Dispersal patterns and behaviour of the nematode Phasmarhabditis hermaphrodita in mineral soils and organic media

Soil Biology & Biochemistry 41 (2009) 1483–1490

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Dispersal patterns and behaviour of the nematode Phasmarhabditis hermaphrodita in mineral soils and organic media Keith MacMillan a, Solveig Haukeland b, Robbie Rae c, Iain Young d, John Crawford d, Simona Hapca d, Michael Wilson a, * a

Institute of Biological and Environmental Sciences, University of Aberdeen, Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, Scotland, UK BIOFORSK, Norwegian Institute for Agricultural and Environmental Research, Plant Health & Plant Protection Division, Department of Entomology & Nematology, Hogskoleveien 7, 1432 As, Norway c Max Planck Institute for Developmental Biology, Department of Evolutionary Biology, Spemannstrasse 35, Tuebingen, Germany d SIMBIOS, University of Abertay Dundee, Bell Street, Dundee DD11HG, Scotland, UK b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 May 2008 Received in revised form 31 March 2009 Accepted 3 April 2009 Available online 3 May 2009

The commercially available parasitic nematode Phasmarhabditis hermaphrodita is an effective biocontrol agent for slugs and particularly Deroceras reticulatum, a widespread pest species. Use of the nematode is currently limited by cost and it may be that by developing a fuller understanding of the ecology and behaviour of this nematode, more cost effective application strategies can be developed. We investigated the ability of two strains of P. hermaphrodita (one newly isolated and one that had been maintained in vitro for >15 years) to move through mineral soils and organic media. Active dispersal of both strains was found to be greatest in organic media (bark chips and leaf litter, and to a lesser extent peat) and the nematode was capable of growth and reproduction in leaf litter. Conversely, active dispersal was poor in mineral soils. Nematodes moved further in a clay loam compared with a sandy loam, and moved more at a bulk density of 1.0 vs. 1.2 Mg m3. However, P. hermaphrodita was capable of moving greater distances in mineral soils by using the earthworm Lumbricus terrestris as a phoretic host. Our data suggest that P. hermaphrodita is a facultative parasite that is adapted to living in leaf litter and organic material where slugs frequently rest. The implications of these findings for using the nematode as a biological control agent for slugs are discussed. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Phasmarhabditis hermaphrodita Nematode Movement Reproduction Mineral soils Organic soils Phoresis

1. Introduction The rhabditid nematode Phasmarhabditis hermaphrodita (Schneider) is a lethal parasite of several species of slug, particularly Deroceras reticulatum, the most widely distributed pest slug in temperate regions (Wilson et al., 1993). It has been formulated as a biocontrol agent (NemaslugÒ) available from Becker Underwood, Littlehampton, UK (Rae et al., 2007). Once applied to soil P. hermaphrodita dauer larvae seek out and infect slug hosts. Death of susceptible hosts usually takes 4–21 days depending on nematode numbers and temperature (Wilson et al., 1993; Tan and Grewal, 2001). The nematode then feeds and reproduces on the decaying slug and on depletion of this food supply, dauer larvae are formed and these actively disperse into the soil in search of fresh hosts (Wilson et al., 1993; Tan and Grewal, 2001). The nematode is

* Corresponding author. Tel.: þ44 0 1224272845; fax: þ44 0 1224272703. E-mail address: [email protected] (M. Wilson). 0038-0717/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2009.04.007

effective in many crops (Rae et al., 2007) and is relatively safe to nontarget species (Wilson et al.,1994a, 2000; Grewal and Grewal, 2003). The dispersal and host finding behaviours of the similar entomopathogenic nematode (EPN) species have been studied in great detail (Schroeder and Beavers, 1987; Portillo-Aguilar et al., 1999; Wilson et al., 2003). These nematodes show limited movement in soils and ability to move varies among species. Some species e.g. Heterorhabditis bacteriophora move deep within the soil whereas other species e.g. Steinernema carpocapsae tend to remain near the soil surface (Campbell et al., 1996). For movement over longer distances, certain EPNs and free-living nematodes are known to utilise a wide range of organisms such as earthworms, isopods, beetles, ants, mites and flies for phoretic dispersal (Epsky et al., 1986; Shapiro et al., 1993, 1995; Lacey et al., 1995; Nickel and Ayre, 1996; Baur et al., 1998; Eng et al., 2005). Unlike the EPNs and due in part to a previous lack of reliable detection and quantification assays for P. hermaphrodita, very little is known about the field behaviour of this nematode (MacMillan et al., 2006). Studies concerning the ecology of P. hermaphrodita have been relatively few,

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and have generally focussed directly on field efficacy (Wilson et al., 1994b; Glen et al., 2000; Ester et al., 2003). However, the results of such studies have suggested that the ability of the nematode to move freely within soil systems is uncertain and that dispersal is at best limited (Wilson et al., 1999a; Bailey et al., 2003). Very little is known about the effect of environmental factors on behaviour and survival of this species in the field. Methods to isolate strains of P. hermaphrodita focus on isolating the nematode from within infected slugs, rather than from soil or vegetation samples, and, as a consequence of the overall lack of field studies, there is no knowledge of the habitat preferences of this nematode. Some work has been done on predation of P. hermaphrodita by micro-arthropods in the field using molecular detection techniques (Read et al., 2006). However, while this study demonstrated the potential for inclusion of P. hermaphrodita within soil matrix foodwebs, the nematodes studied were not naturally endemic and had been manually applied to the soil in advance. In the current study we attempted to elucidate this aspect of P. hermaphrodita ecology by testing the hypotheses that 1) dispersal of P. hermaphrodita will be influenced by the physical properties (texture, bulk density) of mineral soils, 2) dispersal of P. hermaphrodita will be influenced by habitat quality when moving through organic media and 3) presence of the earthworm Lumbricus terrestris in microcosms would increase dispersal of P. hermaphrodita. These hypotheses were tested using two strains of P. hermaphrodita reared under different conditions. One strain (Nemaslug) was a commercially produced strain that had been reared in vitro for many years, whereas the other (Norway) was a recently isolated strain that was reared in vivo in the host slug D. reticulatum. 2. Materials and methods 2.1. Source of invertebrates Two strains of P. hermaphrodita were used in this study. The first (NemaslugÒ) was supplied by Becker Underwood, Littlehampton UK. Before use the product was mixed with tap water to rehydrate the nematodes. The second strain of P. hermaphrodita used was isolated from an Arion subfuscus slug collected in an abandoned garden site in southern Norway by Dr S. Haukeland and cultured on D. reticulatum prior to use. Estimated quantities of nematodes were prepared for experimental use by pipetting measured volumes of a previously quantified uniformly distributed nematode suspension. Repeated laboratory tests of this standard quantification procedure showed that variation levels were acceptable for these assay types. For example, pipetted subsamples of 10 ml solution would typically result in a mean of 103.2  2.6 (mean  1 SE, n ¼ 12) nematodes. Mean counts varied temporally with concentration and mortality of the nematodes in solution, therefore fresh calculations and estimates were made before each inoculation. D. reticulatum slugs were collected as adults from field and garden sites and stored in moist, dark conditions at approximately 6  C. The earthworm species used in the phoretic transport assay was L. terrestris, supplied by Worms Direct, Ulting, U.K. Prior to use, these were stored at 16  C in polythene boxes half filled with moist sandy loam soil. 2.2. Mineral soils and organic media The mineral soils used in this study were chosen to represent a spectrum of agricultural soils and were a sandy loam from Craibstone (73.9% sand, 20% silt, 6.1% clay; Countesswells Series, pH 6.2 and 5% organic matter), and a clay loam from Cruden Bay

(43.4% sand, 32.3% silt, 24.3% clay, Tipperty series, pH 6.4 and 3% organic matter). Both these topsoils were sourced in the North East of Scotland. Soils were sieved to aggregates of <25 mm and sterilised by heating to 120  C overnight. Soils were sealed in polythene bags and stored dry until use. The organic media used were peat, deciduous leaf litter (predominantly beech Fagus sylvatica) and bark chip. The peat and bark were sourced from a local garden supplier, while the leaf litter was collected from the Aberdeen University Botanic Gardens. Leaf litter consisted of whole dead leaves without rotten leaf mould. These media were chosen to give a range of matrix properties when packed. In terms of void space, the organic media ranged from large pores in the bark chip, to medium in the leaf litter and smaller pores in the peat. The organic media were sterilised prior to use by autoclaving at 120  C. They were then oven dried, sieved to <25 mm, sealed in polythene bags and stored dry until use. Samples of all mineral and organic media were checked using Baermann funnels for 24 h to ensure no naturally present nematodes had survived the sterilisation. 2.3. Dispersal of P. hermaphrodita through mineral soil This experiment had a two-way factorial design investigating the effect of soil type (sandy loam vs. clay loam) and presence of bait (with or without slug hosts) on dispersal of P. hermaphrodita through mineral soils at two consistent and reproducible Dry Bulk Densities (1.0 & 1.2 Mg m3). The dispersal assays through the various prepared organic and mineral media all used plastic tubes (length 17.5 cm, diameter 5 cm). Each tube consisted of seven separate 2.5 cm sections bound together with adhesive tape, and sealed at each end with a pierced plastic cap. The central five sections were filled with media, giving an overall media length of 12.5 cm. This left an empty tube section at either end, separated from the media by 1 mm2 plastic gauze stretched across the crosssection of the tube. One section served to accommodate three D. reticulatum as host bait, while the section at the other end served to prevent the P. hermaphrodita from accumulating and dehydrating on the plastic lid as condensation tended to form on this at the initial stages of the experiment. The mineral soils were uniformly moistened by spraying and folding the required quantity of tap water onto the spread out soil in order to achieve a consistent volumetric water content of 0.22 after packing into the tubes. This water content was chosen to equate to a matric potential within the soil of 30 kPa, which has been shown to facilitate normal movement of nematodes in a soil matrix (Wallace, 1968). Gravimetric water content (GWC) of soil was calculated taking into account the required dry bulk density (DBD) to which the soil was to be packed using the following equation:

m ¼ q=ðgd =gw Þ Where m ¼ gravimetric water content in ml H2O g1 soil, q ¼ desired volumetric water content, gd ¼ dry bulk density of packed soil and gw ¼ density of water. The moistened mineral soils were then uniformly packed into the plastic tubes at DBD 1.0 and 1.2 Mg m3 chosen to reflect typical values found in agricultural soil systems. Six tubes were packed for each of the two soil types at each DBD value. An estimated 1963 P. hermaphrodita individuals were applied in suspension to the soil surface on the first section of each tube (application rate of 100 cm2). Three of each six tubes contained three D. reticulatum in the lowest section (þ bait treatment), while the other three acted as no-bait controls ( bait treatment). The tubes were incubated at 16  C for 14 days in a vertical position.

K. MacMillan et al. / Soil Biology & Biochemistry 41 (2009) 1483–1490

The tubes were then carefully separated and the nematodes in each section were recovered by Baermann extraction and counted. Three replicate tubes were used per treatment and the whole experimental procedure was repeated using the Norwegian isolate of P. hermaphrodita. 2.4. Dispersal of P. hermaphrodita through organic media This experiment had a two-way factorial design investigating the effect of media type (leaf litter, bark chips or peat) and presence of bait (with or without slug hosts), on nematode dispersal through organic media in two consistent orientations (vertical & horizontal). Due to the heterogeneous physical structure and hydrophobic properties of the organic media when dry, it was not possible to regulate the volumetric water content as closely as was the case in the mineral soil assays, therefore the organic media was moistened by uniformly spraying the spread out media with tap water and sealing in polythene bags overnight to allow the water to soak into the media. After packing, and prior to nematode application, the tubes of media were allowed to drain naturally to remove any excess water remaining in the media. By following this procedure, we achieved a moist organic media matrix while avoiding possible nematode transport via free water movement through the influence of gravity. The moistened organic media were packed into the tubes at DBD values which conferred sufficient and consistent structure without undue compaction of the media. The values chosen were 0.14 Mg m3 for the peat and bark chip, and 0.04 Mg m3 for the leaf litter. The lower target value for leaf litter reflects the much lower density of this material. Six tubes were packed for each of the three organic media types at each orientation (horizontal and vertical). An estimated 1963 P. hermaphrodita individuals were applied in suspension to the media surface on the first section of each tube (application rate of 100 cm2). Three of each six tubes contained three D. reticulatum in the lowest section (þ bait treatment), while the other three acted as no-bait controls ( bait treatment). The tubes were incubated at 16  C for 14 days in either vertical or horizontal positions according to treatment. Nematodes were recovered and counted as previously described. The experiment used three replicate tubes per treatment and the whole experimental procedure was repeated using the Norwegian isolate of P. hermaphrodita. 2.5. Phoretic dispersal of P. hermaphrodita by L. terrestris The experiment comprised four treatments (upward and downward movement of nematodes, either in the presence or absence of L. terrestris) and each treatment was replicated three times. Twelve tubes (length 24 cm, diameter 3.9 cm) were each filled with 236 g of moist sandy loam soil. Each tube was constructed of six separate 4 cm sections of plastic pipe bound together with adhesive tape. This resulted in a wet bulk density of soil within each tube of 0.82 Mg M3, which represented a loose and uncompacted soil structure in order to facilitate the movement of L. terrestris within the relatively confined volume. Ten thousand P. hermaphrodita, of Nemaslug strain only, were added to the top of six tubes and the bottom of the remaining six. In three of the top treated tubes and in three of the bottom treated, two L. terrestris (mean weight ¼ 4.38  0.15 g) were placed in the section containing the nematodes. The remaining six tubes had no earthworms added and acted as no-carrier controls for both orientation treatments respectively. All tubes were sealed with aluminium foil and incubated at 16  C for 14 days. Each experiment used three replicate tubes per treatment and the whole experiment was replicated five times (n ¼ 15 in total).

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2.6. Survival of nematodes within media We estimated the effects of each different assay soil or media type on the survival of both strains of P. hermaphrodita by calculating mean numbers recovered from each media type, regardless of DBD or orientation, and comparing these with the original application amount of 1963 individuals per tube. 2.7. Data analysis Recovered counts of nematodes from each section were collated and converted into the value of nematode mass centre which gave one quantitative measurement to the nematode movement within each tube. The nematode mass centre corresponded to the average distance within the tube from the initial application point at which the recovered nematodes were found, and therefore gave a measurement of nematode movement which was comparable between assays. This value was calculated based on percentages of nematodes in each section and the distance from the centre of that section to the release point, and was calculated for the dispersal and phoretic assays using the formula:

Masscentre ¼ p1*d1 þ p2*d2 þ p3*d3 þ p4*d4 þ p5*d5 Where pi ¼ proportion of nematodes in section i from the total recovered (i ¼ 1.5), and di ¼ distance from the application point to the centre of section i The resulting data were checked for normality of distribution and, in the case of the dispersal data, were transformed by adding 1 to remove zero values in the cases of no movement from the initial section. The log10 was then taken of those values in order to carry out parametric statistical analyses. To analyse the results of the mineral soil movement assays, a two-way ANOVA was used to evaluate the main and interaction effects of the fixed factors; soil type and bait presence for each DBD and for each of the two strains of P. hermaphrodita used. In the organic media assays, a two-way ANOVA was used to evaluate the main and interaction effects of the fixed factors; media type and bait presence for each orientation and for each of the two strains of P. hermaphrodita used. Tukey’s post-hoc comparison tests were carried out to determine significant differences among means. Survival of nematodes within soils and organic media was quantified by collating and calculating the mean total number of nematodes recovered from each tube after 14 days, irrespective of DBD or orientation. Means were log10 transformed and one-way ANOVAs used to evaluate the effect of soil or media type on nematode recovery for each strain of P. hermaphrodita and in the presence and absence of bait. Tukey’s post-hoc comparison tests were carried out to determine significant differences among means. In the phoretic dispersal assays, successive transformations of the data failed to satisfy the assumptions of normality and equal variances therefore the untransformed data were subjected to the non-parametric Kruskal–Wallis test. This test was used to evaluate the effect of worm presence on nematode movement in either assay direction, and to compare the effect of assay direction on nematode movement in both the presence and absence of worms. All statistical analyses were carried out using MINITABÒ Release 15. 3. Results 3.1. Dispersal of P. hermaphrodita through mineral soil Treatment effects are shown in Fig. 1 (Nemaslug strain) and 2 (Norway strain) and demonstrate that more nematode movement was evident through the clay loam soil. Movement of the commercial NemaslugÒ strain (Fig. 1) and Norway isolate strain,

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K. MacMillan et al. / Soil Biology & Biochemistry 41 (2009) 1483–1490 Table 1 ANOVA results for the effects of soil type (sandy loam and clay loam) and presence of bait and their interactions on the movement of both nematode strains in mineral soil at two dry bulk densities (DBDs) for 14 days. ns ¼ non significant, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical data refer to Figs. 1 and 2. Nemaslug

Soil Bait Soil * Bait

Fig. 1. Mean movement of NemaslugÒ P. hermaphrodita after 14 days in vertical tubes filled with two types of mineral soil, packed at Dry Bulk Densities 1.0 Mg m3 and 1.2 Mg m3, and in the presence or absence of 3 D. reticulatum host slugs as bait. Untransformed mass centre data presented. Error bars represent one SEM, n ¼ 3.

(Fig. 2) was reduced in the sandy loam soil compared to the clay loam. However, the Norway isolate strain showed more movement overall in comparison to the NemaslugÒ strain. Also the Norway strain moved more readily through both types of soil, and the results suggest this strain also fared better with the increased DBD. Figs. 1 and 2 suggest an increase of movement in response to bait slug presence in the low DBD assays, however Table 1 reveals that the presence of D. reticulatum as bait in the tubes did not have a significant effect on the pattern of nematode movement through mineral soil (P > 0.05). The comparison tests confirm that both strains showed significantly more movement through clay loam soil than sandy loam, but that this difference remained significant at the higher DBD only for the Norway strain (P < 0.05).

3.2. Dispersal of P. hermaphrodita through organic media Media type significantly affected nematode movement (P < 0.001) (Table 2) for both strains and at both orientations of assay. In each case, the results from the peat assay showed the most reduced movement compared to the other media types (Figs. 3 and 4). In the Norwegian strain, movement was found to be significantly different in all three media types (P < 0.001). Movement was lowest through peat and highest through leaf litter while, in the NemaslugÒ strain, movement through leaf litter and bark chip was

DF 1 1 1

Norway Isolate

DBD 1.0 Mg m3

DBD 1.2 Mg m3

DBD 1.0 Mg m3

DBD 1.2 Mg m3

F 44.73 1.14 1.14

F 0.56 0.16 0.55

F 29.02 0.05 2.49

F 43.31 0.23 0.04

P *** ns ns

P ns ns ns

P *** ns ns

P *** ns ns

not found to be significantly different (P > 0.05). With the exception of the peat horizontal assays, nematodes in all treatments were found to have reached the tube section furthest from application. Table 2 shows a significant (P < 0.05) effect of bait presence in the NemaslugÒ vertical assays as a whole: dispersal was increased by slug presence. There was no significant interaction found between bait and media type, and bait was not found to have a significant (P < 0.05) effect on nematode movement in the horizontal NemaslugÒ assays, nor in the Norwegian strain assays at any orientation. 3.3. Survival of P. hermaphrodita within media The numbers of nematodes recovered in the leaf litter assays, as detailed in Fig. 5, reveal that in some cases, many more nematodes were recovered than were initially applied. The post-hoc comparison of the means for total number of nematodes per tube revealed that there were significant differences in recovery rates of nematodes from each different soil and media type (P < 0.05). Significantly higher numbers of nematodes of the NemaslugÒ strain were recovered from clay loam soil than from sandy loam, while for the Norway isolate strain the opposite was found. In both strains the recovery of live nematodes was highest from the leaf litter organic medium. In the NemaslugÒ strain the rate was 31–42% of the original application amount, but in the Norway isolate assays, nematodes had reproduced resulting in a >10-fold recovery. Fig. 5 does not indicate any influence of the presence/absence of bait. 3.4. Phoretic dispersal of P. hermaphrodita by L. terrestris L. terrestris had a significant effect on the distribution of P. hermaphrodita recovered from the sections of all assay tubes (Table 3); in both downward and upward assays there was significantly (P < 0.001) more nematode movement in the tubes containing L. terrestris. Virtually no movement was found in those assay tubes containing nematodes alone. The orientation of the assay had an influence on movement patterns, with more overall movement seen in downward assays compared to upward. However, this effect

Table 2 ANOVA results for the effects of organic media type (peat, leaf litter and bark chip) and presence of bait and their interactions on the movement of both nematode strains in organic media stored at two orientations for 14 days. ns ¼ non significant, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical data refer to Figs. 3 and 4. Nemaslug

Fig. 2. Mean movement of Norwegian isolate P. hermaphrodita after 14 days in vertical tubes filled with two types of mineral soil, packed at Dry Bulk Densities 1.0 Mg m3 and 1.2 Mg m3, and in the presence or absence of 3 D. reticulatum host slugs as bait. Untransformed mass centre data presented. Error bars represent one SEM, n ¼ 3.

Media Bait Media * Bait

DF 2 1 2

Norway Isolate

Orientation Vertical

Orientation Horizontal

Orientation Vertical

Orientation Horizontal

F 94.26 8.21 0.78

F 59.55 1.11 0.73

F 25.30 1.15 0.30

F 48.88 0.46 1.45

P *** * ns

P *** ns ns

P *** ns ns

P *** ns ns

K. MacMillan et al. / Soil Biology & Biochemistry 41 (2009) 1483–1490

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applications of nematodes at lower overall numbers at the site of slug feeding or shelter, which is a reasonable argument given that P. hermaphrodita, particularly in the commercial form grown in liquid fermentation vessels, seems unlikely to move far from any point of application in soil. The increased dispersal shown by both strains through the clay soil compared to the sandy soil most likely reflects the nematodes taking advantage of the differing physical soil structure in the clay loam. This soil showed a tendency to form larger and more stable aggregates and thus contained larger macropores within the soil matrix. Potential routes of dispersal through this soil could therefore be assumed to be less tortuous and would subsequently require less time and energy to navigate than those in the sandy soil. Fig. 3. Mean movement of NemaslugÒ P. hermaphrodita after 14 days in vertical and horizontal tubes filled with three types of organic media. Untransformed mass centre data presented. Error bars represent one SEM, n ¼ 3.

was only found to be significant in the presence of L. terrestris (Table 4). 4. Discussion 4.1. Dispersal of P. hermaphrodita in mineral soils The poor ability of P. hermaphrodita to actively disperse within soil systems seems clear when the results of the mineral soil dispersal assays are considered. Movement of the nematode, particularly the NemaslugÒ strain, was severely restricted within the sandy loam soil, even at the relatively low DBD of 1.0 Mg m3. The higher compaction value of DBD 1.2 Mg m3 in both soils resulted in severe restriction of dispersal in both strains. The soils utilised in the study reflect the types of agricultural soil in which P. hermaphrodita is commonly used as a slug biocontrol agent. Additionally, while bulk density values within agricultural soil systems are inherently heterogeneous, normal values for near surface layers are in the range 1.1–1.4 Mg m3, depending on management practice (Unger, 1996; Moret and Arru´e, 2007; Verbist et al., 2007). The bulk density values chosen for the assays were also therefore representative of the range in which P. hermaphrodita is commonly in use. This apparent lack of active dispersal within mineral soils has implications for a biocontrol agent which has been assumed to have the ability to actively seek out host prey within soil systems, and lends extra weight behind the arguments for strategic application, as proposed by Hass et al. (1999), Wilson et al. (1999a), and Grewal et al. (2001). These authors suggest that a more economical application strategy would be to concentrate

Fig. 4. Mean movement of Norway isolate P. hermaphrodita after 14 days in vertical and horizontal tubes filled with three types of organic media. Untransformed mass centre data presented. Error bars represent one SEM, n ¼ 3.

4.2. Dispersal of P. hermaphrodita in organic media The effect of void space on nematode movement rates can also be seen clearly in the organic media assay results. Movement in both strains of P. hermaphrodita was found to be most restricted in the peat assays where the pore sizes within the matrix, while larger than those within the mineral soils, were smaller than those of the leaf litter and bark chip assays. The different patterns of movement measured between the two strains in the bark chip assays could be the result of differing qualities between the strains, however, it seems anomalous that NemaslugÒ would out perform the Norwegian strain in this one assay alone. Another interesting finding from our results was the fact that regardless of the presence/absence of host bait a few centimetres away through a seemingly easily negotiated organic media, the nematodes were found to be freely reproducing on the moist leaf litter itself. Non-parasitic reproduction has been reported for P. hermaphrodita by Tan and Grewal (2001), who found that P. hermaphrodita could reproduce on slug and slug faeces homogenates. This incidence of reproduction was not observed in any other soil or organic media and it is the first time that P. hermaphrodita has been reported to be facultatively reproducing on leaf litter alone, which supports the suggestion by Rae et al. (2006) that this species is capable of growth and reproduction on a wide range of decaying plant and animal material. The occurrence of facultative saprobic reproduction, coupled with the increased dispersal observed in the organic media, lead us to conclude that P. hermaphrodita is perhaps better adapted to living in this medium than in mineral soils, which may explain why efforts to isolate this species of nematode in soils have been unsuccessful. 4.3. Comparison of commercial and Norwegian strains In terms of recorded movement, and indeed in the incidence of reproduction described above, there were differences in performance measured between the two strains of P. hermaphrodita used in the assays. In general, it was observed that the Norway isolate strain exhibited greater dispersal within the various soil and organic matrices used, as seen most clearly in the ability of this strain to penetrate further and in greater numbers through mineral soils. This strain also displayed greater fecundity, as is evident by the far higher rate of reproduction observed in the leaf litter assays compared to that of the commercial NemaslugÒ strain. We do not believe, however, that these differences reflect a real behavioural or evolutionary difference between the strains, but rather a physiological one. The differences in performance are most likely an artefact of the different methods used to prepare the original nematode suspensions. The NemaslugÒ strain was produced in vitro and prepared directly from the partially dehydrated commercial product and therefore was utilised according to manufacturer’s instructions. The Norway isolate was prepared by culturing in vivo

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Fig. 5. Mean recovery of two strains of P. hermaphrodita after 14 days in vertical tubes of mineral soil at two dry bulk densities and in vertical and horizontal tubes of organic media. Mean Log10 transformed counts of nematodes recovered from entire tube presented. Within each category, columns which do not share the same letter differ significantly (P < 0.05). Error bars represent one SEM. n ¼ 6.

in the preferred host slug D. reticulatum. Having observed the increased virility apparent in the dauer larvae of NemaslugÒ after they have been through one ‘‘natural’’ life cycle, the authors believe that the differences in performance between strains merely reflect the difference between in vivo and in vitro production.

4.4. Host finding One surprising result was the fact that these assays did not reveal any real response to the presence of bait, even within the assays where movement was apparently not restricted. The one significant response to bait was in the vertical organic media NemaslugÒ assays, which included some highly variable results and was not reproduced by the Norwegian strain that showed greater dispersal in the same assay. Previous studies of the response of P. hermaphrodita to the presence of host slug cues such as mucus and faeces have reported positive results (Rae et al., 2006; Hapca et al., 2007). However, these studies were carried out on agar plates, and it is apparent from the results in this study that the inclusion of host slugs has largely failed to produce a similar response in this case. The diffusion of chemical cues in soils or organic media may be much more limited by the physical and chemical properties of the surrounding matrix and only where a combination of proximity of cue source and ease of movement coincided, such as in the final Table 3 Mean counts of nematodes recovered from each mineral soil filled tube section after 14 days at two assay directions (downwards and upwards) and in presence or absence of L. terrestris as a potential phoretic agent. In each direction tube section 1 is always the location of initial nematode application. Untransformed data presented (mean  1 SE). Tube section

1 2 3 4 5 6

L .terrestris Present

L. terrestris Absent

Downward assay

Upward assay

Downward assay

Upward assay

346.53  56.80  21.13  8.60  6.60  5.07 

444.73  7.33  2.13  0.73  0.87  0.20 

967.93 0.60 0.00 0.07 0.00 0.00

538.28 0.07 0.07 0.00 0.00 0.00

82.99 11.39 4.25 1.27 1.32 0.86

61.27 2.03 0.54 0.28 0.29 0.14

     

204.76 0.24 0.00 0.07 0.00 0.00

     

section of the organic media assays, might a significantly positive movement towards the host bait be observed. 4.5. Phoretic dispersal of P. hermaphrodita by L. terrestris Although both upward and downward dispersal of P. hermaphrodita was increased by the presence of L. terrestris, the downward dispersal was enhanced to the greatest degree. These directional differences in dispersal patterns could be explained by the movement behaviour of the earthworms themselves. L. terrestris may preferentially occupy the lower parts of a profile of soil, and may tend to avoid the surface. As a consequence, the earthworms in the upward assays may have remained at or near the bottom where they were initially introduced resulting in reduced potential for phoretic transport throughout the soil in this treatment. However, the worms were confined in a relatively small volume of soil, unexposed to light and not subject to any significant moisture gradient. We therefore assume that movement of the earthworms occurred randomly throughout the tube. Such earthworm enhanced dispersal has been reported for nematodes before. For example Shapiro et al. (1993) found dispersal of S. carpocapsae was significantly increased by the presence of L. terrestris and Aporrectodea trapezoids (Duges). However, in a further study using a larger range of entomopathogenic nematodes, Shapiro et al. (1995) also reported directional and species-specific differences in dispersal. The authors found upward dispersal of S. carpocapsae and Steinernema feltiae was increased by L. terrestris while that of Steinernema glaseri was not. They reported that downward dispersal of S. carpocapsae and S. feltiae was not enhanced. The fact

Table 4 Kruskal–Wallis test results for the effects of assay direction (upwards and downwards) and presence of L. terrestris and their interactions on the movement of nematodes in mineral soil for 14 days. ns ¼ non significant, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical data refer to Table 3. 114.50 0.07 0.07 0.00 0.00 0.00

L. terrestris presence in downward assay only L. terrestris presence in upward assay only Orientation in presence of L. terrestris Orientation in absence of L. terrestris

DF

H

P

1 1 1 1

22.19 23.69 19.88 2.60

*** *** *** ns

K. MacMillan et al. / Soil Biology & Biochemistry 41 (2009) 1483–1490

that differences have now been found in either direction, and that there appears to be a species-specific element in evidence, suggests that these dispersal patterns cannot be attributed to earthworm behaviour alone. The authors found nematodes present in the interior and exterior of the worms and believed that direct contact was responsible for the increased dispersal but also speculated that the nematodes may be passed through the worm digestive system and still remain viable upon release (Shapiro et al., 1995). The increased dispersal of P. hermaphrodita by L. terrestris could be attributed to three factors. First, burrows created by earthworms provide tunnels for the nematodes to utilise. Second, nematodes are ingested by the earthworms and subsequently defecated into the surrounding soil either further up or down the soil column. Third, the nematode attaches to the earthworm surface or is carried on small soil particles adhering to the earthworm surface. Shapiro et al. (1995) showed that earthworm burrows alone had no effect on the dispersal of Steinernematid nematodes and Campos-Herrera et al. (2006) reported that passing through the gut of Eisenia fetida had adversely affected the mobility and virulence of nematodes. This could perhaps account for reduced overall numbers recorded in our assay tubes containing L. terrestris. Any impairment to nematode motility would lower the efficiency of the Baermann funnelling technique and lead to reduced recovery. In relation to other studies on nematode dispersal and earthworm associations, the most plausible scenario seems to be attachment to the surface of the earthworm. A behavioural element on the part of the nematode seems to be evident in this process with clear speciesspecific differences being measured both in terms of incidence of phoretic transport and subsequent direction of enhanced dispersal. For nematodes, the main benefits of utilising invertebrates for transportation are dispersal from unsuitable habitats or gaining access to beneficial habitats, protection from predation and increasing chances of parasitism. Other species of both free-living and entomopathogenic nematodes have been shown to utilise a wide range of invertebrates for transport. For example, Eng et al. (2005) found Heterorhabditis marelatus (Liu & Berry) was dispersed by the isopod Porcellio scaber Latreille. Lacey et al. (1995) recorded S. glaseri was dispersed by the Japanese beetle (Popillia japonica Newman). Nematodes such as Caenorhabditis dolichura (Schneider) and steinernematid infective juveniles can attach to ants and are actively dispersed (Nickel and Ayre, 1996; Baur et al., 1998), and nematodes have also been reported to use mites as phoretic hosts (Epsky et al., 1986). In the case of P. hermaphrodita, the use of earthworms as a carrier may allow access to areas where potential slug hosts may be present. The vertical distribution of slugs in soil can vary greatly between species. In a study by South (1964), D. reticulatum were found in the top 2–3 cm of the soil surface yet Arion fasciatus were located from depths between 2.5 and 7.5 cm while eggs of Testacella scutulum have been found at depths of 36 cm (Barnes and Stokes, 1951). Boettgerilla pallens can even be found at depths approaching 60 cm (Barker and Efford, 2004) demonstrating the extreme variation in soil penetration depths between slug species. In this study, we recovered P. hermaphrodita to depths of 24 cm from our downward soil columns and it is highly plausible that P. hermaphrodita can use passing earthworms in order to gain access to lower soil layers were slugs may be present, more explicitly during drier periods. 4.6. Conclusions We have attempted to elucidate certain under-researched aspects of the ecology of P. hermaphrodita and have shown not only that phoretic transport of this nematode is possible but is also likely to be necessary in order for the species to achieve significant

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mobility to function as an effective parasite of host slugs in mineral soil systems. We have also demonstrated that the species may indeed be better adapted to living in loose organic matter as it is capable of greater dispersal in this environment and we have observed that, even in the presence of host slugs, P. hermaphrodita is capable of survival and saprobic reproduction on decaying leaf tissue. In terms of using the nematode as a biological control agent in crops, it may be beneficial to cover the crops with an organic mulch to enable P. hermaphrodita to move and locate potential slug hosts. An example crop could be strawberry, which is frequently attacked by slugs (Cross et al., 2001) and is often grown using straw mulch. The presence of straw may aid movement of the nematodes, and such straw mulches have been shown in the past not to impede the access of entomopathogenic nematodes to host arenas for control of black vine weevil (Wilson et al., 1999b).

Acknowledgements We are grateful to BBSRC and to Kintail Land Research Foundation for funding research on P. hermaphrodita at Aberdeen University and to Becker Underwood for supplying nematodes. We thank Tarin Toledo-Aceves for help with statistical analyses and Ryan Aichison and Irene Rasmussen for technical help with organic media movement experiments. Thanks to the Research Council of Norway for funding research on slugs and P. hermaphrodita at Bioforsk, (the Norwegian Institute for Agricultural and Environmental Research).

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