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42Russian Journal of Nematology, 2010, 18 (1), 9 - 18
Ecological characterisation of Steinernema
australe (Panagrolaimomorpha:
Steinernematidae) an entomopathogenic
nematode from Chile
1 2 Steve Edgington and Simon R. Gowen
1 CABI, Bakeham Lane, Egham, Surrey TW20 9TY, UK [email protected]
2 University of Reading, School of Agriculture, Policy and Development, Whiteknights, Reading, RG6 6AH, UK
Accepted for publication 16 November 2010
Summary. This paper reports on studies of a recently described species of entomopathogenic nematode, Steinernema australe, from Chile. Under laboratory conditions S. australe had a fast life-cycle, with new infective juveniles (IJ) observed after 6 d at 20°C; however, adults and non-infective juveniles also emerged from cadavers and migrated away. The thermal and moisture profiles for infectivity were wide and significant infection occurred at cool, humid conditions, 7°C and 15.8% moisture content (MC). Five days at ca -1°C had no effect on IJ survival, but subsequent host infection was substantially reduced, a possible chilling-injury or phase-switching of the symbiotic bacteria. Steinernema australe infected a wide range of insect pests from Chile, including mobile and sedentary hosts, appearing most effective against Lepidoptera. No nictating or jumping behaviour of IJ was observed. Key words: ecology, freeze, host, moisture, Steinernema australe, temperature.
Manipulation of entomopathogenic nematodes (EPN) in managed systems through inundation, inoculation and conservation can provide effective and environmentally benign methods for controlling insect pests (Koppenhofer & Kaya, 1999; Grewal et al., 2005; Stuart et al., 2006). Key to the success of EPN as a control strategy is an understanding, based on both laboratory and field observations, of the biology, ecology and population dynamics of the EPN. The breadth of type-localities that 65+ valid EPN species, including recent collections in Tibet (Mrâcek et al., 2009) the Sonoran Desert (Mexico) (Stock et al., 2009) and Colombia (Lopez-Nunez et al., 2008), gives reason to suggest a rich biological diversity amongst these organisms, with characteristics of value for controlling insects. Koppenhofer & Kaya (1999) suggest a protocol for ecological characterisation to complement a new EPN species description, providing basic information on host range, foraging strategy and abiotic profiles.
This paper describes a number of studies on the biology and ecology of Steinernema australe Edgington, Buddie, Tymo, Hunt, Nguyen, France, Merino, & Moore, 2009, a recently described species of nematode discovered during surveys in
Chile, and follows basic bionomic observations carried out during the original description (Edgington et al, 2009). Steinernema australe was discovered on Isla Magdalena, an island in the south of Chile, approximately 2 km from the mainland (44° 35' 48.8" S, 72° 57' 35.7" W). Precipitation on the island is high (approximately 4000 mm per annum) with an average annual temperature of around 7°C. Steinernema australe was discovered within 200 m of a beach, below dense undergrowth, in a loamy-sand soil, shaded by trees.
We measured virulence of S. australe in the laboratory to a number of insect species of agricultural importance in Chile, the effect of temperature and moisture on infectivity and development, including exposure to temperatures below 0°C and behavioural aspects such as the ability to attach to an actively mobile host (i.e., the ability to ambush a host).
MATERIAL AND METHODS
Nematode culture. The nematodes used in the study were cultured in late instar waxmoth (Galleria mellonella L.) larvae (obtained from Live Foods Direct, Sheffield, UK). Emergent infective juveniles (IJ) were collected in modified White traps (White,
1927) and stored at 8 ± 2°C (a recognised storage temperature for temperate steinernematids) in tap water, prior to the trials. Only IJ collected within one week of first emergence were used in the trials and IJ in storage for > 20 d were discarded. All experiments were done in the laboratory.
Life cycle. Fifty late instar waxmoth larvae, in a Petri dish (15 cm diameter) lined with moistened filter paper, were exposed at a concentration of 50 IJ/ larva (in 5 ml sterilised tap water) at 20 ± 2°C. There were five Petri dishes in total, i.e., 250 waxmoths. At 0, 1, 4, 12, 24 then every 24 h up to 240 h following inoculation, 10 waxmoth larvae were washed in tap water and dissected individually in 0.5% saline solution (NaCl). Penetration rates, sex ratios and in vivo EPN development were recorded. Fifty larvae were also checked for mortality every 24 h and cadavers transferred to modified White traps to monitor nematode emergence.
To test for self-fertility, one late instar waxmoth larva was placed into a chamber (1.5 cm3) lined with moistened filter paper. One IJ in 10 ^l sterilised tap water was topically applied to the dorsal region of each larva. Control waxmoths received 10 ^l sterilised tap water without IJ. The larvae were maintained at 20 ± 2°C and monitored every 24 h for mortality. Cadavers were transferred to modified White traps to check for nematode emergence. There were 50 waxmoths for each treatment and the trial was carried out twice.
Temperature profile. Each chamber (1.5 cm3) of a 25-chamber bioassay plate was partially filled with 0.5 g sterilised, air-dried sand (medium sized particles, mesh designation -30+50). The test temperatures were 7, 11, 16, 20, 23, 28 and 33°C, with IJ and waxmoth larvae equilibrated for 1 h at these temperatures prior to testing. Twenty five IJ in 50 ^l sterilised tap water were transferred into each chamber, followed by one waxmoth larva. Control chambers received 50 ^l sterilised tap water without IJ. Plates were placed in plastic bags to reduce desiccation and then maintained at one of the test temperatures. Larval mortality and time to first IJ emergence were recorded every 24 h for 20 d, then every 48 h thereafter for another 20 d. The trial was done twice.
Freeze tolerance. Plastic tubes (1.5 ml volume), containing 100 IJ in 1 ml sterilised tap water, were submerged in a water bath at -1 ± 1°C. The water bath contained approximately 30% antifreeze. Control treatment consisted of IJ in sterilised tap water maintained at 8 ± 2°C. Five tubes containing IJ were taken out every 24 h for 120 h (destructive sampling), the IJ left to recover in distilled water for
4 h at room temperature and then the number of live and dead IJ counted. Nematodes that did not respond to gentle probing with a needle were counted as dead. Twenty live IJ were then picked out from pre-selected locations on a counting chamber and applied in 50 ^l sterilised tap water to a chamber (1.5 cm3), lined with filter paper, containing a single waxmoth larva. The chambers were covered and kept at 20 ± 2°C. Mortality of waxmoth larvae was assessed after 144 h. The trial was set up as a randomised block design, with five blocks of five tubes, and was done three times.
Effect of soil moisture. Approximately 50 IJ in 50 ^l sterilised tap water were placed at the bottom of a plastic tube (8 cm height, 1.5 cm diameter, 28 ml volume), which was then part-filled with pre-moistened sand (mesh designation -30+50) to a height of 4 cm, the column being gently compacted by tapping the tube on the bench. The moisture contents (MC) tested were (w/w) 0.2, 4.6, 8.3, 13.4 and 15.8% (saturation point of the sand was approximately 20.0% MC). Moisture contents were assessed using a HG53 Mettler Toledo Moisture Analyser. A disc of wire mesh was placed on the top surface of the sand onto which one late instar waxmoth larva was placed. The tubes were sealed and maintained at 20 ± 2°C for 72 h. Control treatment consisted of larvae maintained on top of the moistened sand column but without the addition of IJ. Mortality of waxmoth larvae was assessed after 72 h, with cadavers dissected in 0.5% NaCl to count the number of IJ that had penetrated. The MC of the sand was checked at 0 and 72 h, including measurements at the top and bottom of the column at 72 h. There were five tubes per treatment at each moisture level. The trial was done three times.
Ambush capacity. A Petri dish (9 cm diameter) was lined with filter paper with a thin layer of sand (10.0% MC w/w) on top. Approximately 500 IJ in 200 ^l sterilised tap water were added to the dish and left at room temperature for 30 min. One late instar waxmoth larva was introduced into each dish and kept active for 20 min by gentle prodding with a pipette tip. After 20 min the larva was washed in 1 ml tap water and a count made of IJ in the wash. There were five waxmoth larvae per trial, i.e., five Petri dishes, and the trial was done three times.
Approximately 100 IJ on a circle of filter paper (0.5 cm diameter) were placed into the middle of a Petri dish (9 cm diameter) containing 2% tap water agar. The dish was left at room temperature for 30 min, following which IJ nictation and jumping was observed, for approximately 1 h, using a stereo microscope.
Laboratory host range. Fourteen insect species, either immature or adult stages, and representing five orders, were used to assess the laboratory virulence of S. australe. All insects were obtained within Chile and tested at the Instituto de Investigaciones Agropecuarias (INIA), Chillán, Chile (Region VII). Adult citrophilus mealybug Pseudococcus calceolariae (Maskell) (Hemiptera: Pseudococcidae), leaf-footed bug Leptoglossus chilensis (Spinola) (Hemiptera: Coreidae), and late instar larvae of G. mellonella (Lepidoptera: Pyralidae), codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae) and tussock moth Orgyia antiqua (Fitch) (Lepidoptera: Lymantriidae) were obtained from laboratory colonies INIA, Chillán, Chile; late instar blackmoth larvae Dallaca pallens (Blanchard) (Lepidoptera: Leporidae) and adult Argentine stem weevil Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae) were obtained from natural pastures in Region X in the south of Chile (approx. latitude 41°S); late instar Mediterranean flour moth larvae Anagasta kuehniella (Zeller) (Lepidoptera: Pyralidae) were obtained from a grain storage house in Region VII (approx. 35°S); late instar chafer larvae Phytolema hermanni (Germain) (Coleoptera: Scolytidae), Brachysternus prasinus (Guerin) (Coleoptera: Scarabaeidae), and adult burrito weevil Aegorhinus nodipennis (Hope) (Coleoptera: Curculionidae) were obtained from blueberry and raspberry crops in Regions VI to X (approx. 34 to 41°S); late instar eucalyptus weevil larvae Gonipterus scutellatus (Gyllenhal) (Coleoptera: Curculionidae) were obtained from a eucalyptus plantation in Region
VII; late instar pear slug larvae Caliroa cerasi (L.) (Hymenoptera: Tenthredinidae) and adult European earwig Forficula auricularia (L.) (Dermaptera: Forficulidae) were obtained from cherry trees and leaf litter respectively, on the INIA estate, Chillán. Each insect was placed into a chamber (1.5 cm high x 3 cm diameter) lined with moistened filter paper. Infective juveniles were applied at doses of 0, 10 and 100 IJ/ insect in 100 ^l sterilised tap water, then left at 20 ± 2°C. Insect mortality was monitored every 24 h for 9 d; cadavers were monitored daily for a further 20 d to check for nematode emergence. The filter paper in each chamber was moistened occasionally. There were between 30 and 60 insects per treatment, depending on circumstances, arranged in three blocks. Adult P. calceolariae were exposed in groups of five per chamber.
Data analysis. In all experiments the results from the repeat trials were similar and were therefore combined. When appropriate, mortality data was corrected for control mortality using Abbott's formula (Abbott, 1925). 'Infectivity' was a function of host mortality and 'development' a function of progeny emergence. Any percentage data were arcsine transformed before significance testing (Dytham, 2003) (the data presented in the paper are pre-transformed data). Analysis of variance with appropriate factors was used to analyse treatment effects in all tests, with Tukey's test and linear regression used to analyse differences and relationships between treatments (Genstat 11th Edition, VSNI). Differences between treatment means (± SE) were considered significant at P < 0.05.
Moisture content % w/w (top to base range, 72 h) Waxmoth infected % (± SE) EPN penetrating (± SE)
0.2 (0.2-0.2) 0a 0 a
4.6 (4.2-4.4) 100 b 20 (± 1.2) b
8.3 (7.5-8.7) 100 b 21 (± 1.1) b
13.4 (12.1-13.7) 100 b 17 (± 1.7) b
15.8 (14.9-16.0) 87 (± 9.1) c 9 (± 2.1)c
Table 1. Infectivity (%) and penetration of waxmoths by Steinernema australe in a 4 cm column of sand of MC 0.2, 4.6, 8.3, 13.4 and 15.8% (w/w). Waxmoths were exposed at a concentration of 50 IJ/ larva for 72 h, with IJ placed at the bottom of the sand column and waxmoths on the top. Included are MC readings taken at end of trial from top and bottom of sand column. Means followed by the same letter within a column are not significantly different (P > 0.05).
Fig. 1. Waxmoth larvae infected by Steinernema australe (%), time until waxmoth death (h) and development of S. australe in vivo, at test temperatures 7, 11, 16, 20, 23, 28 and 33°C. Waxmoth larvae were kept in chambers partially filled with sand and exposed at a concentration of 25 IJ/ larva, for a total of 40 d. Bars indicate means and vertical lines represent the 95% confidence intervals. Means sharing the same letter are not significantly different (P > 0.05).
RESULTS
Life cycle. Infectivity of waxmoth larvae at 20°C was observed after 24 h, with no larvae alive after 48 h. The level of penetration (mean ± SE) after 48 h was 62 ± 4.6% of the original IJ inoculum. First generation adults were observed 48 h following host death (female: male sex ratio of 1.8:1), second generation adults a further 48 h later. The mean time (range) from inoculation until IJ were first observed emerging from cadavers was 192 (144 - 216) h. Second generation adults and non-infective juveniles were observed outside the insect cadaver 144 h following inoculation. There were no indications of self-fertility for S. australe from the 1:1 trials; mortality of waxmoths was 38% vs 4% for IJ and control treatments respectively, but no progeny emerged.
Temperature profile. Steinernema australe infected 76% of the waxmoth larvae at 7°C and 100% at all other test temperatures (Fig. 1A). Temperature had a significant effect on mean time until waxmoth death (F = 824.3; df = 6,331; P < 0.05). Mortality of waxmoths was most rapid at 23°C and slowest at 7°C (33 ± 1.7 h and 418 ± 12.3 h
until death, respectively) (Fig. 1B). No progeny emerged at 7, 28 and 33°C, despite infectivity of > 76%. The percentage of cadavers producing progeny was > 90% at 16 and 20°C, 84% at 23°C and 72% at 11°C (Fig. 1C). There was no mortality of waxmoth larvae in the control treatment at each test temperature. Temperature had a significant effect (F = 333.6; df = 3,169; P < 0.05) on the time until first nematode emergence after inoculation, with IJ observed outside the host 8.3 (± 0.4), 11.1 (± 0.7), 16.5 (± 0.6) and 44.8 (± 1.7) d after inoculation at 20, 23, 16 and 11 °C, respectively.
Freeze tolerance. At -1 ± 1°C IJ survival was similar for all exposure times (F = 1.82, df = 6,84, P > 0.05); there was a significant effect of exposure time at 8 ± 2°C on IJ survival (F = 5.81, df = 6,84, P < 0.05) (y = -0.04 + 95.9x; r2 = 0.26) although survival remained > 90% (Fig. 2). There was a significant effect of exposure time at -1 ± 1°C on subsequent waxmoth infectivity (F = 13.7, df = 6,84, P < 0.05)(y = -0.82 + 107.9x; r2 = 0.77), falling from 100% at 0 h exposure, to approximately 80% at 72 h and 0% after 120 h. Waxmoth infectivity by IJ stored at 8 ± 2°C was 100% throughout the study.
Fig. 2. Survival (%) of Steinernema australe IJ following exposure to -1 ± 1°C and 8 ± 2°C for 0, 24, 48, 72, 96 and 120 h, and the subsequent infectivity of waxmoth larvae. Waxmoths were exposed at a concentration of 20 live IJ/ larva, at 20 ± 2°C for 144 h. Bars and symbols (▲ and X) indicate means and vertical lines represent the 95% confidence intervals.
Table 2. Infectivity (%) of various insect species from Chile by Steinernema australe and subsequent EPN development. Insects were exposed at a concentration of 10 and 100 IJ/ insect for 9 d; IJ emergence from cadavers was monitored daily for 20 d. Stage: L = larva; A = adult. Means ± SE followed by the same letter within a column are not significantly different (P > 0.05).
Order Family Species Stage % Mortality % Cadavers producing progeny
10 IJ 100 IJ 10 IJ 100 IJ
Lepidoptera Pyralidae Galleria mellonella L 83 ± 9.0 a 100 ± 0 a 39 ± 9.4 a 87 ± 1.7abc
Tortricidae Cydia pomonella L 55 ± 12.3 abc 79 ± 7.2 ab 59 ± 7.6 a 92 ± 1.6 ab
Lymantriidae Orgyia antiqua L 49 ± 3.1 abc 95 ± 4.8 ab 46 ± 10.6 a 78 ± 2.8 abcde
Leporidae Dallaca pallens L - 19 ± 2.4 f - 100 ± 0.0 a
Pyralidae Anagasta kuehniella L 51 ± 10.2 abc 91 ± 4.8 ab 42 ± 6.0 a 36 ± 2.9 def
Coleoptera Scolytidae Phytolema hermanni L 13 ± 10.0 cd 40 ± 11.0 cdef 0 ± 0 b 28 ± 14.3 ef
Scarabaeidae Brachysternus L 16 ± 1.0 cd 59 ± 4.8 bcde 32 ± 12.9 ab 49 ± 12.2 bcdef
prasinus
Curculionidae Listronotus A 4 ± 1.1 d 10 ± 6.3 f 0 ± 0 b 0 ± 0 f
bonariensis
Curculionidae Aegorhinus A 14 ± 7.2 cd 23 ± 8.4 ef 0 ± 0 b 33 ± 16.7 def
nodipennis
Curculionidae Gonipterus scutellatus L 23 ± 9.4 cd 73 ± 6.8 abc 0 ± 0 b 14 ± 4.5 f
Hemiptera Pseudococ- Pseudococcus A 37 ± 13.4 bcd 33 ± 6.4 def 0 ± 0 b 0 ± 0 f
cidae calceolariae
Coreidae Leptoglossus chilensis A - 79 ± 11.5 ab - 83 ± 11.8 abcd
Dermaptera Forficulidae Forficula auricularia A - 9 ± 5.9f - 17 ± 16.7 f
Hymenoptera Tenthredinidae Caliroa cerasi L 72 ± 1.7 ab 73 ± 6.8 abcd 3 ± 2.8 b 42 ± 14.4 cdef
Effect of soil moisture. Results from the soil moisture study can be seen in Table 1. There were minor variations in MC in the tubes during the 72 h trial (Table 1). At 0.2% MC there was no infectivity of waxmoth larvae, for all other treatments infectivity was > 87% (F = 115.2, df = 4,70, P < 0.05). Control mortality was 0% for all treatments. There was a significant effect (F = 41.3, df = 4,70, P < 0.05) of MC on the number of IJ penetrating the host; highest penetration was 21 (± 1.1) IJ/ larva at 8.3% MC (approximately 42% of the original IJ inoculum level), falling to 9 (± 2.1) IJ/ larva (approximately 18% of original inoculum) at 15.8% MC.
Ambush capacity. There were no indications of an ability to attach to a mobile waxmoth larvae as no IJ were recovered from the insect cuticle after 20 min exposure. No IJ were seen nictating or jumping when on agar.
Laboratory host range. There were significant differences in corrected mortality among the target species at both 10 and 100 IJ/ target (F = 8.92, df= 10,21, P < 0.05 and F = 19.53, df = 13,26, P < 0.05 respectively) (Table 2). Infectivity > 90% was only observed in the Lepidoptera where O. antiqua and A. kuehniella suffered 95 and 91% mortality, respectively (interpretation excludes G. mellonella); infectivity > 70% was seen in all other orders apart from Dermaptera. A number of target species
showed relatively low levels of infection (< 33% at the highest IJ dose of 100), including four of the five species tested at the adult stage, viz., F. auricularia, L. bonariensis, A. nodipennis and P. calceolariae. Infection amongst the Coleoptera ranged from 10 - 73%, although emergence of new IJ never exceeded 50% of infected cadavers.
DISCUSSION
The results from this study provide baseline data on the biology and ecology of S. australe, thereby complementing the species description by Edgington et al. (2009). A new EPN species description, unless produced solely for taxonomic/systematic reasons, should be complemented by basic bionomic information as this will assist future researchers in selecting the most suitable EPN, optimising field efficacy and developing a suitable production process (see Grewal et al., 2005).
The life-cycle of S. australe was relatively rapid, with IJ emergence observed after only 6 d post inoculation at 20°C. Other temperate Steinernema species emerged from waxmoth larvae after 8, 12 and 14 d at 20°C (Koppenhöfer & Kaya, 1999; Koppenhöfer et al., 2000; Gungor et al., 2006). There was no evidence of self-fertility, something that has only been recorded in S. hermaphroditum
(Stock et al., 2004). It was not uncommon to see second generation males, females and non-infective juveniles of S. australe outside the cadaver, often moving into the water trap and surviving for several days. Adult emergence and migration away from the cadaver has been observed for S. affine (San-Blas pers. comm.), although it would appear to be a relatively rare characteristic for EPN. The IJ stage is morphologically and physiologically adapted to survive outside the host, possessing rich food reserves and being protected by the retained cuticle from the previous stage (O'Leary et al., 1998; Griffin et al., 2005), it is not clear whether adults and non-infective juvenile stages have suitable adaptations to survive for very long outside a host, although it would be surprising if they had.
The ability of S. australe to infect a host at 7°C may reflect an adaptation to its type-locality, a relatively cold island in southern Chile. Infectivity at cold temperatures can be a useful biological control feature. Steinernematid isolates sourced from cold localities in Canada and Scotland were infective at temperatures below 7°C (Mracek et al., 1997; Long et al., 2000), the Scottish isolate since becoming a successful commercial product for use against black vine weevil (Otiorhynchus sulcatus F.). The temperature profile of S. australe narrowed for development over infectivity, a feature not unusual for EPN (Molyneux, 1986; Koppenhofer et al., 2000; Gungor et al., 2006). Infectivity has a wider window of opportunity than development and may also rely less on the proliferation of the symbiotic bacterium, itself restricted by lower temperatures (Wright, 1992). Indeed, EPN infectivity has been known to proceed in the absence of the symbiotic bacterium (see Ciche et al., 2006). Steinernematids are found in some very cold localities including Arctic territories (see Haukeland et al., 2006) where they are exposed to prolonged sub-zero temperatures. Infective juveniles of S. australe were able to survive 5 d at -1°C. Survival mechanisms of EPN to sub-zero conditions include the retained cuticle from the previous stage, the production of trehalose and cold-shock proteins (freeze-avoidance adaptations) or tolerance of a degree of tissue freezing (freeze-tolerance) (Glazer, 2002; Jagdale et al, 2005). Ensheathed IJ of S. australe were used in the present study which may have enhanced their survival at -1°C. However, as exposure time to -1°C increased, the ability of S. australe to infect waxmoth larvae was substantially reduced, even at the near optimum temperature of 20°C. In similar studies, Steinernema and Heterorhabditis species both lost infectivity following exposure to sub-zero temperatures, the
reduction being attributed to sluggish movement and vacuolisation of the IJ (chilling-injuries) (Wharton & Surrey, 1994; Brown & Gaugler, 1998). Environmental factors are known to destabilise the symbiotic bacteria of EPN (Krasomil-Osterfeld, 1995), which in turn may reduce infectivity (Dowds & Peters, 2002); however, whether the bacteria of S. australe were sensitive to temperatures of -1°C is unknown.
Steinernema australe infected over a wide range of moisture levels. The high level of infectivity recorded at 15.8% MC may reflect the large body size of S. australe (IJ body length > 1300 ^m, body diam. 38 ^m), Koppenhofer et al. (1995) suggesting that larger IJ are better adapted to move through thicker films of water as smaller nematodes may start floating. Establishment rates of S. australe at MC > 13% appeared slightly lower than S. thermophilum (now regarded as a junior synonym of S. abbasi (Hunt, 2007)) (Ganguly & Gavas, 2004) and S. monticolum (Koppenhofer et al., 2000), both much smaller nematodes, although both these trials used shorter columns of substrate than the present trial and therefore their motility challenge would have been less. Infective juveniles active at a wide range of moisture levels could be of use in both badly drained (humid) soils and those experiencing considerable fluctuations in moisture levels, although desiccation tolerance of the IJ would be important as a soil dries out.
The natural host range of S. australe is unknown as this EPN was isolated from soil using waxmoth baiting. Reports on host ranges of EPN in their natural, non-agricultural habitats are relatively rare, most host range data generally originating from laboratory studies (and should be referred to as the 'laboratory host range') and/or insects collected from infested agricultural sites (see Peters, 1996). Steinernema australe infected a wide range of insect hosts in the laboratory, although there was clear host specificity. The laboratory host ranges of EPN are well documented. Temperate strains of S. rarum, S. feltiae and S. monticolum could be regarded as generalists with respect to insect order, infecting 11 insect orders between them, yet showing marked specificity at the insect family level (Koppenhofer et al., 2000; de Doucet et al, 1999). Specificity is controlled by a combination of host finding, host acceptance and host suitability, which in turn relies on the host, EPN, bacterial symbiont and environmental conditions (Li et al., 2007; Lewis et al., 2006). During the ambush studies there were no indications that S. australe could attach to a mobile host and no nictation or jumping behaviour was observed. A similar result regarding attachment was
obtained for the cruise-forager S. glaseri (Koppenhöfer & Fuzy, 2003); Campbell & Kaya (2002) observed a lack of jumping behaviour for EPN species over 800 ^m in length (S. australe body length > 1300 ^m). An absence of nictation and/or jumping may preclude ambush foraging. During studies using inter-connected chambers filled with sand, S. australe was observed to travel at least 11 cm, in 48 h, in the absence of host cues (S. Edgington pers. obs.).
The results on the biology and ecology of S. australe presented here, whilst simple and to be treated with caution in making generalisations regarding behaviour in a field setting, can assist future researchers in assessing the use of this EPN as a control organism. The results complement both the formal description of S. australe and considerable EPN research being carried out by staff at INIA on other, locally sourced, EPN isolates as part of the national policy to find alternatives to chemical pesticides.
ACKNOWLEDGEMENT
This study was supported by the Darwin Initiative (project number 15/004), a programme coordinated by the UK Department for Environment, Food and Rural Affairs (Defra). The authors wish to thank Dave Moore (CABI) and Loreto Merino (INIA) for their help with the survey work on Isla Magdalena and support in the laboratory, the insectary team at INIA for providing insect cultures and David Hunt for reviewing the manuscript.
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S. Edgington, S. R. Gowen. Экологическая характеристика Steinernema australe (Panagrolaimomorpha: Steinernematidae) - энтомопатогенной нематоды из Чили. Резюме. Приводятся результаты изучения недавно описанного вида энтомопатогенной нематоды Steinernema australe из Чили. В лабораторных условиях S. australe имеет довольно короткий жизненный цикл. Так, миграция инвазионных личинок (IJ) наблюдается при 20°C уже на 6-й день после заражения; при этом и взрослые нематоды, и питающиеся личинки также покидают труп насекомого. Пределы оптимальных температур и влажности для этих нематод оказываются достаточно широкими. Достаточно высокая степень заражения были получены при низкой температуре и высокой влажности (7°C и 15.8% содержания влаги). Инкубация при температуре около -1°C в течение 5 дней не влияла на выживаемость IJ, однако эффективность заражения хозяев такими личинками существенно снижалась. Предполагается, что причиной могло служить термическое повреждение симбиотических бактерий или воздействие низких температур на смену фаз у бактерий. Steinernema australe заражает широкий круг насекомых вредителей, отмеченных для Чили, включая высокоподвижные и малоподвижные формы, показывая при этом наивысшую эффективность против Lepidoptera. Для этого вида не отмечена способность IJ к прыжкам или никтации.
