Journal of Invertebrate Pathology 92 (2006) 39–44

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Ecological characterization of Steinernema anatoliense (Rhabditida: Steinernematidae)

Didem Simsek Gungor a, Nevin Keskin a, Selcuk Hazir b,¤

a Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe, Ankara, Turkeyb Adnan Menderes University, Faculty of Arts and Science, Department of Biology, 09010 Aydin, Turkey

Received 14 November 2005; accepted 26 January 2006Available online 29 March 2006

Abstract

Our study describes the basic ecological characteristics of the entomopathogenic nematode Steinernema anatoliense including itsresponse to temperature, moisture, and host range. The eVect of temperature and soil moisture on the infection of Galleria mellonella lar-vae by S. anatoliense was determined. The temperature range for infectivity was greater than that for development. The optimal tempera-ture for infection and development was 25 °C. Although S. anatoliense infected the hosts at 10 °C, no reproduction occurred at thistemperature. This nematode species that was isolated from a cold region of Turkey exhibited warm-adapted temperature characteristics.Optimum water content of the soil for S. anatoliense to infect the host was 10%.© 2006 Elsevier Inc. All rights reserved.

Keywords: Entomopathogenic nematode; Steinernema anatoliense; Biological control; Steinernema sp.

1. Introduction

Entomopathogenic nematodes of the genus Steiner-nema are obligate lethal parasites of insects (Burnell andStock, 2000). These nematodes are mutualistically associ-ated with enteric bacteria in the genus Xenorhabdus. Theinfective juvenile (IJ), the resistant stage that survives out-side of a host without feeding, carries the bacterial symbi-ont in its bacterial pouch. When the IJ Wnds a susceptibleinsect host, it enters the insect through natural openings(anus, spiracles or mouth), penetrates into the hemocoel,and releases the symbiotic bacterium. The bacterial cellsmultiply in the hemocoel and kill the host within 48 h bysepticemia. The nematodes feed on the bacterial cells andhost tissues digested by the symbiotic bacterium (Kayaand Gaugler, 1993).

Entomopathogenic nematodes are eVective and safe bio-logical control agents of a number of soil insect pests (Gau-

* Corresponding author. Fax: +90 312 2992028.E-mail address: shazir@adu.edu.tr (S. Hazir).

0022-2011/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.jip.2006.01.008

gler and Kaya, 1990; Koppenhöfer, 2000). Accordingly,there is an intense interest to isolate these nematodes fromdiVerent regions of the world so that they can be used asbiological control agents of insect pests in the area orregion from which they were isolated (Hazir et al., 2003a).Such nematodes will be adapted climatically as well asadapted to the insect pest and will avoid the introduction ofexotic nematodes to the region. Therefore, many surveyshave been conducted in many parts of the world to Wndwell-adapted and more suitable strains and/or species forinundative biological control.

Steinernema anatoliense was isolated during a survey inTurkey (Hazir et al., 2003b) and described as a newentomopathogenic nematode species by Hazir et al.(2003c). However, the description of new species usuallycontains little information on their biology and ecology(Koppenhöfer and Kaya, 1999). Hence, our objectives wereto conduct an ecological characterization of S. anatoliense.We studied the eVects of temperature on infectivity, devel-opment, and reproduction, the eVects of soil moisture oninfectivity and the susceptibility of insects in Wve insect

40 D.S. Gungor et al. / Journal of Invertebrate Pathology 92 (2006) 39–44

orders to S. anatoliense. We also conducted a self-fertiliza-tion test with S. anatoliense.

2. Materials and methods

2.1. Nematode culture

Steinernema anatoliense was reared in the wax moth(Galleria mellonella) larvae (Kaya and Stock, 1997). Nema-tode-killed larvae were placed on White traps (White,1927), and the new generation of IJs emerging from cadav-ers were harvested. Collected IJs were rinsed three times insterile distilled water before being stored at 15 °C and usedwithin a week for the experiments.

2.2. Temperature experiments

Wells of a 24-well tissue culture plate were Wlled with0.5 g of sterilized, air-dried sandy loam soil to which 60�l ofnematode inoculum containing 50 S. anatoliense IJs wasadded to the surface of sand in each well. Two sets of 24-well culture plates were prepared and were placed in incu-bators at 5, 10, 15, 20, 25, 30, 33 or 35 °C. After 1 h acclima-tion, one last instar wax moth larva was added to thesurface of each well. One set of plates was used for theinfectivity study based on time of death and penetrationeYciency. The other set was used for the determination oftime to Wrst emergence of progeny from the cadavers andthe number of emerged IJs at various temperatures. To ver-ify the health of the wax moth larvae, two 24-well cultureplates containing sandy loam soil plus 60�l distilled waterin each well were included in the study as a control(Koppenhöfer and Kaya, 1999). The plates were placed intopolyethylene bags to prevent desiccation. The experimentwas repeated three times.

The following data were recorded: time of death of thewax moth, penetration eYciency of the IJs, the time of Wrst IJproduction and the total number of IJs produced. To deter-mine the time of death, each larva was probed daily, and ifthe larva did not respond to the probe, it was considereddead. To determine penetration eYciency, dead larvae wererinsed in distilled water to remove the external nematodes.Cadavers from 5, 8, 10, and 15°C were placed at room tem-perature (23–24 °C) for two additional days, whereas larvaerecovered at 20, 25, 30, and 33°C were held for 1 day beforedissection (Hazir et al., 2001). The cadavers were dissectedand digested in a pepsin solution (Mauleon et al., 1993) andthe number of nematodes that penetrated was counted at50£ magniWcation. To determine the time of emergence andnumber of emerged IJs, cadavers were placed individually onWhite traps. The cadavers per temperature and traps weremonitored daily. The Wrst day of IJs emerging from thecadaver was recorded. The emerged IJs were collected regu-larly until no more entered the water in the White trap. Thetotal number of emerged IJs was estimated by the countingof subsamples at 50£ magniWcation (Hazir et al., 2001). Theexperiment was repeated three times.

2.3. Host range

Seven insect species representing Wve orders were used todetermine their susceptibility to S. anatoliense. Third-instarhouse Xy, Musca domestica (Diptera: Muscidae), and work-ers of the PaciWc dampwood termite, Zootermopsis angusti-collis (Isoptera: Termopsidae), were tested at the Universityof California, Davis. All other insects were tested at Hacet-tepe University. These included last instar wax moth Galleriamellonella larvae (Lepidoptera: Pyralidae), Cydia pomonellalarvae (Lepidoptera: Tortricidae), adult German cock-roaches, Blatta germanica (Blattodea: Blatellidae), fourth-instar Tenebrio molitor (Coleoptera: Tenebrionidae), nutweevil, Balaninus nucum (Coleoptera: Curculionidae), andthe chestnut weevil, Curculio caryatrypes L. (Coleoptera:Curculionidae). Twenty-four well tissue culture plates with0.5 g sterile sand and 60�l of nematode inoculum containing50 S. anatoliense IJs added to each well were used followingthe protocol above. One insect was added per well.

One 24-well tissue culture plate with 0.5g sterile sand con-taining only 60�l distilled water to which an insect was addedwas included in the study as a control. The plates were placedinto polyethylene bags to prevent desiccation and kept atroom temperature (23–24°C). All bioassays were examineddaily for 10 days and mortality recorded. The cadavers weretransferred to White traps to observe if S. anatoliense couldsuccessfully reproduce in the hosts (Koppenhöfer and Kaya,1999). Host range experiments were repeated twice.

2.4. Life cycle

To determine the life cycle of S. anatoliense, nematode-killed Galleria larvae were dissected daily and the stage ofdeveloped nematodes was recorded. This study was con-ducted at room temperature (23–24 °C). Generally, steiner-nematids are amphimictic, but GriYn et al. (2001) found aself-fertile, steinernematid nematode in Indonesia. There-fore, to test whether S. anatoliense was self-fertile, only oneIJ was placed in each well of 24-well tissue culture platesand one Galleria larvae was added to each well. Nematode-killed larvae were placed individually on a White trap andmonitored daily to determine whether progeny emergedfrom the cadavers. As a control group, two IJs added toeach well of 24-well tissue culture plates and one G. mello-nella larvae was added to each well. When the experimentwas terminated, the cadavers were dissected and examinedto conWrm nematode infection and presence of progeny.This experiment was repeated twice.

2.5. Soil moisture

To determine the eVect of soil moisture on nematodeinfection, dry sand to which water was added at variousamounts was used. The water content of the sand was at 1,3, 5, 8, 10, 15, and 20% moisture (w/v). Twenty microlitersof nematode inoculum containing 100 IJs was placed on thebottom of 24-well tissue culture plates and covered with

D.S. Gungor et al. / Journal of Invertebrate Pathology 92 (2006) 39–44 41

one of the moisten sand. Then one last instar wax mothlarva was added to each well. To prevent desiccation, the lidof the plates was covered with ParaWlm (Susurluk et al.,2001). After 3 days at 25 °C, the cadavers were dissectedand digested in pepsin solution to determine the number ofnematodes that penetrated per larva (Mauleon et al., 1993).

2.6. Standing and jumping behavior

Agar (2%) in a petri dish (100£15 mm) was sprinkledwith 0.5 g of sand particles (<80 �m in diameter) onto thesurface. Water was removed from the nematode suspensionby vacuum Wltration and approximately 100 IJs were trans-ferred to the agar surface with a probe (Campbell andKaya, 2000). The behavior of the IJs was monitored using adissecting microscope at 50£ magniWcation.

2.7. Statistics

Data on time of death, the number of nematodes thatpenetrated, time of emergence and reproduction of IJs wereanalyzed using analysis of variance (ANOVA), and meansseparation was with Tukey’s test (SAS Institute, 1996).

3. Results

3.1. Temperature experiments

Temperature had a signiWcant eVect on the time of hostdeath by S. anatoliense (FD211; dfD4,17; P < 0.05)(Fig. 1). However, at 5 °C the time of death could not beaccurately determined because the Galleria larvae did notrespond to probing even though they appeared to be alive.Larval mortality occurred most rapidly at 30 and 33 °C (2days), whereas it occurred most slowly at 10 °C (17 days).The time of death was not signiWcantly diVerent at 20, 25,30, and 33 °C (P > 0.05). At 15 °C, the time of death was sig-niWcantly longer than one group including 20, 25, 30 and33 °C and signiWcantly shorter than 10 °C (FD211;dfD4,17; P < 0.05) (Fig. 1). No infection was observed at35 °C. The control larvae had very low mortality (<5%).

Infective juveniles of S. anatoliense penetrated in rela-tively high numbers (>34) into the wax moth larvae at

Fig. 1. Time to death of wax moth host held at diVerent temperatures. Thesame lowercase letter above the bars indicates no signiWcant diVerence(P > 0.05; Tukey’s test).

0

4

8

12

16

20

5 10 15 20 25 30 33 35

Temperature

Mea

n tim

e of

dea

th (

days

) a

b

c c c c

25 °C. There was no signiWcant diVerence in penetrationeYciency at 15, 20, 25 or 30 °C (P > 0.05). However, at 10and 33 °C, S. anatoliense showed signiWcantly less penetra-tion eYciency (<13) (FD17.46; dfD 4,16; P < 0.05) than theother temperatures (Fig. 2).

No IJs emerged at 5, 10, and 33 °C. S. anatolienseshowed the fastest emergence time at 25 °C (average 6 days)and the slowest at 15 °C (average 17 days) (Fig. 3). Therewas no signiWcant diVerence in the time of Wrst progenyemergence at 30 °C, which averaged 9 days and at 20 °C

Fig. 2. Number of penetrated nematodes into wax worm hosts at diVerenttemperatures. The same lowercase letter above the bars indicates no sig-niWcant diVerence (P > 0.05; Tukey’s test).

0

10

20

30

40

5 10 15 20 25 30 33 35

Temperature

Num

ber

of p

enet

rate

d ne

mat

odes

a

b b bb

a

Fig. 3. Time of Wrst progeny emergence from host cadaver at diVerent tem-peratures. The same lowercase letter above the bars indicates no signiW-cant diVerence (P > 0.05; Tukey’s test).

0

4

8

12

16

20

5 10 15 20 25 30 33 35

Temperature

Tim

e of

fir

st p

roge

ny (

days

) a

b

c

b

Fig. 4. Total number of emerged IJs per wax moth larva at diVerent tem-peratures. The same lowercase letter above the bars indicates no signiW-cant diVerence (P > 0.05; Tukey’s test).

0

20

40

60

80

100

120

140

160

180

5 10 15 20 25 30 33 35

Temperature (˚C)

Tot

al n

umbe

r of

em

erge

d IJ

s (x

1000

)

a

a

ab

b

42 D.S. Gungor et al. / Journal of Invertebrate Pathology 92 (2006) 39–44

which averaged 8 days (P > 0.05). However, signiWcantlyfaster emergence was observed at 25 °C compared with theother four temperatures. Additionally, signiWcant sloweremergence was observed at 15 °C compared with the othertemperatures (FD 75; dfD3,13; P < 0.05) (Fig. 3).

The highest number of emerged IJs was observed at25 °C (average 188,700), and the lowest number wasobserved at 30 °C (average 116,600). The number ofemerged IJs at 25 °C was signiWcantly higher than 15 and30 °C (FD4.34; dfD3,13; P < 0.05) (Fig. 4).

3.2. Host range

Steinernema anatoliense infected all insect species usedin the study under laboratory conditions (Table 1). Themost susceptible species were Galleria mellonella andCydia pomonella followed by T. molitor, Z. angusticollis,and M. domestica, whereas the least susceptible specieswas adult B. germanica. No mortality was observed in thecontrols.

3.3. Life cycle

Steinernema anatoliense completed its life cycle in 7 daysat room temperature (23–24 °C). No progeny was observedfrom the cadavers when they contained one IJ. After wait-ing for 18 days, none of the nematode-killed insects pro-duced progeny and when the cadavers were dissected onlyone adult nematode was found. Thirteen cadavers had asingle giant female whereas 10 cadavers had a single male.Accordingly, S. anatoliense is an amphimictic speciesbecause in the control group where two IJs were added,progeny was obtained from 8 of 24 cadavers (33%).

3.4. Soil moisture

Starting from 1% soil moisture, the penetrationeYciency of S. anatoliense increased until 10%, but athigher soil moisture levels, the nematode were not able toinfect the insect host. The highest number of nematodesthat penetrated was obtained at 10% soil moisture (Fig. 5).

3.5. Standing and jumping behavior

Infective juveniles of S. anatoliense did stand on theirtails and demonstrated body-waving and jumping behav-

ior. Standing IJs raised more than 95% of their body oV thesubstrate and maintained a straight posture. The IJ canstand on a sand particle by balancing on a bend in its tail.Then its head region made contact with its ventral surfaceand the IJ jumped out of the Weld of view. We observed thatapproximately seven nematodes jumped in 10 min althoughthe majority of them either stood or body waved on thesand particle.

4. Discussion

This study provides the ecological characteristics of S.anatoliense, which is important information for its use as abiological control agent. S. anatoliense was isolated fromgrassland in Kars region located in eastern Turkey (Haziret al., 2003b). This area has a harsh temperate climate withnearly 5 months of winter at negative temperatures. It snowsapproximately for 4 months. For example, during 1999 to2001, average temperature was ¡4.2 °C from November toMarch, 8.1 °C in April and May, 17.5 °C from June, toAugust and 10.6 °C in September and October (TurkishMeteorology and Statistics Department). Based on Kars’ cli-matic information, we expected that S. anatoliense would bea cold-adaptedspecies. However, the data of the infectivityand reproduction of S. anatoliense at diVerent temperaturesshowed that this new species didn’t infect the wax moth lar-vae below 10 °C and no new progeny emerged at 10 °C andwas very similar to S. carpocapsae (Grewal et al., 1994; Moly-neux et al., 1986). The infectivity range of S. anatoliense wasbetween 10 and 33 °C (optimum 15–25°C) and reproductionrange was between 15 and 30 °C (optimum 15–25 °C).Koppenhöfer et al. (2000) reported that S. monticolum

Fig. 5. Penetration eYciency of S. anatoliense at diVerent sand moisturelevels.

0

10

20

30

40

50

60

1 3 5 8 10 15 20

Moisture %

Num

ber

of n

emat

odes

that

pen

etra

ted

Table 1Infectivity of Steinernema anatoliense to various insect species at room temperature

Order Family Species Stage % Mortality

Coleoptera Tenebrionidae Tenebrio molitor L5 88Coleoptera Curculionidae Balaninus nucum Larva 35Coleoptera Curculionidae Curculio caryatrypes Larva 43Lepidoptera Pyralidae Galleria mellonella L4 100Diptera Muscidae Musca domestica L3 75Isoptera Termopsidae Zootermopsis angusticollis Worker 62Blattodea Blatellidae Blatta germanica Adult 26

D.S. Gungor et al. / Journal of Invertebrate Pathology 92 (2006) 39–44 43

infected wax moth larvae between 8 and 30 °C (optimum 12–25 °C) and produced progeny between 12 and 25 °C (opti-mum 15–25°C). With S. rarum, 100% infectivity of wax mothlarvae occurred from 15 to 33 °C and reproduction occurredfrom 15 to 30 °C (Koppenhöfer and Kaya, 1999).

Steinernema feltiae, which is a very common species inmany parts of the world, is known as a cold-adapted spe-cies that can infect their host from 8 to 28 °C and produceoV-spring from 8 to 25 °C (Grewal et al., 1994, 1996; Haziret al., 2001; Wright, 1992). S. kraussei is a cold-adaptedspecies and an isolate from Iceland infects Otiorhynchussulcatus larvae from 6 to 10 °C (Willmott et al., 2002). Onthe other hand, S. riobrave and S. glaseri are warm-adapted species with infectivity occurring from 10 to39 °C and 10 to 37 °C, respectively (Grewal et al., 1994). S.riobrave reproduced at a range from 20 to 35 °C and S.glaseri from 12 to 32 °C (Grewal et al., 1994). The lowesttemperatures for the nematode reproduction are 8 °C forS. feltiae and 10 °C for S. aYne, whereas the lowest tem-perature is 15 °C for S. anatoliense. Some Steinernemaspecies such as S. feltiae (Grewal et al., 1994, 1996; Haziret al., 2001) and S. monticolum (Koppenhöfer et al., 2000)did not reproduce at 30 °C, but S. anatoliense is similar toother warm-adapted Steinernema spp. that produced oV-spring at this temperature.

Many reports suggested that the original climaticlocality of the nematodes correlate with their tempera-ture range (Kung et al., 1991; Molyneux, 1985). ButMenti et al. (2000) mentioned that this is not always thecase because when they compared two S. feltiae and twoHeterorhabditis megidis isolates recovered from Greeceand UK, they didn’t Wnd obvious diVerence between theisolates. In addition, the cold-adapted S. feltiae has beenisolated from tropical Hawaii, and it still retains its cold-adapted traits (Hazir et al., 2001). GriYn (1993) reportedthat Heterorhabditis spp. isolated from the north coast ofScotland performed worse than isolates of the same inter-breeding group from the south coast of Scotland at lowtemperatures. Dunphy and Webster (1986) speculatedthat long period cultural practices may have reduced thegenetic variability of nematode stocks and/or their sym-biotic bacteria and changed their ability to permanentlyadapt to diVerent temperatures. S. anatoliense had beencultured in the laboratory condition for 2 years beforeinitiating this study. This may be the reason for ourresults; however, other species have retained their tem-perature tolerances even after being reared in the labora-tory for many generations. A new isolate of S. anatoliensefrom Kars may conWrm whether it is a warm-adaptedspecies or not.

Our laboratory data have provided some basic ideaabout the life cycle of S. anatoliense in its natural area.Because of S. anatoliense ability to jump, we speculate thatit has a cruising or intermediate foraging strategy (Camp-bell and Kaya, 2002; Campbell et al., 2003) and that itprobably infects its hosts at or near the soil surface.Because the average soil temperature is above 20 °C at 5, 10,

and 20 cm depth during the summer months, this is the besttime for infection and development of S. anatoliense. Thisnew species may continue to infect and reproduce activelyin the fall, but during the harsh winters, the IJs of S. anatol-iense may migrate deeper in the soil to around 100 cmwhere the temperature averages 5.8 °C. Molyneux et al.(1986) suggested that at low temperatures, some nematodespecies remained in the host’s hemocoel as IJs and theycould remain without killing the host until temperaturesrise allowing bacterial growth and nematode reproduction.S. anatoliense probably uses both strategies to survive thewinters in the Kars region.

Steinernema anatoliense showed a wide host range inthe laboratory and can reproduce in the tested insectsexcept for B. germanica and C. caryatrypes. The mortal-ity caused by S. anatoliense to T. molitor, M. domestica,Z. angusticollis, and B. germanica suggests that it is morevirulent than S. rarum, S. monticolum and S. scarabaei at50 IJs/insect. For example, S. anatoliense showed 88, 75,62, and 26% mortality against these insect hosts, respec-tively, whereas S. monticolum showed 65, 62, 19, and 6%,S. rarum showed 84, 13, 35, and 18% and S. scarabeishowed 20, 17, 20, and 0%, respectively (Koppenhöferand Fuzy, 2003; Koppenhöfer and Kaya, 1999;Koppenhöfer et al., 2000).

Soil moisture is the another important factor for sur-vival and infectivity of entomopathogenic nematodes.Nematodes need a thin water Wlm on the surface of soilparticles for movement and infectivity (Grant and Villani,2003). Susurluk et al. (2001) showed that the most suitablewater content of the soil for H. bacteriophora and S. fel-tiae to invade the host was 10%, but when water contentwas 20%, the number of invading IJs decreased sharply.SigniWcantly, more IJs of S. anatoliense invaded into waxmoth larvae at 10% water content. The number of estab-lished IJs declined above and below of 10%, and no infec-tivity was observed at 20%. The size of S. anatoliense IJ issimilar to S. carpocapsae (Poinar, 1990) and S. rarum(Doucet, 1986). Koppenhöfer et al. (1995) speculated thatthe eVect of soil moisture on nematode infectivity was cor-related with nematode size. That is, the smaller nematodespecies has diYculty in moving in soil pores with increas-ing presence of water.

In summary, our data show that S. anatoliense is awarm-adapted species that was isolated from a very tem-perate region of Turkey. This would suggest that this spe-cies can survive well in this region. It may also suggest thatthis nematode may have originally come from a warmerregion of Turkey.

Acknowledgments

We thank Dr. Harry Kaya, University of California-Davis, for providing laboratory space and insects to S.H.and editing the manuscript, Dr. Oner Kocak for providinginsect hosts in Turkey. We acknowledge Wnancial supportfor the study from Hacettepe University.

44 D.S. Gungor et al. / Journal of Invertebrate Pathology 92 (2006) 39–44

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