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Diversity and phylogenetic relationships of entomopathogenic nematodes

(Steinernematidae and Heterorhabditidae) from the Sky Islands of Southern
Arizona

Article in Journal of Invertebrate Pathology · July 2006

DOI: 10.1016/j.jip.2006.02.001 · Source: PubMed

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 Journal of Invertebrate Pathology 92 (2006) 66–72
www.elsevier.com/locate/yjipa

Diversity and phylogenetic relationships of entomopathogenic

nematodes (Steinernematidae and Heterorhabditidae)
from the Sky Islands of southern Arizona
S. Patricia Stock a,¤, Joanna C. Gress b
a
Department of Entomology, University of Arizona, 1140 E. South Campus Dr., Tucson, AZ 85721-0036, USA
b
Department of Plant Sciences, University of Arizona, 1140 E. South Campus Dr., Tucson, AZ 85721-0036, USA

Received 30 December 2005; accepted 3 February 2006

Available online 22 March 2006

Abstract

A survey for entomopathogenic nematodes was conducted in oak-juniper woodlands of four mountain ranges (Santa Rita, Santa Cat-
alina, Pinaleño, and Chiricahuas), in southeastern Arizona. From a total of 120 soil samples, 23.3% were EPN-positive. Of them 78.5%
were positive for Steinernema spp. and 21.5% were positive for Heterorhabditis spp. An integrated approach, combining both traditional
(morphological) and molecular methods, was used for examining the diversity of species of these entomopathogenic nematodes. Two
named-species S. oregonense and S. riobrave are reported for the Wrst time in Arizona, expanding their currently known geographic range.
In addition to this, three undescribed Steinernema and three Heterorhabditis spp. were recovered. Insular evolution, in part, could account
for the geographic distribution of entomopathogenic nematodes in Arizona.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Arizona; Diversity; Distribution; Insect; Pathogens; Nematodes; Oak woodlands; Semi-desert; Desert habitats

1. Introduction host. Following entry through the cuticle and/or natural

openings (i.e., mouth, anus, and spiracles), the IJs release
Entomopathogenic nematodes (EPN) in the Steinerne- the symbiotic bacteria into the insect hemocoel, multiply
matidae and Heterorhabditidae, have emerged as excellent and kill the host, usually within 24–48 h (Ciche and Ensign,
biological control agents of insect pests (Gaugler, 2002). 2003; Poinar, 1990). Nematodes feed on symbiotic bacteria
Moreover, interest in EPN has increased rapidly in recent and complete 1–3 generations in the host cadaver. As food
years, with approximately one hundred laboratories repre- resources are depleted, new IJs are produced and disperse
senting sixty countries worldwide conducting research with in search of new hosts.
these beneWcial organisms (Stock, 2005). These nematodes Numerous surveys have provided evidence of the omni-
are symbiotically associated with bacteria in the family presence of these nematodes in natural and agricultural
Enterobacteriaceae that are pathogenic to insects; steiner- soils (Hominick, 2002) and new species and isolates are
nematids are associated with Xenorhabdus spp., and het- described every year. In contrast to human modiWed areas,
erorhabditids with Photorhabdus spp. (Boemare, 2002). The natural habitats are likely uncontaminated by introduced
third-stage infective juvenile (IJ) is the only free-living stage nematodes and oVer increased opportunities for Wnding
that persists in the soil in search of a susceptible arthropod native nematode species. In this respect, and with particular
reference to EPN, the isolation of native species and/or
populations provides a valuable resource not only from a
*
Corresponding author. Fax: +1 520 621 1150. biodiversity perspective but also from a more applied
E-mail address: [email protected] (S.P. Stock). standpoint. Indigenous EPN may be more suitable for

0022-2011/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jip.2006.02.001
 S.P. Stock, J.C. Gress / Journal of Invertebrate Pathology 92 (2006) 66–72 67

inundative release against local insect pests because of 2. Material and methods
adaptation to local climate and other population regulators
(Bedding, 1990). 2.1. Soil sampling
A survey of EPN in natural habitats in California pro-
vided valuable information regarding the diversity and dis- Soil samples were collected from oak woodlands in four
tribution of these nematodes (Stock et al., 1999). Of all mountain ranges of the Coronado National Forest in
habitats sampled in this study, oak woodlands were the southern Arizona: Santa Catalina, Pinaleño, Santa Rita,
most resourceful for the recovery of EPN (Stock et al., and Chiricahua, between July and November 2002 (Fig. 1).
1999). Indeed, oak woodlands showed the highest recovery At each mountain range, three sampling sites were chosen
rate (100%) in all geographic regions sampled. Several pop- based on accessibility to the area. At each site, 10 random
ulations of two steinernematid species, Steinernema carpo- samples were taken, at least 100 m apart from each other.
capsae (Weiser) and Steinernema kraussei (Travassos) were Each sample (ca. 1 kg of soil), consisted of a composite of 5
isolated from the Sierra Nevada foothills, low coastal sub-samples taken along a 2 m transect at 40 cm intervals
mountains, Great Valley, Southwest mountains, and valleys from the base of an oak tree to the furthest point in the
across the state. transect. Samples were taken with either a hand shovel or a
As in California, the diversity of oak species in Arizona is soil corer, and the Wrst upper 20 cm of soil, including litter
broad. Several Quercus sp. such as Emory oak (Quercus emo- cover, were collected. Each sample (composite of 5 sub-
ryi Torr.), Mexican blue oak (Q. oblongifolia Torr.), silverleaf samples) was placed in heavy-duty polyethylene bags, and
oak (Q. hypoleucoides Camus), desert shrub oak (Q. turbi- closed tightly to prevent water loss. Samples were kept in
nella Greene), and Arizona white oak (Q. arizonica Sargent), coolers (ca. 15 °C) and transported to the laboratory where
have been reported from diVerent mountain ranges in south- they were processed.
eastern Arizona (Borelli et al., 1994; Comeaux, 1981). How- Vegetation (shrubs and trees) associated to each sam-
ever, in contrast to California where oaks form a continuum pling site was collected for further identiWcation. Field tax-
along and across the state, the distribution of oak woodlands onomic keys and identiWcation guides were considered for
in southeastern Arizona is fragmented. Oaks are mainly con- this purpose (Bowers, 1993; Elmore, 1976).
Wned to higher elevations and have a scattered distribution in For each sampling site, a portion of the soil from each
the diVerent mountain ranges (Brown, 1982; Brown and sample (ca. 100 g) was combined and analyzed for the fol-
Lowe, 1980). Studies conducted on Arizona’s fragmented lowing physical and chemical characteristics; pH, organic
woodlands suggest that they had a greater degree of connec- matter, sand, silt, and clay content. Soil samples were sub-
tivity during late Pleistocene to early Holocene, roughly mitted to the Soil, Water and Plant Analysis Laboratory,
10,000 years ago, when the climate was cooler and more University of Arizona.
meseic (Van Devender and Spaulding, 1990). At this time,
woodland habitats spread contiguously across lower eleva- 2.2. Nematode recovery
tions, eVectively linking mountain ranges. A warming and
drying climate has since caused oak woodlands to move ups- EPN were recovered from soil samples by the modiWed
lope (1500–2500 m elevation) and has fragmented them on insect baiting technique (see Kaya and Stock, 1997). Sam-
the isolated ranges. This climate-induced habitat fragmenta- ples were thoroughly mixed and tap water (amount
tion has created what is now referred to as the “Sky Islands” depending on the dryness of the sample) was added to
of southern Arizona with populations of plants and animals moisten the soil. Five last instar Galleria mellonella (L.)
dependent on the woodland habitat subdivided into their larvae (Lepidoptera: Pyralidae) were placed in 200 ml plas-
present disjunct distributions. tic containers (5 containers/sample) with soil obtained from
The particular distribution of oak woodlands in this each sample. Containers were covered with a lid, turned
region provides an interesting framework for studying the upside down, and kept at room temperature (20 § 3 °C).
diversity and population structure of EPN associated to After 7–9 days, all insects were recovered, and parasitized
these habitats. Additionally, investigating EPN diversity in cadavers, recognized by the change of their color (usually
southern Arizona is a particularly timely study. Urban red/purple for heterorhabditids, and ocher/brown/black for
expansion and mesquite invasion of many oak woodlands steinernematids), were placed in modiWed White traps
in the foothills has brought dramatic changes to these natu- (Kaya and Stock, 1997) to allow the emergence of the infec-
ral ecosystems (Comeaux, 1981). Consequently, detailed tive-stage juveniles. Emerging nematodes were pooled from
characterization of the biodiversity in these areas is critical the modiWed White traps and were thoroughly rinsed to
so that appropriate actions may be taken to ensure the remove insect debris, if any, and were used to infect fresh
proper management and future health of these ecosystems. G. mellonella larvae to conWrm Koch’s postulates for path-
In this study, and for the Wrst time, the occurrence and ogenicity and for future identiWcation and establishment of
distribution of EPN in four mountain ranges in southeast- cultures. All nematode cultures were stored in 100 ml tissue
ern Arizona is reported. Phylogenetic relationships of the culture Xasks at 10–15 °C.
recovered species in relation to related taxa is analyzed and Soil samples with negative nematode recovery were
discussed. baited two additional times with either more G. mellonella
68 S.P. Stock, J.C. Gress / Journal of Invertebrate Pathology 92 (2006) 66–72

Fig. 1. Outlines of areas covered by oak vegetation (after Brown and Lowe, 1980) on southern Arizona mountain ranges. 䉬, steinernematid isolates; [ ],
heterorhabditid isolates.

larvae or other available insects (i.e., white grubs, meal- The resulting sequences were compared to a library of more
worms, house crickets) to conWrm results of the Wrst test. than 40 EPN species.

2.3. Nematode identiWcation 3. Results

2.3.1. Morphological characterization 3.1. IdentiWcation of isolates

Nematodes were examined live or heat-killed in 60 °C
Ringer’s solution. The heat-killed nematodes were placed in EPN were recovered from all mountain ranges sampled.
triethanolamine–formalin (TAF) Wxative (Kaya and Stock, From a total of 120 soil samples, 23.3% were EPN-positive.
1997) and processed to anhydrous glycerine for mounting Of them 78.5% (22 samples) were identiWed as Steinernema
(Seinhorst, 1959). Observations were made from live and spp. and 21.5% (6 samples) as Heterorhabditis spp.
mounted specimens using an Olympus IX81 microscope Sequence analysis of 28S rDNA and morphological
equipped with diVerential interference contrast optics. examination indicated two of the steinernematid isolates
Specimen measurements were made using AnalySIS Image recovered, ML-10 and GR-22, were Steinernema riobrave
software (Soft Imaging System Corp., CA, USA). For mor- Cabanillas, Poinar and Raulston and Steinernema oregon-
phological characterization of the isolates, 25 Wrst-genera- ense Liu and Berry, respectively. The remaining Steiner-
tion males and 25 IJs were randomly selected from diVerent nema isolates were depicted as three new undescribed
G. mellonella cadavers. species, hereafter refer to as Steinernema sp. 1 (ML18-20
According to their morphological traits, isolates were isolates), Steinernema sp. 2 (SR isolates) and Steinernema
placed into similar species-groups using taxonomic criteria sp. 3 (CH isolates) (Table 1).
suggested by Stock and Kaya (1996) and Hominick et al. Steinernema sp. 1 resembles most taxa in the interme-
(1997). dium-group, particularly in the body size of the third-stage
infective juveniles which average length is between 600 and
2.3.2. Molecular characterization 800 m. Both Steinernema sp. 2 and sp. 3 are more morpho-
Isolates were molecularly characterized by analysis of logically similar to the feltiae-group species, which are char-
the LSU (large subunit) and ITS-1 (internal transcribed acterized by having third-stage infective juveniles with an
spacer region 1) rDNA sequences for steinernematids and average body size of 800–1000 m (Stock and Hunt, 2005).
heterorhabditids, respectively, according to procedures Sequence analysis of the ITS-1 spacer region of rDNA
described by Stock et al. (2001) and Adams et al. (1998). and morphological examination denoted the heterorhabditid
 S.P. Stock, J.C. Gress / Journal of Invertebrate Pathology 92 (2006) 66–72 69

Table 1
Soil characteristics of EPN-positive sites
Mountain Elevation pH % Organic Soil type Number of positive Nematode species
range range (m) matter samples/total number
Sand (%) Silt (%) Clay (%)
of samples
Santa Catalina 1400–2000 7.5 2.8 78.8 18.5 4.15 3/30 Steinernema n. sp. 1
3/30 Steinernema riobrave
Santa Rita 1600–1800 7.4 3.4 71 23.5 4.6 6/30 Steinernema n. sp. 2
2/30 Heterorhabditis sp. 1
2/30 Heterorhabditis sp. 2
Pinaleño 1600–1750 7.4 3.4 75.1 22.0 2.9 1/30 S.oregonense
Chiricahua 1750–1950 7.5 3.2 62.5 31 6.4 9/30 Steinernema sp. 3
2/30 Heterorhabditis sp. 3

isolates recovered as three new unidentiWed species, Heteror- wood (Cornus stolonifera Michx.), and thimbleberry (Rubus
habditis sp. 1 (CH isolates), Heterorhabditis sp. 2 (SR-2 iso- parviXorus Nutt.) among other native bushes and shrubs.
lates), and Heterorhabditis sp. 3 (SR-3 isolates) (Table 1). Steinernema and Heterorhabditis isolates collected in the
Morphological traits of third-stage infective juveniles (i.e., Santa Rita mountain range (Madera Canyon) were associ-
total body size) and males (i.e., tail and spicule morphol- ated with silverleaf (Q. hypoleucoides) and Arizona white
ogy) of Heterorhabditis sp. 1 and 2 relate these two new spe- oaks (Q. arizonica) and madrone (Arbutus arizonica Gray).
cies to the bacteriophora-group. Whereas Heterorhabditis EPN isolates recovered in the Chiricahua mountain
sp. 3 resembles most H. marelatus. Formal description of range were associated with Arizona white and Emory oaks,
all new taxa is currently being done. Arizona cypress (Cupressus arizonica Greene) and alligator
juniper (Juniperus deppeana Von Steudal).
3.2. Soil types and elevation
3.4. Phylogenetic relationships
Steinernematids were found in all four mountain
ranges sampled, but heterorhabditids were only limited to Phylogenetic analysis of 28S rDNA sequences depicted
the Santa Rita and Chiricahua mountains (Fig. 1). All three distinct Steinernema spp. Steinernema sp. 1 isolates
EPN-positive samples were recovered at an elevation (ML18, ML19, and ML20) from Mt. Lemmon (Santa Cata-
range between 1400 and 2000 m. Steinernema riobrave was lina mountain range) were related to the clade formed by
found at the lowest elevation (1400 m) and Steinernema Steinernema aYne (Bovien) and Steinernema intermedium
sp. 1 was collected at the highest elevation surveyed (Poinar). Relationship of this new species with this clade
(2000 m). Both isolates were recovered in Mt. Lemmon in was highly supported by bootstrap resampling (Fig. 2).
the Santa Catalina mountain range. Heterorhabditis iso- Santa Rita and Chiricahua steinernematid isolates
lates were recovered at an elevation range between 1600 were placed in a major clade that comprises Steinernema
and 1800 m. species known to have medium size infective juvenile
Soil types were classiWed as sandy and sandy-loam with stages. The Chiricahua isolates (Steinernema sp. 3) was
a pH slightly alkaline (7.4–7.5) and an organic matter con- found to be closely related to a clade that comprises Stein-
tent ranging from 2.8 to 3.4% (Table 1). ernema oregonense Liu and Berry (including isolate PI-1)
and Steinernema kraussei (Steiner) (Fig. 2). Although
3.3. Associated vegetation position of this new species in this clade was not resolved
by maximum parsimony analysis. Evolutionary relation-
Associated vegetation to EPN-positive samples varied ships of Steinernema sp. 2 (SR5 and SR6 isolates) were
among the diVerent mountain ranges and in relation to also not resolved in this analysis.
the elevation where samples were taken. For example, Ste- Phylogenetic analysis of ITS-1 rDNA sequences indi-
inernema sp. 1 isolates collected on Mt. Lemmon (Santa cated the three Heterorhabditis spp. recovered represent
Catalina mountains) were associated with Emory oak (Q. three uncharacterized species. Heterorhabditis sp. 1, repre-
emoryi Torr.) and single-leaf pinyon (Pinus monophylla sented by the Chiricahua isolates was found to be more
Torr.). Steinernema riobrave was found in association with closely related to Heterorhabditis bacteriophora Poinar,
mix vegetation of desert shrub live oak (Quercus turbinella 1976 and to Heterorhabditis sp. 2 (Santa Rita isolates).
Greene), velvet mesquite (Prosopis velutina Wooton), and Heterorhabditis sp. 3 was placed in a clade that includes
Fremont cottonwood (Populus fremontii Zapata). H. megidis and H. zealandica (Fig. 3).
Steinernema oregonense was recovered in Wet Canyon,
Mt. Graham (Pinaleño mountain range) along a riparian 4. Discussion
area. This area supports great biological diversity and is
characterized by a mixture of water birch (Betula occiden- Until now only a few EPN species have been isolated
talis Hook), Gambel oak (Q. gambelii Nutt.), red-osier dog- from desert or semi-desert habitats worldwide. For exam-
70 S.P. Stock, J.C. Gress / Journal of Invertebrate Pathology 92 (2006) 66–72

S. anatoliense
100
S. websteri

S. kraussei

S. oregonense
82 72
S. oregonense (PI-1 islate)

Steinernema sp.3 (CH isolates)

97 S.feltiae

83 Steinernema sp. (Costa Rica-Li isolate)

Steinernema sp.2 (SR isolates)

S. sangi

S. kushidai

100 S. cubanum

100 100 S. longicaudum

95 S. glaseri

63 S. arenarium

54 S. diaprepesi

95 S. puertoricense

100 S. hermaphroditum

S. scarabaei
60
Steinernema sp. (CFVII isolate)
100 98
100 S. karii

Steinernema sp. (T29 isolate)

Steinernema sp. (Costa RIca-CR-9 isolate)

100 S. bicornutum

S. ceratophorum

S. abbasi

S. riobrave

S. rarum

S. affine

S. intermedium

Steinernema sp. 1 (ML isolates)

S. monticolum

63 S. siamkayai
56 S. carpocapsae

S. scapterisci

Fig. 2. Strict consensus of 28s rDNA sequences tree showing evolutionary relationships of Steinernema spp. Tree length D 768, CI D 0.76. Numbers
indicate bootstrap values.

ple, Glazer et al. (1993) identiWed one steinernematid (S. from the Negev desert in Israel. Biological characterization
carpocapsae) and two heterorhabditid spp. (H. bacterio- of these isolates demonstrated these nematodes were highly
phora Poinar and H. megidis Poinar, Jackson, and Klein) tolerant to temperatures above 30 °C and that they were
 S.P. Stock, J.C. Gress / Journal of Invertebrate Pathology 92 (2006) 66–72 71

Heterorhabditis sp. 1(CH isolates) Interestingly, each of the surveyed mountain ranges held
100
a unique group of Steinernema spp. and/or Heterorhabditis
Heterorhabditis sp. 2 (SR-2 isolates)
83 spp., with no evidence of having a taxon from one Sky
H. bacteriophora Island being present on another mountain range (at least for
the ones considered in this study). The concept of insular
H. baujardi
100 evolution could explain the geographic distribution of Ari-
94 H. mexicana zonan EPN. BrieXy, it could be hypothesized that perhaps
H. indica
these new species were at a time sympatric populations rep-
100 resenting one or two EPN species, with a wide distribution
H. marelatus in an extensive oak woodland region. A speciation event
99
H. megidis
perhaps occurred when climate warming and drying frag-
100
mented and restricted oak woodlands, therefore conWning
99 H. zealandica and isolating EPN upland and to a particular Sky Island.
Heterorhabditis sp. 3 (SR-3 isolates) EPN populations recovered in this study represent new
germplasm that is now available for a wide range of studies.
Pellioiditis typica Moreover, these new species may oVer a more suitable
Fig. 3. Strict consensus of ITS-1 rDNA sequences tree showing phyloge- alternative for inundative release against a variety of native
netic relationships of Heterorhabditis spp. Tree length D 753, CI D 0.82. and/or introduced pests in southwestern USA. This aspect
Numbers indicate bootstrap values. is of particular interest in desert or semi-desert habitats
where hot and dry weather conditions coupled with inten-
very successful in killing a number of local insect pests sive agricultural systems and conventional pest control
(Glazer et al., 1993; Glazer and Goldenberg, 1993). Fur- practices, contribute to periodic and often unpredictable
thermore, the genetic basis of heat tolerance of the Israeli outburst in insect pest populations.
H. bacteriophora strain was studied based on cross-breed-
ing experiments and selection of molecular markers, sug- Acknowledgments
gesting potential for strain improvement (Segal and Glazer,
1998; Shapiro et al., 1997). This study was funded in part by a University of
More recently, three other EPN species, S. abbasi Elawad, Arizona Faculty Small Grant to S.P.S. and by the NSF-
Ahmad, and Reid, S. thermophilum Ganguly and Singh and IGERT Fellowship (Evolutionary, Functional and Compu-
H. taysearae Shamseldean, Abou-El-Sooud, Abd-Elgawad, tational Genomics Program) to J.C.G.
and Saleh, were recovered from semi-arid regions in the Sul-
tanate of Oman, India, Palestine, and Egypt, (Ganguly and
Singh, 2000; Sansour et al., 2003; Shamseldean et al., 1996). References
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