Pergamon J. hvm. Bid. Vol. 22, No. 2. pp. 143-149, 1997

0 1997 Elwier Science Ltd. All rights reserved Printed in Great Britain

PII: SO306-4565(97)00005-3 0306-4565/97 $17.00 + 0.00

POLYMORPHISM IN HEAT SHOCK PROTEIN GENE (hsp70) IN ENTOMOPATHOGENIC NEMATODES (RHABDITIDA)

GHAZALA HASHMI*, SARWAR HASHMI, SEN SELVAN, PARWINDAR GREWAL and RANDY GAUGLER

Department of Entomology, Rutgers University, New Brunswick, NJ 08903, U.S.A.

(Received 24 November 1996; accepted IO February 1997)

Abstract-l. All organisms tested respond to a sudden increase of temperature by synthesizing heat-shock proteins, which helps organisms to survive high temperature. A correlation with increased thermotolerance and production of majot: 70 kDa protein has been observed in many organisms.

2. Many studies have been designed on a large number of animal species to assess their adaptation to different thermal environments. Genetic analysis of the hsp70 gene in entomopathogenic nematodes inhabiting different environments may provide insight into the physiological roles of hsps in these nematodes and could be useful for ecological studies.

3. To assess variation among species of entomopathogenic nematodes for thermotolerance, we initiated a search for the molecular organization of heat-inducible hsp70 genes in these nematodes. Five Heterorhabditis species/isolates with different temperature optima for survival and one warm-adapted species of Steinernema were tested

4. PCR and RFLP analyses of hsp70 in Heterorhabditis species and S. scapterisci demonstrated a putative homology with the Caenorhabditis eregans hsp70 A gene, thus indicating evolutionary conserved nature among different nematode species.

5. RFLPs with the hsp70 A gene probe revealed different banding patterns for Heterorhabditis species and isolates.

6. This is the first report on the identification of any hsp70 gene in entomopathogenic nematodes. 7. Our observation establishes a unique correlation between geographical distribution and

polymorphisms for hsp70 A gene in these nematodes. 0 1997 Elsevier Science Ltd

Key Word 1nde.x: Entomopathogenic nematode, heat shock protein gene, /up70 A polymorphisms

INTRODUCITON

During heat shock, organisms express a set of proteins, the hsps, which helps the organism survive at temperatures slightly above their normal growth temperature (Ashbumer and Bonner, 1979; Schlessinger et al., 1982; Craig, 1985; Lindquist, 1986; Lindquist and Craig, 1988). Although the functional significance of heat shock response is poorly understood, there are indications that it leads to thermotolerance (Ritossa, 1962; Lindquist, 1986). The mechanism of thermotolerance is based on the synthesis of heat shock proteins (especially, 70-kD family) encoded by the heat shock genes. Tran- scription of heat-shock cognate (hsc) proteins

‘To whom correspondence should be addressed. E-mail: Hashmi@rci.rutgers.edu Tel: (908) 932-9459 Fax: (908) 932-7229.

(hsc70) in several organisms to occur at normal temperatures, and is not increased by heat shock (Ingolia and Craig, 1982; Ingolia et al., 1982). The presence of cognates may be responsible for tolerance of high temperatures in organisms native to warmer areas. In a recent review, Colman et al. (1995) argue that the variations in hsps reported in several organisms of diverse environmental extremes can be correlated, and may be involved in ecological variation in thermotolerance. For example, the mechanism suggested for thermal adaptation of desert ants, Cataglyphis bombycina and C. bicolor

is that these animals synthesize heat shock proteins for protection before even leaving their nests (Gehring and Wehner, 1995). Analysis of six closely related species of desert fishes of the genus Poeciliopsis inhabiting diverse river systems indi- cated a high level of diversity in heat shock protein isoforms (White et al., 1994).

The various hsp70 related genes are organized into

143

144 G. Hashmi PI (I/

a hsp70 multigene family, that has been identified

in many organisms including D. meianogaster

(Lindquist and Craig, 1988), human (Mues e/ ul.,

1986) Sacchuronr_rces cerrisiue (Craig, 1989) and

C. elegans (Snutch et al., 1988; Heschl and Baillie,

1989a. HeschI and Baillie, 1989b). Snutch e! a/. (1988)

have isolated six distinct members of the C. elegans

70-kDa heat-shock protein gene family (hsp70

family). Each member exists as a single copy element

in the C. elegans genome. One member, the Irsp70

A gene of C. eleguns, is highly heat inducible and

is homologous to Drosophilu and yeast heat-shock

inducible genes (Snutch et al., 1988).

The soil-inhabiting entomopathogenic nematodes.

Heterorhabditis and Steinernema, have great poten-

tial for the biological control on many important

insect pests (Gaugler and Kaya, 1990; Kaya and

Gaugler, 1993). An outstanding characteristic of

these two groups of nematode is the ability to carry

and release Photorhabdus or Xenorhabdus bacteria in

the body cavity of insects, thus killing the host and

providing nourishment for the developing nematode

in the host (Akhurst and Boemare, 1990). The only

free-living stage is an infective juvenile. Although

variation exists among species of entomopathogenic

nematodes for temperature tolerance (Griffin and

Downes, 1991; Grewal ef ul.. 1993). the persistence of

a free-living stage in soil, their infectivity (Blackshaw

and Newell, 1987; Griffin and Downes, 1991)

development, maturation. and reproduction are

influenced by temperature (Dunphy and Webster,

1986; Grewal et al., 1993). The phenomenon of

thermotolerance is studied in one species Heterorhub-

ditis bacteriophora and correlated with the expression

of 70 kDa protein (Selvan et al., 1996).

Entomopathogenic nematodes isolated from di-

verse geographical regions and climates provide

an opportunity to compare adaptations of mor-

phologically similar animals to a wide variety of

temperature regimes. Adaptation of these nematodes

to various temperatures may be correlated to the

increased diversity of hsp70 genes. We studied the

polymorphism for heat inducible hsp70 A gene in

five closely related species of entomopathogenic

nematodes as an initial step in the analysis of

variation among species of these nematodes for

temperature tolerance. Because genetic analysis of

functional genes does offer the advantage to

determine the relative levels of gene expression. We

used polymerase chain reaction (PCR) for ampli-

fication and restriction fragment length polymor-

phism (RFLP) to determine diversity of hsp70 A

gene in geographically separated isolates of Het- erorhnbditis species and a warm adapted species

Steinernema scapterisci.

MATERIALS AND METHODS

Culture and isolation qf’ nematodes

Nematode species and isolates used in this study

and their geographical origin are listed in Table I.

Heterorhabditis and Steinernema species were main-

tained on lipid agar media seeded with their

symbiotic bacteria (Photorhabdus luminiscens for H.

spp. and Xenorhabdus nematophilus for S. scapter-

ix?). All stages of nematodes were collected by

centrifugation at 3000 g for 10 min in EN buffer

( 100 mM NaCI, IO mM EDTA) (Sulston and

Hodgkin, 1988) followed by three washes with

distilled sterile water. Finally, the nematodes were

collected in I .5 ml eppendorf tubes and stored frozen

at ~ 20‘C until used for DNA extraction.

DNA extruction

Genomic DNA was extracted from nematodes

according to the method of Sulston and Hodgkin

(1988). The nematode pellet was ground in liquid

nitrogen and treated with proteinase K buffer

containing 100 mM NaCI, 100 mM Tris HCl at

pH 8.5, 50 mM EDTA at pH 7.4, I% SDS, I %

fl mercaptoethanol. 100 pg/ml proteinase K for

one hour at 65°C. The DNA was extracted with

equal volumes of phenol, phenol-chloroform, and

choloroform-isoamyl alcohol (24: I) and was pre-

cipitated in 100% ethanol overnight at - 2o’C.

DNA pellet was then dissolved in IX TE buffer

(IO mM Tris-HCI pH 8.0, I mM EDTA at

pH 8.0).

Southern unalysis

Genomic DNA of five species/isolates of Het- erorhabditis and S. scapterisci (Table I), was digested

with five restriction enzymes: EcoRl, Barn HI, Dra

I, Sal 1 and HindIII. Each lane was loaded with equal

amount of DNA (Spg/lane) and samples were

electrophoresed on a 0.8% agarose gel for 1622 h at

20 V. Phage 1 DNA digested with Hind111 was

included as a molecular weight marker. DNA

fragments were transferred (Southern, 1975) onto

Hybond N + membrane (Amersham, Arlington

Table I. List of nematode species with their isolate

designation and geographic origin. used in this study

Biological species Isolate Geographic

designation origin

Hetrrorhabditis bacreriophora H. species H. species H. indicus

HP88 U.S.A.

El Spain Moldavia Moldovia EMS-13 Egypt

H. ntegidis S. seaprerisci

HOI

Colon U.S.A.

Argentina

hsp70 gene in insect nematodes 145

Heights IL.). Blots were treated with hybridization buffer (Snutch and Baillie, 1984), and membrane was

hybridized with hsp70 A gene from C. elegnns

(provided by D. L. Baillie). To prepare the probes, the recombinant plasmids were purified and digested to release the inserts, which were then isolated by electroelution from agarose gels (Maniatis et al., 1982). Plasmids were cut with Eco Rl /BamH 1 and an internal 1.5 kb fragment was excised from agarose gel. DNA probes were made by random primer labeling (Feinberg and Vogelstein, 1983) with

[a - “PI using a commercially available kit Prime-a- gene (Promega Corporation, Madison WI). Blots were hybridized for 12-16 h in hybridization buffer

containing approximately 5 ng labeled DNA/ml. Blots were washed at room temperature twice for 15 min each with ZXSSC/O.l% SDS and then with 1 XSSC/O. 1% SD for 30 min. Hybridized DNA was

detected by exposing blots to Kodak XAR-5 film for 48-72 h at - 70°C.

PCR analysis

The reaction mixture (100 pl) contained 10 mM Tris HCl at pH 8.3, 50 mM KCI 1.5 mM Mg Cl*, .OOl% (w/v) gelatin, 200 pM each of dNTP, 0.5 pM primer, 50-100 ng of DNA and 0.5 units of Taq DNA polymerase (Perkin Elmer Cetus) overlaid with mineral oil (United States Technologies, Inc., Alameda, CA). Degenerate primers to amplify about 2.7 kb fragment of hsp70 gene of C. elegans were synthesized (Bio-Synthesis, Inc., Lewisvelle, TX) by the sequence available in Gene Bank, and used in each PCR reaction. The sequence of these primers was as follows: GCGAACATTCTCTGCGAGG and GCGTAATAACGGTTTGGGTGG. Amplification

conditions were as described previously (Hashmi et al., 1996). The amplification products were electrophoresed on a 0.8% agarose gel at lV/cm for 1622 h and visualized with ethidium bromide staining. These gels were blotted onto nylon membrane (Southern, 1975) and hybridized with the hsp70 A gene from C. elegans as described above except that the 6.6 kb EcoRl fragment containing hsp70 A gene, released from the plasmid was used as probe.

Heating protocol. A heat exposure protocol (Gaugler and Hashmi, 1996) was used to determine the thermotolerance in two species H. bacteriophoru

and H. indicus. We selected these two nematode species because the upper thermal limit and temperature optima is lower for former (Grewal et al., 1994) while the later is a warm temperature adapted nematode. In our heating protocol, the infective juveniles (500) of each species were subjected

to three different heat treatments as follows: 1) two hours of exposure to 35°C acclimatized to 25°C overnight; 2) two hours at 35°C acclimatized for one

hour at 25°C and then incubated for one hour at 38°C followed by 25°C overnight and 3) two hours at 35°C acclimatized for one hour at 25°C and then incubated for one hour at 40°C followed by 25°C overnight. Nematode survived the treatment were counted. There were 10 replicates in each treatment and experiments were repeated four times. Data were analyzed using student’s t-test, and presented as mean + standard error.

RESULTS

We searched for DNA polymorphism for hsp70 A gene among five closely-related species of nematodes by comparing southern blot hybridization pattern of restriction enzyme digested nematode’s genomic DNAs. Genomic DNA hybridized to C. elegans hsp70 A probe under moderate stringency conditions. All restriction enzymes used in this study yielded

RFLP patterns showing polymorphisms. In some species, hybridization to DNA fragments of discrete

sizes gave a discrete banding pattern. In other species there was no distinct banding pattern, suggesting that a different number of copies are dispersed throughout the genome of different species/isolates.

Detailed qualitative comparisons of the hybrid-

ization pattern of the five Heterorhabditis species/ isolates and S. scapterisci with the C. elegans hsp70

A gene were made, in order to assess the RFLPs among species of nematodes. Most isolates showed RFLP, which varied from fragment to fragment. Figure 1 compares the representative banding patterns of EcoRl and Hi&II digested genomic DNA following hybridization with radiolabelled 1.5 kb fragment of the hsp70 A C. elegans gene. EcoRl digest of H. bacteriophora HP88 produced one faint band of about 2 kb (data not shown), while H. megidis did not produce a hybridization signal. Isolate El produced a strong fragment of about 9.0 kb with EcoRl (lane A) and a 6.6 kb with Hind111 (lane B). It is evident that none of the EcoRl fragments are the same size between Heterorhabditis

isolates. S. scapterisci hybridized with C. eleguns hsp70 A probe with about 10.0 kb fragment of EcoRl and HindIII (lane C and D). Egyptian strain (EMS-13) of H. indicus hybridized to three frag- ments of 3.0, 10 and 1.5 kb when digested with EcoRl and a single 9.0 kb fragment with Hind111

(lane E and F). Two fragments of about 9.5 kb were hybridized with isolate Moldovia (G and H).

The isolate HP88 of H. bacteriophora is susceptible to high temperature. Genomic DNA of this isolate

146 G. Hashmi et al.

ABCDEFGH Kb

_ 9.4

- 6.5

- 43

Fig. 1. Southern blot of Hc/rrorhabdiri.s species. DNA was isolated from nematodes. digested with EcoR I (I) and Hind111 (2). blotted and probed with 1.5 kb EcoRI/BamIII fragment of hsp70A. Lane A = El

digested with I. B = El digested with 2, C = Strinemma scapferisci digested with I. D = S.scopterisci digested with 2, E = H. indicus digested with 1. F = H.itzdicus digested with 2, G = Moldovia digested

with I. H = Moldavia digested with 2.

produced a faint band with only one restriction

enzyme. To determine the true nature of the ksp70 A

gene in this isolate, PCR analysis was performed with

the two primers specifically designed for C. rlegans

hsp70 gene. PCR analysis of DNA of H. bacterio-

phora HP88 showed amplification of two fragments

(1.0 and 2.3 kb) (Fig. 2). A 2.7 kb fragment was

amplified when hsp70 gene of C. elegans was used as

template (Fig. 2. lane A). In southern hybridization,

the 2.3 kb fragment of H. bacteriophora hybridized

with the C. elegans hsp70 A probe. While the minor

fragments did not show any hybridization.

Two species H. bacteriophora and H. illdicus

ABC DM

used in this study showed great variability in

hybridization patterns with hsp70 gene. To gain

information whether this variation would reflect in

their thermotolerance a heat exposure protocol was

designed. Although both species survived at 35’C

(Fig. 3), only 15% H. bacteriophra survived at

one hour 38°C heat treatment. All H. indicus sur-

vived at one hour 38’C indicating that this nematode

was more tolerant to high temperature than

H. bacteriophora. The survival of both species was

less than 5% at 40’ C.

DISCUSSION

Heat shock of H. bacteriophora induces the

synthesis of a 70 kDa protein (Selvan et al., 1996).

The genomic DNA of Heterorhabditis species/strains

and S. scapterisci which hybridizes to the C. elegans

hsp70 A gene probe corresponds to the nematodes

hsp70 gene. However, the hsp70 gene of entomo-

pathogenic nematodes is polymorphic among species

and seems to be a putative homologue of hsp70 A

gene of C. elegans. Further sequence analysis will

reveal the true nature of this homology.

We compared the lrsp70 A gene in five closely

related species and isolates of Heterorhubditis, S.

scapterisci, and C. elegans using southern blot

hybridization. We found that each nematode species

has a distinctive DNA banding pattern for the hsp70

A gene. However, polymorphism exists for the hsp70 Fig. 2. PCR amplification of Heterorhabditis bacteriophora HP88 genomic DNA and Caenorhabditis elegans hsp70

A gene among geographically separated isolates of

gene, with hsp70 gene specific primers. A = C. elegans Heterorhabditis species. Differences in the expression

hsp70 gene, B-D = different samples of H. bacteriophora of heat shock proteins have been reported in closely

HPSS. Molecular weight markers are Hind111 digested i. related species which occupy different environmental

DNA. niches. White et al. (1994) analyzed the heat shock

HSp70 gene in insect nematodes 147

Fig. 3. Survival of H. bacteriophora and H. indicus after heat treatment. Third stage infective juveniles

of both species were treated with 35, 38 and 40°C following a heat treatment protocol (see materials and

methods).

proteins of six closely related species of desert fishes, Poeciliopsis spp., collected from four river systems and demonstrated the existence of biochemical diversity in hsp proteins among species. The five nematode species analyzed herein were isolated from different geographical origin (Table 1) where average daily temperature varies from 25 to 40°C. Optimum temperature for infection, establishment and repro- duction of H. bacteriophora. H. megidis and S. scupterisci indicate well defined thermal niches (Grewal et al., 1994). Nematode adaptation to a particular temperature environment may be a contributing factor for the existence of polymorphism in hsp70 gene within closely related species. Whether these differences are correlated with the differences in hsp70 gene product in these nematodes remains to be determined.

Although variation exists among Heterorhabditis

spp. and isolates they all show homology with the heat-inducible hsp70 A gene of C. elegans. Variation among entomopathogenic nematode species for temperature tolerance has been reported (Molyneux, 1986; Griffin and Downes, 1991; Kung et al., 1991; Grewal et al., 1994). Our data on the variation of heat tolerance between H. bacteriophora and H. indicus,

correlates with the variation of hsp70 gene in these nematodes. The functional nature of the heat shock gene in entomopathogenic nematodes needs to be determined before any correlation between the gene expression and thermotolerance in these organisms can be made. The present study however, suggests that polymorphic forms of the hsp70 gene may be responsible in part for the differences in temperature optima of these nematodes.

Members of the hsp70 family are the most highly conserved gene known (Lindquist, 1986; Lindquist and Craig, 1988) and the possibility of variation in

hsp70 gene product among closely related species has been discussed (White et al., 1994). The existence of high levels of polymorphism of hsp70 gene in Hetcrorhabditis spp. either reflects mutational roots within this region or reflects divergence due to effect of varying degrees of stress on species/strains caused by different environmental conditions of their habitat. Hashmi et al. (1996) reported a high level of DNA polymorphisms among Heterorhabditis and Steinernema species isolated from various parts of the world identified with RAPD markers. The hsp70 polymorphisms is seen with at least four restriction enzymes may be due to a DNA rearrangement rather than to a single base pair change. The hsp70 gene is known to be inducible in all cells which are transcriptionally competent (Dura, 1981; Bensaude et al., 1983) and in C. elegans, the region of this gene accumulates mutations at a rate of 10 to 20 fold higher than other regions of the genome (Snutch and Baillie, 1984). Thus, it is reasonable to assume that the Heterorhabditis hsp70 A gene region also contains mutational hot spot. Independent mutations of the

same genes in different species provide multiple mutant alleles that can be used to seek correlation between a specific phenotype (heat tolerance) and genotype. We are now focusing on the isolation of Heterorhabditis hsp70 A gene from a genomic library using C. elegans hsp70 A probe. Sequence analysis of the gene should provide information into the conservation of sequences responsible for the regulation of this gene.

In conclusion, the variability observed in hsp70 A gene among different geographical isolates of entomopathogenic nematodes signals important physiological differences. Our data also provide a foundation to study thermotolerance and heat shock proteins in entomopathogenic nematodes.

148 G. Hash] mi er al.

Acknowledgemmrs-We thank Dr David Baillie for

providing hsp70 A probe, Dr M. Shamseldean for providing

Egyptian strain of H. brdirus. We also thank Dr

Tom Leustek for reviewing the manuscript and Ana

Monroy and Dan Collins for technical assistance. This

publication is a New Jersey Agricultural Experiment Station

Publication #D-08255- 16-95. supported by state funds and

by U. S. Hatch Act, and U. S. Department of Agriculture

National Agricultural Research Project (No.58-319R-3-

001).

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