects and humans,resulted in decreased formation of small aggregates ofyeast cell wall preparations (zymosan beads) and increasedsensitivity of larvae to natural infection with Heterorhab-ditis nematodes and their symbiotic Photorhabdus bacte-ria, whereas PO was not involved in the immune responseto these pathogens [83]. The immune function of clotformation was also confirmed by data on the role of eico-sanoids in protecting Drosophila larvae against infectionswith Heterorhabditis and Photorhabdus. In particular,injection of eicosanoid biosynthesis inhibitors prior to


nematode infection significantly increased insect mortali-ty, while it was further shown that two clotting factors,gp150 and fondue, when silenced in hemocytes and fatbody, respectively, are required for efficient response to thenematode–bacteria complex [84]. Interestingly, Photo-rhabdus has been shown to produce a hydroxystilbenecompound that inhibits activated Manduca PO and leadsto decreased host resistance, and a metalloprotease thatinduces a melanization response on injection in Manduca[85,86]. In addition, certain Xenorhabdus metabolites haverecently been found to inhibit PO of the diamondbackmoth, Plutella xylostella [87]. However, it is currentlyunknown whether axenic Heterorhabditid nematodesare able to interfere with the insect proPO activatingsystem using a similar molecule.

In mosquitoes, field studies recently showed thatAnopheles punctulatus employs the melanization reactionas a defense against invasion by W. bancrofti microfilariae[88]. Knockout of proPO1 inhibits melanization of micro-filariae in A. subalbatus and proPO2 transcripts wereshown to increase in abundance in blood-fed and micro-filariae-injected mosquitoes [89,90]. Also in A. punctulatus,a hemocyte-produced glucan recognition protein, which isprobably involved in the melanization of B. malayi para-sites, has been identified [91]. The transcriptional responseof A. subalbatus to B. malayi has revealed differentialtranscription of a large number of serine proteases, serpinsand phenylalanine hydroxylase, which are implicated inthe biosynthesis of melanin [31]. However, characteriza-tion of an EST library for the same mosquito–parasitesystem identified few hits representing the melanizationpathway among the different clusters, which was attribut-ed to methodology bias related to construction of thelibrary or inoculation of the mosquitoes [32]. Finally, arecent microarray analysis investigating the global tran-scriptional response of A. aegypti mosquitoes to B. malayiinfection showed increased abundance for transcripts ofseveral serine protease genes and putative melanization-related components, which further indicates a potentialrole for the PO system in the interaction between filarialnematodes and their mosquito vectors [28].

Concluding remarks and future perspectivesInsects are excellent models for studying host–nematodeinteractions and immune responses. Identification of hostimmune reactions or immune-related molecules will great-ly benefit human and animal health by assisting strategiesfor controlling insect vectors of human parasitic nematodesor agricultural nematode pests. From a medical point ofview, future studies could focus on how filarial worminfections are sensed, how their presence is communicatedboth within and among cells and tissues of the host, andhow they can be efficiently eradicated. It would also beparticularly interesting to compare the immune responsesto nematodes of vertebrate and invertebrate hosts. In thefuture, whole genome sequencing of nematode parasites ofinsects will certainly play an important role in the devel-opment of insect–nematode model systems for studyinghost defense mechanisms. Such genomic information willpotentially reveal the number and nature of genes that areinvolved in parasite–host interactions and molecules that

could participate in the invasion of the host, evasion orabrogation by the parasite of host immune responses, orproduction of compounds with antiparasite activity by thehost. Functional genomics analysis, in combination withgenetic and RNAi screens in model insects, such as Dro-sophila, will offer a powerful approach for elucidating theregulation of host immune genes responsible for antine-matode reactions in insects that will in turn help us toinvestigate and understand the evolutionary conservationof antiparasitic immune responses in vertebrate animals.

AcknowledgmentsWe apologize that owing to space limitations we are unable to citeadditional references. We thank members of the Department of BiologicalSciences at The George Washington University for their continuous help,support, critical reading of the manuscript and constructive criticism.

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Insect immune responses to nematode parasites