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Facultative scavenging as a survival strategy of entomopathogenic nematodes
International Journal for Parasitology 38 (2008) 85–91 www.elsevier.com/locate/ijpara
Facultative scavenging as a survival strategy of entomopathogenic nematodes Ernesto San-Blas *, Simon R. Gowen School of Agriculture, Policy and Development, University of Reading, Earley Gate, Reading RG6 6AR, UK Received 22 February 2007; received in revised form 11 June 2007; accepted 12 June 2007
Abstract Entomopathogenic nematodes cannot be considered only as parasitic organisms. With dead Galleria mellonella larvae, we demonstrated that these nematodes use scavenging as an alternative survival strategy. We consider scavenging as the ability of entomopathogenic nematodes to penetrate, develop and produce offspring in insects which have been killed by causes other than the nematode– bacteria complex. Six Steinernema and two Heterorhabditis species scavenged but there were differences among them in terms of frequency of colonisation and in the time after death of G. mellonella larvae that cadavers were penetrated. The extremes of this behaviour were represented by Steinernema glaseri which was able to colonise cadavers which had been freeze-killed 240 h earlier and Heterorhabditis indica which only colonised cadavers which had been killed up to 72 h earlier. Also, using an olfactometer, we demonstrated that entomopathogenic nematodes were attracted to G. mellonella cadavers. Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Steinernema; Heterorhabditis; Cadaver; Olfactometer; Galleria mellonella
1. Introduction Entomopathogenic nematodes (EPNs) have been considered obligate parasites of insects because they can invade the insect body and subsequently release a symbiotic bacterium which kills the hosts by septicaemia within 96 h. In soil, the infective juveniles of an EPN are initially a non-feeding stage but can be considered as parasites when they invade the insect body; after killing the host, they start feeding in the resultant soup of bacteria and insect tissue and can be compared with any bacteria-feeding nematode, both morphologically and physiologically (Yeates et al., 1993). One of the major reasons for the increase in the usage of EPNs for biological control of pests in recent years is that they can be mass-produced relatively easily by industrial techniques (Woodring and Kaya, 1988). Those tech*
Corresponding author. Tel.: +44 0 118 378 8484; fax: +44 0 1189352421. E-mail address: [email protected] (E. San-Blas).
niques do not need to be carried out with insects; instead some production protocols have been developed using media which contain a wide range of proteins and carbohydrates to first grow the symbiotic bacteria and then the nematodes. [Some media include potato mash (McCoy and Glaser, 1936), dog food (Hara et al., 1981), pig kidney-fat (Bedding, 1981), poultry offal (Bedding, 1984) or whey (Chavarrı´a-Herna´ndez et al., 2006). Due to the number of production options, it may be supposed that if the right conditions are met, EPNs could use any resource available in nature to complete their life cycles; that means the nematodes can be scavengers and survive using this behaviour and would not need to find and kill a live host. Scavenging can be divided into two categories: obligate and facultative. Obligate scavenging occurs when an organism can only survive by feeding on dead material. Oncholaimus campylocercoides, which lives in hydrothermal vents, can take advantage of the number of organisms dying due to the high concentration of hydrogen sulphide present in that environment and feeds only on dead material
0020-7519/$30.00 Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.06.003
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(Thiermann et al., 1997). The second category, facultative scavenging, occurs where predator organisms take advantage of the opportunity to feed on cadavers that they may encounter. Some benthic nematodes such as Enoploides longispiculosus (Moens et al., 1999, 2005), Ascolaimus, Axonolaimus and Daptonema (Urban-Malinga et al., 2006), have been reported as scavengers. Prein (1988) described the occurrence of a mass aggregation and attraction of Pontonema vulgare after mortality of fishes due to anoxic conditions. Juveniles of Enchelidiidae and Oncholaimidae were classified as predatory-scavenger nematodes by Jensen (1987, 1988). For example, Andocholaimus yhalassophygas (Oncholaimidae) can complement their diet, based on dissolved organic matter, by scavenging on carcasses (Lo´pez et al., 1979). Some nematodes from the family Diplogasteridae can be attracted by insect cadavers, consume them and lay their eggs inside the cadavers (Poinar, 1979). Also, Capinera et al. (1982) observed that EPNs could feed upon pieces of earthworm body tissue and even complete their life cycles, producing small numbers of offspring. Although EPNs have never been reported as scavenger organisms, some evidence of the use of insect cadavers by EPNs has been reported. Some Steinernema species successfully completed life cycles in dead Hylobius pales larvae (Jackson and Moore, 1968), and Pye and Burman (1978) showed the same rate of infection of Steinernema carpocapsae in Hylobius abietis which had been previously killed by chloroform or heat, as that found in live insects. The nematodes could also complete their life cycles and produce offspring in H. abietis cadavers. This work attempts to determine if EPNs can colonise and complete their life cycles within cadavers of Galleria mellonella and if so, observe whether the nematodes are attracted to the cadavers or if they encounter them by chance through random movement. 2. Materials and methods 2.1. Nematode cultures The species Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, Steinernema kraussei, Steinernema affine, Steinernema riobrave, Heterorhabditis indica and Heterorhabditis bacteriophora were cultured in the fourth instar larvae of G. mellonella (Lepidoptera: Pyralidae) (Livefoods Direct Ltd. Sheffield, UK) following the technique of Dutky et al. (1964); the larvae were kept at 10 °C until the day of the experiment (never more than 1 week). The incubation temperature for culturing nematodes was based on whether they originated from tropical or temperate climates. Therefore all Steinernema spp. except S. riobrave were incubated at 20 °C whereas Heterorhabditis spp. and S. riobrave were incubated at 28 °C. The infective juveniles were collected using White traps (White, 1927) and were stored at 10 °C (all Steinernema spp. except S. riobrave) and at 15 °C (Heterorhabditis
spp. and S. riobrave) until the day of the experiments (1 week maximum). 2.2. Capacity and frequency of scavenging One hundred and ten G. mellonella were killed by freezing and separated into groups of 10 at room temperature. The first group was immediately placed in a 9 cm diameter Petri dish with a WhatmanÒ No. 1 filter paper and 1 ml of a suspension containing 1,000–2,000 infective juveniles was added depending of the size of the species: S glaseri, S. kraussei – 1,000 nematodes, S. feltiae, S. affine, S. riobrave – 1,500 nematodes, the rest 2,000 nematodes. This difference in the dosage was used to avoid intra-specific competition and density dependent effects (Kaya and Koppenho¨fer, 1996). The second group was placed in another Petri dish with the same number of nematodes 24 h after death, the third group 48 h after death and so on. The control treatment was 10 live G. mellonella. The Petri dishes were incubated at 20 or 28 °C depending on the nematode species as described above. After 5 days, all the G. mellonella were dissected, subjected to a pepsin digestion (Glazer and Lewis, 2000), and nematodes were counted. The experiments were repeated three times. An extra replication of the experiment was performed in which the G. mellonella were left in the incubator for 14 days and then placed in White traps. Completion of the nematode life cycles was confirmed by direct microscopic observation and by the number of new infective juveniles that emerged after 2 days in the White traps. The analysis of the number of individuals found in the G. mellonella cadavers was done using SASÒ software; the data was transformed (square root) and then a one-way ANOVA analysis was performed. A Tukey grouping test was performed when differences were significant (P < 0.05). 2.3. Attraction of entomopathogenic nematodes to G. mellonella cadavers An olfactometer constructed with polyvinyl chloride pipes (Fig. 1), was filled with sterilised silver sand (Westland horticulture, Dungannon, UK) initially adjusted to 15% w/w water content. Four G. mellonella, previously killed by freezing, were set in one of the extremes of the olfactometer in a chamber made with 4 cm of the anterior part of a 50 ml syringe (diameter = 2.5 cm), and isolated from the main arm with a 10 lm mesh, allowing the diffusion of any gaseous compound but avoiding passage of the nematodes; the control chamber was made in the same way in the opposite arm but left empty. Both extremes and the centre opening were sealed with NescofilmÒ sealing film (Azwell Inc., Osaka, Japan). The olfactometer was then placed in the incubator at 20 or 28 °C (as above) for 48 h to create a gradient of a possible attractant compounds. Between 500 and 1,000 (as above) S. feltiae, S. carpocapsae, S. kraussei, S. glaseri, S. affine, S. riobrave, H. bacteriophora and H. indica were placed in the central part of the
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Fig. 1. Detail of olfactometer constructed from polyvinyl chloride pipes.
olfactometers which were then returned to the incubators for a further 48 h, except for S. glaseri which was returned for only for 8 h because they were very active and escaped from the olfactometer in less than 24 h (data not shown). Each section of the olfactometer was separated, nematodes were recovered from the sand by incubation (Whitehead and Hemming, 1965) and counted under the stereomicroscope. The experiment was repeated 10 times, the percentages of the responding nematodes that moved to one or the other arms were calculated and a two samples paired t-test was performed (a = 0.05). 3. Results 3.1. Capacity and frequency of scavenging The capacity and frequency of scavenging varied between species of the EPNs tested (Table 1). The number of S. feltiae found colonising G. mellonella cadavers in the treatments at 0, 24 and 48 h was the same (102.2 ± 9.16, 61.1 ± 6.71 and 75.05 ± 6.51, respectively) as those in the control treatment (91.95 ± 7.9). At 72 h there was a significant increase of the number of nematodes found in the cadavers (125.9 ± 10.6). The number of nematodes found in G. mellonella killed 96 h before exposure dropped to 39.6 ± 11.9 and at 120 h the number recorded was 0.2 ± 0.15. No nematodes were found in the treatments with larvae that had been dead for longer than 120 h. The numbers of individuals of S. carpocapsae that scavenged were similar to those in the control group (129.6 ± 13.9) except for the last two groups. Similar numbers entered G. mellonella cadavers for up to 144 h after being freeze-killed but numbers of nematodes scavenging cadavers after 168 and 192 h were 43.42 ± 7.06 and 28.75 ± 6.4, respectively. Increasing numbers of S. kraussei scavenged G. mellonella cadavers from 0 h (40.50 ± 3.75) until a maximum at 120 h (89.3 ± 8.39), greater than the numbers in the control group (27.84 ± 3.42). After 120 h, the number declined to 11.03 ± 1.61 at 144 h and 2.39 ± 0.66 per cadaver at 216 h. Steinernema glaseri colonised all treatment groups. At 0 h, the number of nematodes that scavenged was lower (20.75 ± 3.28) than those recorded in the control group (34.79 ± 4.47), but at 48 h, the total number of nematodes in G. mellonella was similar to that of the control group (26.53 ± 3.16). The number of individuals per cadaver
declined in the remaining treatments, to a minimum of 6.45 ± 1.47 at 216 h. The greatest numbers of S. affine (39 ± 5.45 and 41 ± 6.44) entered cadavers at 0 and 24 h, which were significantly greater than the control group (14.55 ± 2.8). In the other treatments, the numbers found in the cadavers were statistically similar until 144 h. The number of S. riobrave scavenging was lower than the number recorded in the control group (113.53 ± 6.35) in all the treatments. The number of nematodes that scavenged varied from 88.6 ± 14.2 at 0 h to 1.4 ± 0.82 at 192 h. The numbers of H. bacteriophora individuals that scavenged showed no differences between the control group (47.7 ± 4.66) and the treatments at 0, 24 and 48 h; the numbers declined to 11.7 ± 6.47 at 72 h and to 13.8 ± 0.36 at 96 h. Of all the nematodes tested, H. indica colonised cadavers the least but nevertheless, they did scavenge. The number of nematodes entering dead G. mellonella varied from 14.89 ± 1.61 at 0 h to 3.35 ± 1.32 at 72 h; these values were lower than the control group (53.8 ± 5.85). The percentage of dead G. mellonella colonised in treatments with different times of exposure after being freezekilled was variable among species (Figs. 2 and 3). Heterorhabditis bacteriophora and H. indica colonised 20% and 50%, respectively, of G. mellonella which had been killed 96 and 72 h before. S. feltiae colonised up to 20% of G. mellonella which had been dead for 120 h; S. affine colonised 30% of cadavers at 168 h and S. riobrave colonised 20% of larvae that had been dead for 192 h. Steinernema carpocapsae and S. kraussei entered larvae that had been dead for even longer, colonising more than 60% of the cadavers at 192 h or 60% at 216 h, respectively. Steinernema glaseri continued to scavenge between 90% and 100% of G. mellonella cadavers which had been killed 240 h before and even longer (data not shown). All nematodes tested completed their life cycles and new infective juveniles were recorded emerging from G. mellonella cadavers (Table 2). The number of individuals emerging was similar to controls in all species but declined in the last treatments where the scavenging was lower. 3.2. Attraction of EPNs to G. mellonella cadavers In terms of attraction of the nematodes to the G. mellonella cadavers, the response varied among species (Fig. 4). The percentage of S. feltiae attracted by dead G. mellonella
nd nd nd
nd
nd nd nd
nd
Fig. 2. Percentage of insects colonised by different entomopathogenic nematodes in freeze-killed Galleria mellonella at different times of exposure from time of death (0 h) (n = 30). n Steinernema carpocapsae; r Steinernema affine; m Steinernema glaseri; d Heterorhabditis bacteriophora.
nd = nematodes not detected in cadavers. Values represent mean ± SEM. Different letters within rows indicate significant differences (P < 0.05).
nd nd 3.35 ± 1.32c 14.89 ± 1.61b 14.45 ± 5.87bc 7.50 ± 2.07c 53.8 ± 5.85a
47.70 ± 4.66a 36.60 ± 3.48a 29.05 ± 3.72a 32.70 ± 5.97a 11.70 ± 6.47b 13.80 ± 9.36b nd
nd
nd
nd 15.90 ± 5.25c 22.60 ± 7.41c 5.30 ± 1.98cd 1.400 ± 0.82d nd 113.53 ± 6.35a 88.6 ± 14.2b
44.79 ± 8.73c 82.6 ± 14.8b
89.2 ± 19.5b
30.0 ± 10.2c
nd nd nd 14.55 ± 2.80b 39.00 ± 5.45a 41.00 ± 6.44a 24.75 ± 4.32ab 27.45 ± 6.67ab 25.63 ± 6.18ab 13.50 ± 4.91b 8.70 ± 3.81b 4.11 ± 2.90c
2.39 ± 0.66d nd 48.29 ± 6.79bc 63.95 ± 9.35b 73.23 ± 8.04b 89.3 ± 8.39a 11.03 ± 1.61d 4.17 ± 0.84d 8.15 ±1.64d 69.13 ±6.04b 27.84 ± 3.42cd 40.50 ± 3.75c
6.45 ± 1.47c 10.05 ± 1.54c 10.55 ± 1.15c 9.05 ± 2.30c 12.80 ± 2.11c 9.30 ± 2.61c 11.65 ± 1.92c 34.79 ± 4.47a 20.75 ± 3.28b 21.00 ± 2.99b 26.53 ± 3.16ab 10.00 ± 1.65c
nd 131.3 ± 11.7a 148.4 ± 21.4a 143.8 ± 13.2a 43.42 ± 7.06b 28.75 ± 6.4b nd 116.4 ± 16.4a 123.7 ±11.6a 95.2 ± 9.42a 129.6 ± 13.09a 98.5 ± 13.0a
240 h 216 h 192 h
nd
168 h
nd
144 h
nd 0.2 ± 0.15d
120 h 96 h 72 h 48 h 24 h 0h Control
91.95 ± 7.90b 102.20 ± 9.16b 61.10 ± 6.71bc 75.05 ± 6.51b 125.9 ± 10.6a 39.6 ± 11.9c
Steinernema feltiae Steinernema carpocapsae Steinernema glaseri Steinernema kraussei Steinernema affine Steinernema riobrave Heterorhabditis bacteriophora Heterorhabditis indica
Table 1 Numbers of individuals in dead Galleria mellonella larvae exposed to nematodes for up to 240 h after death
nd
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nd
88
Fig. 3. Percentage of insects colonised by different entomopathogenic nematodes in freeze-killed Galleria mellonella at different times of exposure from time of death (0 h) (n = 30). h Steinernema feltiae; n Steinernema riobrave; s Steinernema kraussei; } Heterorhabditis indica.
was 22.42 ± 2.33%, which was higher than the percentage that moved towards the control section of the olfactometer (12.76 ± 2.42%). More S. glaseri moved to the section of the olfactometer where the G. mellonella cadavers were placed (29.72 ± 3.47%), compared with the number of nematodes found in the control section (7.52 ± 2.84%), the highest response among the Steinernematid species. Both Heterorhabditids tested showed a positive movement towards G. mellonella cadavers; the percentage of H. indica found in the G. mellonella section of the olfactometer was higher than the portion of nematodes found in the control section (48.68 ± 5.72% and 23.46 ± 3.67%, respectively) and the percentage values for infective juveniles of H. bacteriophora which moved towards the G. mellonella cadavers was 36.31 ± 3.01%, whereas the percentage of these
E. San-Blas, S.R. Gowen / International Journal for Parasitology 38 (2008) 85–91
Fig. 4. Percentage of nematodes (mean ± SEM) which moved in the olfactometer towards the control arm (empty bars) or to Galleria mellonella cadavers arm (solid bars). Steinernema feltiae (Sf), Steinernema carpocapsae (Sc), Steinernema riobrave (Sr), Steinernema kraussei (Sk), Steinernema affine (Sa), Steinernema glaseri (Sg), Heterorhabditis indica (Hi) and Heterorhabditis bacteriophora (Hb). Different letters within species indicate significant differences (P < 0.05).
nematodes which moved to the control section of the olfactometer was 20.85 ± 3.39%. Steinernema affine, S. riobrave, S. carpocapsae and S. kraussei did not show a positive movement to the G. mellonella cadavers.
4. Discussion The EPNs used scavenging as an alternative survival strategy and completed their life cycles in G. mellonella cadavers. In some species, there was no difference between the number of individuals representing a normal infection process (control groups) and the number of nematodes colonising cadavers (scavenging). We suppose that this behaviour could be repeated in nature if similar conditions existed such as the presence of a insect which had been dead for a short time. In fact, the possibilities of finding freeze-killed insects in nature are remote but there are many other viable options occurring. Despite the fact that sometimes EPNs are not able to share the host with another entomopathogenic pathogen organism
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such as Beauveria bassiana (Barbercheck and Kaya, 1991), larvae infected by granulosis virus can be used by EPNs as suitable hosts (Kaya and Brayton, 1978). There are other sources of dead insects such as adults after mating, drowned larvae and insects partially eaten by other predators, all of which could be used by nematodes as food resources, but further investigation is required. The majority of the species tested showed higher colonisation rates (number of individuals and percentage of insects colonised) in larvae that had been dead for up to 120 h, although some species scavenged larvae that had been dead for longer (S. carpocapsae up to 144 h or S. glaseri up to 240 h). The possibility of successful scavenging could be affected by the time of larval death, because EPNs have to colonise, release the bacteria and avoid competition from other saprophytic organisms. Also, it is important that the cadaver is at a distance that can be reached by the nematodes. Steinernema glaseri, S. kraussei and S. carpocapsae infected more than 90% of the G. mellonella that had been dead for 240, 192 and 168 h, respectively (Figs. 2 and 3) which makes them better scavengers in terms of the time of resource utilisation than H. indica, H. bacteriophora and S. feltiae which colonised up to 90% of the cadavers dead for 24, 48 and 96 h, respectively. According to these results, and despite the fact that all species scavenged, Heterorhabditis spp. seem to be less disposed to perform scavenging than species of Steinernema, but other Heterorhabditid species need to be tested. Probably one of the key factors for the success of this behaviour is that neither nematodes nor bacteria have to fight the insect immune system and the possibilities of survival might therefore be increased. In fact, some insects which have been thought of as non-susceptible to Steinernematid nematodes such as wireworms (Agriotis spp.), could be a source of colonisation if they die, but further investigation is required. The apparent failure of nematodes to colonise older cadavers of G. mellonella may be due to the fact that nematodes had entered but had failed to develop due to unfavourable conditions. It seems that the EPNs can identify the cadaver as a possible host by volatile cues, even though
Table 2 Numbers of Galleria mellonella (out of 10) exposed to nematodes for up to 240 h after death
Steinernema feltiae Steinernema carpocapsae Steinernema glaseri Steinernema kraussei Steinernema affine Steinernema riobrave Heterorhabditis bacteriophora Heterorhabditis indica
Control
0h
24 h
48 h
72 h
96 h
120 h
144 h
168 h
192 h
216 h
240 h
10+++ 10+++ 10++ 10+++ 10+++ 10+++ 10+++ 10+++
10+++ 10+++ 10++ 10+++ 10+++ 10+++ 10+++ 10+++
10+++ 10+++ 10++ 10+++ 10+++ 10+++ 10+++ 9+++
10+++ 10+++ 10++ 10+++ 10+++ 10+++ 10+++ 7+++
9+++ 10+++ 10++ 10+++ 10++ 10+++ 5+++ 5+++
7+++ 10+++ 10++ 10+++ 9++ 10+++ 3+++ 0
4++ 10+++ 10++ 10+++ 5+ 7++ 0 0
0 10++ 9++ 10+++ 3+ 8++ 0 0
0 7++ 10++ 9+++ 3+ 4+ 0 0
0 5++ 10++ 9+++ 0 1+ 0 0
0 0 9++ 6++ 0 0 0 0
0 0 10++ 0 0 0 0 0
Galleria mellonella were incubated for 14 days after nematode exposure and placed in White traps. Infective juvenile nematodes that emerged after 2 days were recorded. Range of total number of infective juveniles recovered per G. mellonella (+++, >5,000; ++, 1,000–4,999; +, <1,000).
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some species did not show a particular attraction to cadavers of G. mellonella in the olfactometers. However, all species tested were capable of colonising cadavers, which could suggest that either the random movements of the nematodes are partially responsible for the first contact, or that the attraction process starts closer to the cadavers (depending on the concentration of the volatile signals or other types of cues) for the species that did not respond in the olfactometers. In any case, further experimentation is needed to elucidate the nature of these compounds. On the other hand, the nematodes which were attracted by the cadavers have been considered, according to the foraging strategy classification, as ‘‘cruisers’’ e.g. S. glaseri (Lewis et al., 1993), H. bacteriophora (Campbell and Gaugler, 1993) and H. indica or ‘‘intermediates’’ e.g. S. feltiae (Lewis, 2002). This positive attraction can be compared with the observation of Lei et al. (1992) who, despite the fact that they did not test any scavenging behaviour in EPNs, reported that Heterorhabditis zealandica was attracted by Delia radicum cadavers rather than encountering them randomly. The use of scavenging as an alternative way for surviving could explain the long-term persistence of EPNs in the soil, although such behaviour has never been included as a variable to be considered in the theory of population dynamics of these organisms. This alternative survival strategy suggests that EPNs should not be considered only as obligate parasites (Hazir et al., 2001; Burnell and Stock, 2000) but as parasite-scavenger nematodes. Acknowledgements The authors thank Barbara Pembroke from the University of Reading for valuable discussion and recommendations for this paper and Mercedes Andrade for statistical advice. References Barbercheck, M.E., Kaya, H.K., 1991. Competitive interactions between entomopathogenic nematodes and Beauveria bassiana (Deuteromycotina: Hyphomycetes) in soilborne larvae of Spodoptera exigua (Lepidoptera: Noctuidae). Environ. Entomol. 20, 707–712. Bedding, R.A., 1981. Low cost in vitro mass production of Neoaplectana and Heterorhabditis species (Nematoda) for field control of insect pests. Nematologica 27, 109–114. Bedding, R.A., 1984. Large scale production, storage, and transport of the insect-parasitic nematodes Neoaplectana spp. and Heterorhabditis spp.. Ann. Appl. Biol. 104, 118–120. Burnell, A.M., Stock, S.P., 2000. Heterorhabditis, Steinernema and their bacterial symbionts-lethal pathogen of insects. Nematology 2, 31–42. Campbell, J.F., Gaugler, R., 1993. Nictation behaviour and its ecological implications in the host search strategies of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae). Behaviour 126, 155–169. Capinera, J.L., Blue, S.L., Wheeler, G.S., 1982. Survival of earthworms exposed to Neoaplectana carpocapsae nematodes. J. Invertebr. Pathol. 39, 419–421.
Chavarrı´a-Herna´ndez, N., Espino-Garcı´a, J.J., Sanjuan-Galindo, R., Rodrı´guez-Herna´ndez, A.I., 2006. Monoxenic liquid culture of the entomopathogenic nematode Steinernema carpocapsae using a culture medium containing whey kinetics and modeling. J. Biotechnol. 125, 75–84. Dutky, S.R., Thompson, J.V., Cantwell, G.E., 1964. A technique for the mass propagation of the DD-136 nematode. J. Insect Pathol. 6, 417–422. Glazer, I., Lewis, E.E., 2000. Bioassays for entomopathogenic nematodes. In: Navon, A., Ascher, K.R.S. (Eds.), Bioassays of Entomopathogenic Microbes and Nematodes. CAB International, Wallingford, UK, pp. 229–247. Hara, A.H., Lindegren, J.E., Kaya, H.K., 1981. Monoxenic mass production of the entomogenous nematode, Neoaplectana carpocapsae Weiser, on dog food/agar medium. Advances in Agricultural Technology, Western Series-16, USDA/SEA, Oakland, CA, pp. 1–8. Hazir, S., Stock, S.P., Kaya, H.K., Koppenho¨fer, A.M., Keskin, N., 2001. Developmental temperature effects on five geographic isolates of entomopathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae). J. Invertebr. Pathol. 77, 243–250. Jackson, G.J., Moore, G.E., 1968. Infectivity of nematodes, Neoaplectana species, for the larva of the weevil Hylobius pales, after rearing in species isolation. J. Invertebr. Pathol. 14, 194–198. Jensen, P., 1987. Feeding ecology of free-living aquatic nematodes. Mar. Ecol. Prog. Ser. 35, 187–196. Jensen, P., 1988. Nematode assemblages in the deep-Sea benthos of the Norwegian Sea. Deep-Sea Res. 35, 1079–1222. Kaya, H.K., Brayton, M.A., 1978. Interaction between Neoaplectana carpocapsae and a granulosis virus of the armyworm Pseudaletia unipuncta. J. Nematol. 10, 350–354. Kaya, H.K., Koppenho¨fer, A.M., 1996. Effects of microbial and other antagonistic organism and competition on entomopathogenic nematodes. Biocontrol Sci. Technol. 6, 357–371. Lei, Z., Rutherford, T.A., Webster, J.M., 1992. Heterorhabditid behavior in the presence of the cabbage maggot, Delia radicum, and its host plants. J. Nematol. 24, 9–15. Lewis, E.E., 2002. Behavioural ecology. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CAB International, Wallingford, UK, pp. 205–223. Lewis, E.E., Gaugler, R., Harrison, R., 1993. Response of cruiser and ambusher entomopathogenic nematodes (Steinernematidae) to host volatile cues. Can. J. Zool. 71, 765–769. Lo´pez, G., Riemann, F., Schrage, M., 1979. Feeding biology of the brackish-water oncholaimid nematode Adoncholaimus thalassophygas. Mar. Biol. 54, 311–318. McCoy, E.E., Glaser, R.W., 1936. Nematode culture for Japanese beetle control. New Jersey Department of Agriculture Circular. New Jersey Department of Agriculture, pp. 1–10. Moens, T., Bouillon, S., Gallucci, F., 2005. Dual stable isotope abundances unravel trophic position of estuarine nematodes. J. Mar. Biol. Assoc. UK 85, 1401–1407. Moens, T., Verbeeck, L., Vinex, M., 1999. Feeding biology of a predatory and a facultatively predatory nematode (Enoploides longispiculosus and Adoncholaimus fuscus). Mar. Biol. 134, 585–593. Poinar Jr., G.O., 1979. Nematodes for biological control of insects. CRC Press, Boca Raton, Florida, USA. Prein, M., 1988. Evidence for scavenging lifestyle in the free-living nematode Potonema vulgare (Enoplida, Oncholaimidae). Kieler Meeresforsch 6, 389–394. Pye, A.E., Burman, M., 1978. Neoaplectana carpocapsae: infection and reproduction in large pine weevil larvae, Hylobius abietis. Exp. Parasitol. 46, 1–11. Thiermann, F., Akoumianaki, I., Hughes, J.A., Giere, O., 1997. Benthic fauna of a shallow-water gaseohydrothermal vent area in the Aegean Sea (Milos, Greece). Mar. Biol. 128, 149–159. Urban-Malinga, B., Hedtkamp, S.I.C., van Beusekom, J.E.E., Wiktor, J., We˛slawski, J.M., 2006. Comparison of nematode communities in
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Woodring, L.J., Kaya, H.K., 1988. Steinernematid and Heterorhabditid nematodes: a handbook on biology and techniques. Southern Coop. Ser. Bull. 331, 1–21. Yeates, G.W., Bongers, T., de Goede, R.G.M., Freckman, D.W., Georgieva, S.S., 1993. Feeding habits in soil nematode families and genera-an outline for soil ecologists. J. Nematol. 25, 315–331.
Facultative scavenging as a survival strategy of entomopathogenic nematodes Ernesto San-Blas *, Simon R. Gowen School of Agriculture, Policy and Development, University of Reading, Earley Gate, Reading RG6 6AR, UK Received 22 February 2007; received in revised form 11 June 2007; accepted 12 June 2007
Abstract Entomopathogenic nematodes cannot be considered only as parasitic organisms. With dead Galleria mellonella larvae, we demonstrated that these nematodes use scavenging as an alternative survival strategy. We consider scavenging as the ability of entomopathogenic nematodes to penetrate, develop and produce offspring in insects which have been killed by causes other than the nematode– bacteria complex. Six Steinernema and two Heterorhabditis species scavenged but there were differences among them in terms of frequency of colonisation and in the time after death of G. mellonella larvae that cadavers were penetrated. The extremes of this behaviour were represented by Steinernema glaseri which was able to colonise cadavers which had been freeze-killed 240 h earlier and Heterorhabditis indica which only colonised cadavers which had been killed up to 72 h earlier. Also, using an olfactometer, we demonstrated that entomopathogenic nematodes were attracted to G. mellonella cadavers. Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Steinernema; Heterorhabditis; Cadaver; Olfactometer; Galleria mellonella
1. Introduction Entomopathogenic nematodes (EPNs) have been considered obligate parasites of insects because they can invade the insect body and subsequently release a symbiotic bacterium which kills the hosts by septicaemia within 96 h. In soil, the infective juveniles of an EPN are initially a non-feeding stage but can be considered as parasites when they invade the insect body; after killing the host, they start feeding in the resultant soup of bacteria and insect tissue and can be compared with any bacteria-feeding nematode, both morphologically and physiologically (Yeates et al., 1993). One of the major reasons for the increase in the usage of EPNs for biological control of pests in recent years is that they can be mass-produced relatively easily by industrial techniques (Woodring and Kaya, 1988). Those tech*
Corresponding author. Tel.: +44 0 118 378 8484; fax: +44 0 1189352421. E-mail address: [email protected] (E. San-Blas).
niques do not need to be carried out with insects; instead some production protocols have been developed using media which contain a wide range of proteins and carbohydrates to first grow the symbiotic bacteria and then the nematodes. [Some media include potato mash (McCoy and Glaser, 1936), dog food (Hara et al., 1981), pig kidney-fat (Bedding, 1981), poultry offal (Bedding, 1984) or whey (Chavarrı´a-Herna´ndez et al., 2006). Due to the number of production options, it may be supposed that if the right conditions are met, EPNs could use any resource available in nature to complete their life cycles; that means the nematodes can be scavengers and survive using this behaviour and would not need to find and kill a live host. Scavenging can be divided into two categories: obligate and facultative. Obligate scavenging occurs when an organism can only survive by feeding on dead material. Oncholaimus campylocercoides, which lives in hydrothermal vents, can take advantage of the number of organisms dying due to the high concentration of hydrogen sulphide present in that environment and feeds only on dead material
0020-7519/$30.00 Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.06.003
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(Thiermann et al., 1997). The second category, facultative scavenging, occurs where predator organisms take advantage of the opportunity to feed on cadavers that they may encounter. Some benthic nematodes such as Enoploides longispiculosus (Moens et al., 1999, 2005), Ascolaimus, Axonolaimus and Daptonema (Urban-Malinga et al., 2006), have been reported as scavengers. Prein (1988) described the occurrence of a mass aggregation and attraction of Pontonema vulgare after mortality of fishes due to anoxic conditions. Juveniles of Enchelidiidae and Oncholaimidae were classified as predatory-scavenger nematodes by Jensen (1987, 1988). For example, Andocholaimus yhalassophygas (Oncholaimidae) can complement their diet, based on dissolved organic matter, by scavenging on carcasses (Lo´pez et al., 1979). Some nematodes from the family Diplogasteridae can be attracted by insect cadavers, consume them and lay their eggs inside the cadavers (Poinar, 1979). Also, Capinera et al. (1982) observed that EPNs could feed upon pieces of earthworm body tissue and even complete their life cycles, producing small numbers of offspring. Although EPNs have never been reported as scavenger organisms, some evidence of the use of insect cadavers by EPNs has been reported. Some Steinernema species successfully completed life cycles in dead Hylobius pales larvae (Jackson and Moore, 1968), and Pye and Burman (1978) showed the same rate of infection of Steinernema carpocapsae in Hylobius abietis which had been previously killed by chloroform or heat, as that found in live insects. The nematodes could also complete their life cycles and produce offspring in H. abietis cadavers. This work attempts to determine if EPNs can colonise and complete their life cycles within cadavers of Galleria mellonella and if so, observe whether the nematodes are attracted to the cadavers or if they encounter them by chance through random movement. 2. Materials and methods 2.1. Nematode cultures The species Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, Steinernema kraussei, Steinernema affine, Steinernema riobrave, Heterorhabditis indica and Heterorhabditis bacteriophora were cultured in the fourth instar larvae of G. mellonella (Lepidoptera: Pyralidae) (Livefoods Direct Ltd. Sheffield, UK) following the technique of Dutky et al. (1964); the larvae were kept at 10 °C until the day of the experiment (never more than 1 week). The incubation temperature for culturing nematodes was based on whether they originated from tropical or temperate climates. Therefore all Steinernema spp. except S. riobrave were incubated at 20 °C whereas Heterorhabditis spp. and S. riobrave were incubated at 28 °C. The infective juveniles were collected using White traps (White, 1927) and were stored at 10 °C (all Steinernema spp. except S. riobrave) and at 15 °C (Heterorhabditis
spp. and S. riobrave) until the day of the experiments (1 week maximum). 2.2. Capacity and frequency of scavenging One hundred and ten G. mellonella were killed by freezing and separated into groups of 10 at room temperature. The first group was immediately placed in a 9 cm diameter Petri dish with a WhatmanÒ No. 1 filter paper and 1 ml of a suspension containing 1,000–2,000 infective juveniles was added depending of the size of the species: S glaseri, S. kraussei – 1,000 nematodes, S. feltiae, S. affine, S. riobrave – 1,500 nematodes, the rest 2,000 nematodes. This difference in the dosage was used to avoid intra-specific competition and density dependent effects (Kaya and Koppenho¨fer, 1996). The second group was placed in another Petri dish with the same number of nematodes 24 h after death, the third group 48 h after death and so on. The control treatment was 10 live G. mellonella. The Petri dishes were incubated at 20 or 28 °C depending on the nematode species as described above. After 5 days, all the G. mellonella were dissected, subjected to a pepsin digestion (Glazer and Lewis, 2000), and nematodes were counted. The experiments were repeated three times. An extra replication of the experiment was performed in which the G. mellonella were left in the incubator for 14 days and then placed in White traps. Completion of the nematode life cycles was confirmed by direct microscopic observation and by the number of new infective juveniles that emerged after 2 days in the White traps. The analysis of the number of individuals found in the G. mellonella cadavers was done using SASÒ software; the data was transformed (square root) and then a one-way ANOVA analysis was performed. A Tukey grouping test was performed when differences were significant (P < 0.05). 2.3. Attraction of entomopathogenic nematodes to G. mellonella cadavers An olfactometer constructed with polyvinyl chloride pipes (Fig. 1), was filled with sterilised silver sand (Westland horticulture, Dungannon, UK) initially adjusted to 15% w/w water content. Four G. mellonella, previously killed by freezing, were set in one of the extremes of the olfactometer in a chamber made with 4 cm of the anterior part of a 50 ml syringe (diameter = 2.5 cm), and isolated from the main arm with a 10 lm mesh, allowing the diffusion of any gaseous compound but avoiding passage of the nematodes; the control chamber was made in the same way in the opposite arm but left empty. Both extremes and the centre opening were sealed with NescofilmÒ sealing film (Azwell Inc., Osaka, Japan). The olfactometer was then placed in the incubator at 20 or 28 °C (as above) for 48 h to create a gradient of a possible attractant compounds. Between 500 and 1,000 (as above) S. feltiae, S. carpocapsae, S. kraussei, S. glaseri, S. affine, S. riobrave, H. bacteriophora and H. indica were placed in the central part of the
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Fig. 1. Detail of olfactometer constructed from polyvinyl chloride pipes.
olfactometers which were then returned to the incubators for a further 48 h, except for S. glaseri which was returned for only for 8 h because they were very active and escaped from the olfactometer in less than 24 h (data not shown). Each section of the olfactometer was separated, nematodes were recovered from the sand by incubation (Whitehead and Hemming, 1965) and counted under the stereomicroscope. The experiment was repeated 10 times, the percentages of the responding nematodes that moved to one or the other arms were calculated and a two samples paired t-test was performed (a = 0.05). 3. Results 3.1. Capacity and frequency of scavenging The capacity and frequency of scavenging varied between species of the EPNs tested (Table 1). The number of S. feltiae found colonising G. mellonella cadavers in the treatments at 0, 24 and 48 h was the same (102.2 ± 9.16, 61.1 ± 6.71 and 75.05 ± 6.51, respectively) as those in the control treatment (91.95 ± 7.9). At 72 h there was a significant increase of the number of nematodes found in the cadavers (125.9 ± 10.6). The number of nematodes found in G. mellonella killed 96 h before exposure dropped to 39.6 ± 11.9 and at 120 h the number recorded was 0.2 ± 0.15. No nematodes were found in the treatments with larvae that had been dead for longer than 120 h. The numbers of individuals of S. carpocapsae that scavenged were similar to those in the control group (129.6 ± 13.9) except for the last two groups. Similar numbers entered G. mellonella cadavers for up to 144 h after being freeze-killed but numbers of nematodes scavenging cadavers after 168 and 192 h were 43.42 ± 7.06 and 28.75 ± 6.4, respectively. Increasing numbers of S. kraussei scavenged G. mellonella cadavers from 0 h (40.50 ± 3.75) until a maximum at 120 h (89.3 ± 8.39), greater than the numbers in the control group (27.84 ± 3.42). After 120 h, the number declined to 11.03 ± 1.61 at 144 h and 2.39 ± 0.66 per cadaver at 216 h. Steinernema glaseri colonised all treatment groups. At 0 h, the number of nematodes that scavenged was lower (20.75 ± 3.28) than those recorded in the control group (34.79 ± 4.47), but at 48 h, the total number of nematodes in G. mellonella was similar to that of the control group (26.53 ± 3.16). The number of individuals per cadaver
declined in the remaining treatments, to a minimum of 6.45 ± 1.47 at 216 h. The greatest numbers of S. affine (39 ± 5.45 and 41 ± 6.44) entered cadavers at 0 and 24 h, which were significantly greater than the control group (14.55 ± 2.8). In the other treatments, the numbers found in the cadavers were statistically similar until 144 h. The number of S. riobrave scavenging was lower than the number recorded in the control group (113.53 ± 6.35) in all the treatments. The number of nematodes that scavenged varied from 88.6 ± 14.2 at 0 h to 1.4 ± 0.82 at 192 h. The numbers of H. bacteriophora individuals that scavenged showed no differences between the control group (47.7 ± 4.66) and the treatments at 0, 24 and 48 h; the numbers declined to 11.7 ± 6.47 at 72 h and to 13.8 ± 0.36 at 96 h. Of all the nematodes tested, H. indica colonised cadavers the least but nevertheless, they did scavenge. The number of nematodes entering dead G. mellonella varied from 14.89 ± 1.61 at 0 h to 3.35 ± 1.32 at 72 h; these values were lower than the control group (53.8 ± 5.85). The percentage of dead G. mellonella colonised in treatments with different times of exposure after being freezekilled was variable among species (Figs. 2 and 3). Heterorhabditis bacteriophora and H. indica colonised 20% and 50%, respectively, of G. mellonella which had been killed 96 and 72 h before. S. feltiae colonised up to 20% of G. mellonella which had been dead for 120 h; S. affine colonised 30% of cadavers at 168 h and S. riobrave colonised 20% of larvae that had been dead for 192 h. Steinernema carpocapsae and S. kraussei entered larvae that had been dead for even longer, colonising more than 60% of the cadavers at 192 h or 60% at 216 h, respectively. Steinernema glaseri continued to scavenge between 90% and 100% of G. mellonella cadavers which had been killed 240 h before and even longer (data not shown). All nematodes tested completed their life cycles and new infective juveniles were recorded emerging from G. mellonella cadavers (Table 2). The number of individuals emerging was similar to controls in all species but declined in the last treatments where the scavenging was lower. 3.2. Attraction of EPNs to G. mellonella cadavers In terms of attraction of the nematodes to the G. mellonella cadavers, the response varied among species (Fig. 4). The percentage of S. feltiae attracted by dead G. mellonella
nd nd nd
nd
nd nd nd
nd
Fig. 2. Percentage of insects colonised by different entomopathogenic nematodes in freeze-killed Galleria mellonella at different times of exposure from time of death (0 h) (n = 30). n Steinernema carpocapsae; r Steinernema affine; m Steinernema glaseri; d Heterorhabditis bacteriophora.
nd = nematodes not detected in cadavers. Values represent mean ± SEM. Different letters within rows indicate significant differences (P < 0.05).
nd nd 3.35 ± 1.32c 14.89 ± 1.61b 14.45 ± 5.87bc 7.50 ± 2.07c 53.8 ± 5.85a
47.70 ± 4.66a 36.60 ± 3.48a 29.05 ± 3.72a 32.70 ± 5.97a 11.70 ± 6.47b 13.80 ± 9.36b nd
nd
nd
nd 15.90 ± 5.25c 22.60 ± 7.41c 5.30 ± 1.98cd 1.400 ± 0.82d nd 113.53 ± 6.35a 88.6 ± 14.2b
44.79 ± 8.73c 82.6 ± 14.8b
89.2 ± 19.5b
30.0 ± 10.2c
nd nd nd 14.55 ± 2.80b 39.00 ± 5.45a 41.00 ± 6.44a 24.75 ± 4.32ab 27.45 ± 6.67ab 25.63 ± 6.18ab 13.50 ± 4.91b 8.70 ± 3.81b 4.11 ± 2.90c
2.39 ± 0.66d nd 48.29 ± 6.79bc 63.95 ± 9.35b 73.23 ± 8.04b 89.3 ± 8.39a 11.03 ± 1.61d 4.17 ± 0.84d 8.15 ±1.64d 69.13 ±6.04b 27.84 ± 3.42cd 40.50 ± 3.75c
6.45 ± 1.47c 10.05 ± 1.54c 10.55 ± 1.15c 9.05 ± 2.30c 12.80 ± 2.11c 9.30 ± 2.61c 11.65 ± 1.92c 34.79 ± 4.47a 20.75 ± 3.28b 21.00 ± 2.99b 26.53 ± 3.16ab 10.00 ± 1.65c
nd 131.3 ± 11.7a 148.4 ± 21.4a 143.8 ± 13.2a 43.42 ± 7.06b 28.75 ± 6.4b nd 116.4 ± 16.4a 123.7 ±11.6a 95.2 ± 9.42a 129.6 ± 13.09a 98.5 ± 13.0a
240 h 216 h 192 h
nd
168 h
nd
144 h
nd 0.2 ± 0.15d
120 h 96 h 72 h 48 h 24 h 0h Control
91.95 ± 7.90b 102.20 ± 9.16b 61.10 ± 6.71bc 75.05 ± 6.51b 125.9 ± 10.6a 39.6 ± 11.9c
Steinernema feltiae Steinernema carpocapsae Steinernema glaseri Steinernema kraussei Steinernema affine Steinernema riobrave Heterorhabditis bacteriophora Heterorhabditis indica
Table 1 Numbers of individuals in dead Galleria mellonella larvae exposed to nematodes for up to 240 h after death
nd
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nd
88
Fig. 3. Percentage of insects colonised by different entomopathogenic nematodes in freeze-killed Galleria mellonella at different times of exposure from time of death (0 h) (n = 30). h Steinernema feltiae; n Steinernema riobrave; s Steinernema kraussei; } Heterorhabditis indica.
was 22.42 ± 2.33%, which was higher than the percentage that moved towards the control section of the olfactometer (12.76 ± 2.42%). More S. glaseri moved to the section of the olfactometer where the G. mellonella cadavers were placed (29.72 ± 3.47%), compared with the number of nematodes found in the control section (7.52 ± 2.84%), the highest response among the Steinernematid species. Both Heterorhabditids tested showed a positive movement towards G. mellonella cadavers; the percentage of H. indica found in the G. mellonella section of the olfactometer was higher than the portion of nematodes found in the control section (48.68 ± 5.72% and 23.46 ± 3.67%, respectively) and the percentage values for infective juveniles of H. bacteriophora which moved towards the G. mellonella cadavers was 36.31 ± 3.01%, whereas the percentage of these
E. San-Blas, S.R. Gowen / International Journal for Parasitology 38 (2008) 85–91
Fig. 4. Percentage of nematodes (mean ± SEM) which moved in the olfactometer towards the control arm (empty bars) or to Galleria mellonella cadavers arm (solid bars). Steinernema feltiae (Sf), Steinernema carpocapsae (Sc), Steinernema riobrave (Sr), Steinernema kraussei (Sk), Steinernema affine (Sa), Steinernema glaseri (Sg), Heterorhabditis indica (Hi) and Heterorhabditis bacteriophora (Hb). Different letters within species indicate significant differences (P < 0.05).
nematodes which moved to the control section of the olfactometer was 20.85 ± 3.39%. Steinernema affine, S. riobrave, S. carpocapsae and S. kraussei did not show a positive movement to the G. mellonella cadavers.
4. Discussion The EPNs used scavenging as an alternative survival strategy and completed their life cycles in G. mellonella cadavers. In some species, there was no difference between the number of individuals representing a normal infection process (control groups) and the number of nematodes colonising cadavers (scavenging). We suppose that this behaviour could be repeated in nature if similar conditions existed such as the presence of a insect which had been dead for a short time. In fact, the possibilities of finding freeze-killed insects in nature are remote but there are many other viable options occurring. Despite the fact that sometimes EPNs are not able to share the host with another entomopathogenic pathogen organism
89
such as Beauveria bassiana (Barbercheck and Kaya, 1991), larvae infected by granulosis virus can be used by EPNs as suitable hosts (Kaya and Brayton, 1978). There are other sources of dead insects such as adults after mating, drowned larvae and insects partially eaten by other predators, all of which could be used by nematodes as food resources, but further investigation is required. The majority of the species tested showed higher colonisation rates (number of individuals and percentage of insects colonised) in larvae that had been dead for up to 120 h, although some species scavenged larvae that had been dead for longer (S. carpocapsae up to 144 h or S. glaseri up to 240 h). The possibility of successful scavenging could be affected by the time of larval death, because EPNs have to colonise, release the bacteria and avoid competition from other saprophytic organisms. Also, it is important that the cadaver is at a distance that can be reached by the nematodes. Steinernema glaseri, S. kraussei and S. carpocapsae infected more than 90% of the G. mellonella that had been dead for 240, 192 and 168 h, respectively (Figs. 2 and 3) which makes them better scavengers in terms of the time of resource utilisation than H. indica, H. bacteriophora and S. feltiae which colonised up to 90% of the cadavers dead for 24, 48 and 96 h, respectively. According to these results, and despite the fact that all species scavenged, Heterorhabditis spp. seem to be less disposed to perform scavenging than species of Steinernema, but other Heterorhabditid species need to be tested. Probably one of the key factors for the success of this behaviour is that neither nematodes nor bacteria have to fight the insect immune system and the possibilities of survival might therefore be increased. In fact, some insects which have been thought of as non-susceptible to Steinernematid nematodes such as wireworms (Agriotis spp.), could be a source of colonisation if they die, but further investigation is required. The apparent failure of nematodes to colonise older cadavers of G. mellonella may be due to the fact that nematodes had entered but had failed to develop due to unfavourable conditions. It seems that the EPNs can identify the cadaver as a possible host by volatile cues, even though
Table 2 Numbers of Galleria mellonella (out of 10) exposed to nematodes for up to 240 h after death
Steinernema feltiae Steinernema carpocapsae Steinernema glaseri Steinernema kraussei Steinernema affine Steinernema riobrave Heterorhabditis bacteriophora Heterorhabditis indica
Control
0h
24 h
48 h
72 h
96 h
120 h
144 h
168 h
192 h
216 h
240 h
10+++ 10+++ 10++ 10+++ 10+++ 10+++ 10+++ 10+++
10+++ 10+++ 10++ 10+++ 10+++ 10+++ 10+++ 10+++
10+++ 10+++ 10++ 10+++ 10+++ 10+++ 10+++ 9+++
10+++ 10+++ 10++ 10+++ 10+++ 10+++ 10+++ 7+++
9+++ 10+++ 10++ 10+++ 10++ 10+++ 5+++ 5+++
7+++ 10+++ 10++ 10+++ 9++ 10+++ 3+++ 0
4++ 10+++ 10++ 10+++ 5+ 7++ 0 0
0 10++ 9++ 10+++ 3+ 8++ 0 0
0 7++ 10++ 9+++ 3+ 4+ 0 0
0 5++ 10++ 9+++ 0 1+ 0 0
0 0 9++ 6++ 0 0 0 0
0 0 10++ 0 0 0 0 0
Galleria mellonella were incubated for 14 days after nematode exposure and placed in White traps. Infective juvenile nematodes that emerged after 2 days were recorded. Range of total number of infective juveniles recovered per G. mellonella (+++, >5,000; ++, 1,000–4,999; +, <1,000).
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some species did not show a particular attraction to cadavers of G. mellonella in the olfactometers. However, all species tested were capable of colonising cadavers, which could suggest that either the random movements of the nematodes are partially responsible for the first contact, or that the attraction process starts closer to the cadavers (depending on the concentration of the volatile signals or other types of cues) for the species that did not respond in the olfactometers. In any case, further experimentation is needed to elucidate the nature of these compounds. On the other hand, the nematodes which were attracted by the cadavers have been considered, according to the foraging strategy classification, as ‘‘cruisers’’ e.g. S. glaseri (Lewis et al., 1993), H. bacteriophora (Campbell and Gaugler, 1993) and H. indica or ‘‘intermediates’’ e.g. S. feltiae (Lewis, 2002). This positive attraction can be compared with the observation of Lei et al. (1992) who, despite the fact that they did not test any scavenging behaviour in EPNs, reported that Heterorhabditis zealandica was attracted by Delia radicum cadavers rather than encountering them randomly. The use of scavenging as an alternative way for surviving could explain the long-term persistence of EPNs in the soil, although such behaviour has never been included as a variable to be considered in the theory of population dynamics of these organisms. This alternative survival strategy suggests that EPNs should not be considered only as obligate parasites (Hazir et al., 2001; Burnell and Stock, 2000) but as parasite-scavenger nematodes. Acknowledgements The authors thank Barbara Pembroke from the University of Reading for valuable discussion and recommendations for this paper and Mercedes Andrade for statistical advice. References Barbercheck, M.E., Kaya, H.K., 1991. Competitive interactions between entomopathogenic nematodes and Beauveria bassiana (Deuteromycotina: Hyphomycetes) in soilborne larvae of Spodoptera exigua (Lepidoptera: Noctuidae). Environ. Entomol. 20, 707–712. Bedding, R.A., 1981. Low cost in vitro mass production of Neoaplectana and Heterorhabditis species (Nematoda) for field control of insect pests. Nematologica 27, 109–114. Bedding, R.A., 1984. Large scale production, storage, and transport of the insect-parasitic nematodes Neoaplectana spp. and Heterorhabditis spp.. Ann. Appl. Biol. 104, 118–120. Burnell, A.M., Stock, S.P., 2000. Heterorhabditis, Steinernema and their bacterial symbionts-lethal pathogen of insects. Nematology 2, 31–42. Campbell, J.F., Gaugler, R., 1993. Nictation behaviour and its ecological implications in the host search strategies of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae). Behaviour 126, 155–169. Capinera, J.L., Blue, S.L., Wheeler, G.S., 1982. Survival of earthworms exposed to Neoaplectana carpocapsae nematodes. J. Invertebr. Pathol. 39, 419–421.
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