Vol.3, No.4, 326-333 (2013) Open Journal of Animal Sciences
http://dx.doi.org/10.4236/ojas.2013.34049
A comparative analysis of entomoparasitic
nematodes Heterorhabditis bacteriophora and
Steinernema carpocapsae
Rinu Kooliyottil*, Devang Upadh ya y, Floyd Inman III, Sivanadane Mandjiny, Len Holmes
Sartorius Stedim Biotechnology Laboratory, Biotechnology Research and Training Center, University of North Carolina at Pembroke,
Pembroke, USA; *Corresponding Author: [e-mail]
Received 13 September 2013; revised 15 October 2013; accepted 26 October 2013
Copyright © 2013 Rinu Kooliyottil et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Heterorhabditis bacteriophora and Steinernema
carpocapsae are microscopic entomoparasitic
nematodes (EPNs) that are attractive, organic
alternatives for controlling a wide range of crop
insect pests. EPNs evolved with parasitic adap-
tations that enable them to “feast” upon insect
hosts. The infective juvenile, a non-feeding, de-
velopmentally arrested nematode stage, is des-
tined to seek out insect hosts and initiates pa-
rasitism. After an insect host is located, EPNs
enter the insect body through natural openings
or by cuticle penetration. Upon access to the
insect hemolymph, bacterial symbionts (Photor-
habdus luminescens for H. bacteriophora and
Xenorhabdus nematophila for S. carpocapsae)
are regurgitated from the nematode gut and ra-
pidly proliferate. During population growth, bac-
terial symbionts secrete numerous toxins and
degradative enzymes that exterminate and bio-
convert the host insect. During development
and reproduction, EPNs obtain their nutrition by
feeding upon both the bioconverted host and
proliferated symbiont. Throughout the EPN life
cycle, similar characteristics are seen. In gen-
eral, EPNs are analogous to each other by the
fact that their life cycle consists of five stages of
development. Furthermore, reproduction is much
more complex and varies between genera and
species. In other words, infective juveniles of S.
carpocapsae are destined to become males and
females, whereas H. bacteriophora develop into
hermaphrodites that produce subsequent gen-
erations of ma les and fe males. Othe r differ ence s
include insect host range, population growth
rates, specificity of bacterial phase variants, etc.
This review attempts to compare EPNs, their
bacterial counterparts and symbiotic relation-
ships for further enhancement of mass produc-
ing EPNs in liquid media.
Keywords: Entomoparasitic Nematodes (EPNs);
Heterorhabditis Bacteriophora; Steinernema
Carpocapsae; Photorhabdus Luminescens;
Xenorhabdus Nematophila; Symbiosis;
Mass Product i o n
1. INTRODUCTION
Entomoparasitic nematodes (EPNs) Heterorhabditis
bacteriophora and Steinernema carpocapsae are utilized
as biocontrol agents against various insect pests of agri-
cultural significance (Figure 1). EPN is an attractive
organic alternative to chemical insecticides as they do
not pose a threat to the env ironment. Additionally, EPNs
are particularly safe for use around humans, livestock,
and plants [1]. The close symbiotic relationship between
EPNs and their bacterial counterparts contributes to the
safety and efficacy of their use as biological control
agents.
Figure 1. Brightfield micrograph depicting
adults of H. bacteriophora under 40× mag-
nification.

R. Kooliyottil et al. / Open Journal of Animal Sciences 3 (2013) 326-333 327
Symbiotic bacteria Photorhabdus spp. and Xenorhab-
dus spp. provide Heterorhabditis and Steinernema nema-
todes, respectively, with diverse services within the host
insect: 1) produce virulence factors that neutralize and
kill the insect host [2-4]; 2) bioconvert the host into nu-
tritional components; 3) serve as the main food source
for their nematode partners [5,6]; and 4) produce antim-
icrobials (Figure 2) which prevent putrification of the
insect host by competing microbes [7-10]. These bacte-
rial based services ultimately produce an optimal envi-
ronment for nematode growth and reproduction [11].
These symbiotic associations are useful in managing
insect pests that are destructive to many commercially
viable plants and crops [12,13]. Heterorhabditis spp.
usually performs better than St e i nern e m a spp. [14];
which is a consequence of their predatory lifestyles. Het-
erorhabditis bacteriophora is considered to be a bur-
rowing “cruiser” nematode that “seeks” out its insect
host by borrowing into the soil. To the contrary, Stein-
ernema carpocapsa e is an “ambushing” nematode that
attacks insect hosts that are “passing” by [14,15].
Mass production of Heterorhabditis bacteriophora and
St e i ner n e ma carpocapsae on a large scale is difficult and
cumbersome either in vivo or in vitro due to various ob-
stacles (Figure 3). Production of EPNs can be achieved
in vivo; however, commercial scale production is imprac-
ticable due to high production costs and low nematode
yields per gram of insect biomass [16,17]. EPN produc-
tion with in vitro solid technology gives rise to higher
nematode yields per gram of solid media when compared
to in vivo technologies. However, costs associated with
solid media technologies are much higher than in vivo
technologies. The high production cost is mainly associ-
ated with labor, materials and storage area [18,19]. To
many researchers, in vitro liquid technologies should be
used in commercial production of EPNs for international
markets because these technologies are considered to be
Figure 2. Antibiotic activity from permeate
obtained from a culture of P. luminescens.
Moraxella (Branhamella) catarrhalis is the
test subject.
Figure 3. Mass production of H. bacteriophora
in a 10 L bioreactor. Note the red pigmentation
produced by its bacterial symbiont P. lumines-
cens.
the most cost-efficient process when compared to other
methods. Although mass production in submerged cul-
ture offers cost-efficiency, capital and technical expertise
is still required [20]. Problems arising during nematode
mass production are due to many different factors. Some
of these factors include phase shifting of the bacterial
symbiont, low percentages of nematode copulation, in-
oculum (bacterial/nematode) concentrations and fermen-
tation parameters (oxygen concentration, pH, tempera-
ture, agitation, etc).
Due to the lack of biological knowledge of EPNs and
their bacterial symbionts, optimization of liquid mass
production technologies is hindered. Furthermore, the
lack of knowledge involving the symbiotic relationship
between EPNs and their respective bacterial symbionts
poses more difficulty in establishing and optimizing
standardized production protocols. Lastly, understanding
the triangular relationship between EPNs, bacterial sym-
bionts and host insects will further promote the devel-
opment and optimization of media and fermentation pa-
rameters for maximizing nematode yields. This review
will 1) identify the biological differences in the life cy-
cles of H. ba cteriophora and S. carpocapsae; 2) describe
the nutritional relationship between EPNs and their bac-
terial symbionts; and 3) briefly describe potential bio-
logical processes occurring during host interaction that
may benefit mass production processes.
2. NEMATODE BIOLOGY AND
LIFE CYCLE
The third stage infective juvenile (IJ) of Heterorhabdi-

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328
tis and Steinernema nematodes occurs free in the soil and
their roles are to seek out and infect host insect larva.
Steinernema nematodes typically enter the insect host
through natural body openings (mouth, anus and spira-
cles). Furthermore, Heterorhabditis nematodes gain ac-
cess to the insect host in a similar fashion as St e i ner nema
spp.; however, Heterorhabditis spp. can also gain entry
by penetrating the insect’s cuticle utilizing a dorsal tooth.
Within their anterior intestine, IJs carry a lethal dose of
their bacterial symbiont and when stimulated within the
insect host, are released by the nematode p artner into the
insect hemolymph (i.e., initiation of recovery).
Upon bacterial release, bacterial proliferation occurs
and as a result, secretion of insect toxins and degradating
enzymes occur that kill and bioconvert the insect cadaver
within 24 - 48 hours. As the bacterial population reaches
stationary phase, produ ction of secondary metabolites, in
particular antimicrobials, and an unknown “food signal”
is initiated. The secreted antim icrobials are speculated to
be a defense mechanism used to ward off competing mi-
crobes that may cause the cadaver to putrefy [21,22].
Additionally, researchers suggest that unidentified food
signals induce IJs to shed their protective sheaths and
continue development to complete their life cycle [23,24].
After the sheaths are shed, IJs become feeding stage 3
nematodes (J3), develop to the fourth juvenile stage (J4)
and ultimately to adulthood.
The first generation of offspring depends on the nema-
tode genus (Heterorhabditis, St einernema). For Heter or-
habditis spp., the first generation of offspring emerges as IJs
[25]. These IJs were developed in utero of the parental her-
maphrodite in a process known as endotokia matricida.
Endotokia matricida (Figure 4) occurs as a result of
self-fertilization; whereby fertilized eggs hatch into juve-
niles of Stage 1 (J1) within the hermaphroditic nematode
[25]. After hatching, J1s feed upon the maternal nematode
and continue to develop to Stage 2 (J2). During this devel-
opmental stage, nutrients become limited within the her-
maphrodite that signals the J2 nematodes to develop into
IJs. As development of IJs is completed, the IJs emerge
from the maternal nematode by bursting through the her-
maphroditic cuticle and into the protected environment.
After their hermaphroditic emergence, IJs continue their
life cycle due to the presence of residual food signals.
This subsequent generation of IJs develop into adult
nematodes that are sexual reproductive (male/females);
however, if females do not mate with the opposite sex,
offspring may be produced by parthenogenesis or
hermaphroditically [17,26,27]. Furthermore, subsequent
generations will continue within the insect cadaver until
all nutrients and symbiotic bacteria are consumed. It is
during this point in the reproduc tive life cycle that nutria-
ent stress will induce J2 nematodes to develop and tran-
sition into IJs that will ultimately emigrate from the
Figure 4. Hermaphrodite of H. bacteriophora
exhibiting endotokia matricida. Nematodes
seen in the body cavity will exit as IJs.
cadaver to search for new insect hosts [25,28].
In St e i ner n e m a spp., IJs undergo the same infective
behavior as Heterorhabditis spp. with the exceptions of
cuticle penetration and the initial round of recovery. In a
population study conducted by Wang and Bedding, the
researchers found that, upo n recovery, IJs of St e i ner nema
spp. develop into reproductive males and females (i.e.
amphimictic reproduction) which also occurs in first and
second generation offspring [25]. However, all eggs
produced by third generation females were found to de-
velop via endotokia matricida. Unlike H. bacteriophora,
juvenile stages resulting from endotokia matricida in S.
carpocapsae do not develop into IJs until they exit the
maternal nematode. As a response to nutrient depletion,
endotokia matricida occurs in Steinernema spp. due to
cessation of egg-laying [29].
Differences in life cycle and reproductive biology in-
fluence the yield of the two genera in liquid culture. Het-
erorhabditids exhibit a “Y” or “
 ” type copulation on
solid media. In contrast to steinernematids, heterorhabdi-
tids are unable to attach to each other in liquid culture
due to sheer caused by agitation and/or aeration [17].
Maximizing reproductive mating is a crucial factor that
must be considered for mass production as the number of
offspring produced from copulation is at least 10-fold
higher than production by endotokia matricida. Opti-
mizing mass production parameters for copulation can be
achieved through bioreactor design and optimization of
agitation and aeration [30]. However, maximizing mating
has some limitations in Heterorhabditis spp. in liquid
culture because the first generation is exclusively her-
maphrodites where the amphimictic forms are not pro-
duced until subsequent generations [17].
Maximizing heterorhabditid yields in liquid culture
will greatly depend on the concentration of nematode
inoculum and the degree of recovery. Percentage recov-
ery is found to be less in liquid media compared to in
vivo conditions [23,31-34]. Additionally, recovery of
EPNs is mostly dependent on the food signal secreted by

R. Kooliyottil et al. / Open Journal of Animal Sciences 3 (2013) 326-333 329
the associated bacterial symbiont during late exponential
growth [23,35]. Maximum average yields of EPNs re-
ported were in shake flask batch cultures were 300,000
and 320,000 IJs per ml for H. ba cteriophora and S. car-
pocapsae, respectively [36]. Furthermore, in a recent
study, fermentat ion modes (b a t c h an d fe d-batc h) for mass
producing S. carpocapsae were compared and found that
fed-batch modes produced an 8.8-fold higher IJ yield
than batch modes [37] .
3. NUTRITIONAL RELATIONSHIP
BETWEEN NEMATO DES AND THEIR
BACTERIAL SYMBIONTS
Mass production in liquid media, regardless of the
culturing vessel, requires the nematode culturing media
to be conditioned [38]. Conditioning of the liquid me-
dium refers to the inoculation of the appropriate bacterial
symbiont. This step is crucial as the bacterial symbionts:
1) convert the complex medium into easily accessible
components for both itself and partner nematodes; 2)
secrete necessary metabolites, (food signals, antibiotics,
pigments, etc.); band 3) serves as the main food source
for the developing nematodes [38]. The nutritional rela-
tionship is highly specific for Heterorhabditis, because
these nematodes cannot be cultured under axenic condi-
tions or on other bacteria. On the other hand, Stein-
ernema are less fastidious can reproduce in the absence
of their symbiotic partner; however, nematodes yields are
severely decreased [24]. The successful development of
St e i ner n e ma spp. in axenic, in vivo conditions or plated
on non-symbiotic bacteria (i.e. Escherichia coli) have
been reported [6,24,39-41]. Add itional research has been
performed that shows Steinernema spp. is unable to de-
velop when given Photorhabdus lumines cens as the bac-
terial food source [39]. Furthermore, this finding sug-
gests possible nematicidal properties of Photorhabdus
spp.; which may also indicate that Xenorhabdus spp. may
also exhibit similar properties.
There are two forms or variants of entomopathogens
(Xenorhabdus spp. and Photorhabdus spp.) that exhibit
different phenotypes and metabolic profiles based upon
several factors [42]. These variants can either be isolated
as primary wild type (Phase I) or secondary form (Phase
II) [43,44]. EPNs require Phase I forms as these variants
are extremely metabolically active and produce a battery
of different substances and traits (enzymes, antimicrobi-
als, insect toxins, bioluminescence, etc). The phase II
form is commonly seen in the laboratory on routine cul-
turing media. This transitioning between Phase I and
Phase II states is known as phase variation. This biologi-
cal phenomenon ensures the survival of a bacterial cell
and/or population in unfavorable conditions [38]. Phase
variation naturally occurs in many enteric bacteria such
as E. coli and Salmon ella spp. The role of phase variation
and the genetic mechanisms involved with it in Photor-
habdus and Xenorhabdus have not been identified. Fur-
thermore, researchers suggest that deteriorating envi-
ronmental conditions (pH, nutrient exhaustion, osmolar-
ity, etc.) may be responsible for triggering thi s effect [45].
Reports indicate the presence of two types of pro-
teinaceous crystalline inclusions in the cytoplasm of both
bacterial symbionts X. nematophila and P. luminescens
[46,47]. Although the functions of the inclusion bodies
are unknown, it is hypothesized that these proteins may
be involved in nematode nutrition or insect pathogenicity
as they represent 40% of the total cellular pro tein [48,49].
The genes responsible for producing inclusion bodies
have been identified (cipA and cipB). Further research
shows that the inactivation of these genes alters the cha-
racteristics of the Phase I variant of P. luminescens
thereby rendering the symbiont incompetent to support
nematode growth and reproduction [49,50]. Additional
research has shown that Ste i nern e ma nematodes can feed,
develop and reproduce on E. coli cultures that express at
least one of the Cip proteins from Xenorhabdus spp. [49].
However, in a similar experiment, E. coli cultures ex-
pressing Xenorhabdus Cip proteins did not support the
development or reproduction of H. bacteriophora [40].
These results suggest that nutrient requirements are dif-
ferent for Heterorhabditis spp. and St e i n erne m a spp.
[49].
4. STABILITY OF NEMATODES AND
BACTERIAL SYMBIONTS DURING IN
VITRO CULTURE
Trait deterioration is a major concern to industrial
producers of entomopathogenic nematodes. Bilgrami et
al. reported trait changes as a result of continuous sub-
culturing in S. carpocapsae and H. bacteriophora [11].
These investigators studied trait stability of P. lumines-
cens and X. n ematophila after serial in vitro subculturing
and demonstrated that phase variation (Phase I to Phase
II) in P. luminescens and X. nematophila strains occurred
within ten subculturing cycles [11]. Furthermore, pheno-
typic variation was controlled in X. nematophila strains
by selection of primary variants; however, trait change
was not detected after prolonged culturing. When phe-
notypic variation in P. luminescens was controlled,
changes in the primary variant were observed. These
observations include cellular morphology, size of inclu-
sion bodies, and prevalence of inclusion bodies [51].
Bilgrami et al. noted the stability of the virulen ce in S.
carpocapsae after subculturing for prolonged periods of
time when compared to H. bacteriophora [11]. Gaugler
et al. also reported the virulence stability in S. carpo-
capsae by correlating stability with numerous nematode
passages in G. mellonella [52]. The basis for virulence
stability in S. carpocapsae may lie in the phase stability

R. Kooliyottil et al. / Open Journal of Animal Sciences 3 (2013) 326-333
330
of its associated bacterial symbiont X. nematophila [11].
In vitro subculturing of P. luminescens resulted in vast
changes including alterations in growth rates, cell size,
number and size of inclusion bodies, virulence and pig-
mentation [11]. In comparison to P. lumin es cen s, X.
nematophila did not result in measurable changes to any
of the traits tested [11]. However, Bilgrami et al. are
critical to the findings of Wang and Grewal involving
stress tolerance reduction, storage stability and reproduc-
tion in H. bacteriophora after three passages in G. mel-
lonella [11,53]. These findings are noteworthy because
present strains in mass production are subcultured many
times before their adoption for commercial use. Such
stability can make a difference between successful and
unsuccessful production runs.
5. HOST INTERACTION
St e i ner n e ma spp. typically searches for insect hosts on
or near the soil surface. This group of EPNs is usually
referred to as “ambush” predators. They generally remain
inactive until a mobile insect host passes by [54]. Am-
bushing in S. carpocapsae also consists of an unusual
jumping behavior in which the IJ nictates, curls into a
loop, and propels itself into the air. Jumping is unique to
Steinernematids and is considered a specialized evolu-
tionary adaptation that facilitates attachment to passing
hosts [54]. Hetero rhabditis spp. dwells into the soil in
search of subterranean, sedentary hosts and commonly
categorized as “cruisers”. Heterorhabditis nematodes are
highly mobile that can respond and target insect hosts
over long-range chemical cues [55]. Host volatiles, such
as CO2, can stimulate both H. bacteriophora and S. car-
pocapsae [56-59]. Hallem et al. investigated the response
of H. b acteriophora and S. carpocapsae IJs to host odors
by using CO2 exposure studies [60]. They found that IJs
of both species were strongly attracted to increasing CO2
concentrations. In the same study, Hallem et al. demon-
strated that the BAG sensory neurons are required for
CO2 attraction [60]. BAG-ablated H. bacteriophora IJs
do not chemotax towards the insect host G. mellonella,
demonstrating a critical role of BAG neurons in host
localization. Since BAG neurons are sensory neurons
that detect CO2, it seems that CO2 is an essential cue for
host attraction [61]. In contrast, ablation of the BAG neu-
rons did not significantly affect the ability of S. carpo-
capsae IJs to jump in response to G. mellonella volatiles,
demonstrating that other neurons besides BAG or possi-
bly other host cues are sufficient to mediate host attrac-
tion [60] .
6. CONCLUSION
Understanding the biology of both the nematodes and
bacterial partner is important for mass production. The
differences in nematode life cycles and bacterial symbio-
sis play major roles in final nematode yields. The time
and concentration of the nematode inoculum along with
nematode recovery greatly affect final yield [62,63].
Mass production strategies involving S. carpocap sae, H.
bacteriophora and their bacterial symbionts have been
developed by many while studying characteristics of
both symbiotic partners in liquid culture [37,38,64,65].
Inman III and Holmes have described the role of treha-
lose, a non-reducing sugar found in abundance within
insect hemolymph that seems to aid in maintainence of
Phase I variant of P. luminescens over extended periods
of time [66]. Research is on-going to increase: 1) the
stability of the bacto-helminthic complex; 2) final nema-
tode yields; and 3) the cost-effectiveness of liquid mass
production technologies.
7. ACKNOWLEDGEMENTS
The authors thank the following participants for their partial financial
support: UNC Pembroke (UNCP) Office of Academic Affairs, UNCP
Department of Chemistry and Physics, UNCP Thomas Family Center
for Entrepreneurship, Robeson County Farm Bureau, North Carolina
Biotechnology Center (NCBC), National Collegiate Inventors and
Innovators Alliance (NCIIA), UNCP Undergraduate Research and
Creativity Center (PURC), and the North Carolina Space Grant Con-
sortium.
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