European Journal of Soil Science, June 1998,49, 237–241

Nematode (Caenorhabditis elegans) movement in sand asaffected by particle size, moisture and the presence ofbacteria (Escherichia coli)

I . M . Y O U N G a, B . S . G R I F F I T H Sa, W. M . R O B E R T S O Nb & J . W. M CN I C O L c

aSoil–Plant Dynamics Unit,bNematology Unit &cBiomathematics and Statistics Scotland, Scottish Crop Research Institute, DundeeDD2 5DA, UK

Summary

The movement of bacterial-feeding nematodes (Caenorhabditis elegans) through sand was investigatedusing a range of sand sizes, equilibrated at a range of matric potentials, in the presence or absence ofan attractant source (Escherichia coli)at the distal end of a column. In the presence ofE. coli there wassignificantly greater movement of the nematode population towards theE. coli population, and the extentof the movement depended on the matric potential of the sand. Over time, an increasing proportion ofthe C. eleganspopulation responded to the presence of theE. coli. The processes controlling theseeffects are discussed with respect to taxis and kinesis mechanisms of the nematode population, and withregard to the diffusive characteristics of the physical structure of the sand.

Introduction

Nematodes are an important component of the soil biomass,being the most abundant metazoan. They are a major trophiccomponent, feeding on bacteria, fungi, protozoa, nematodesand plants, and important in nutrient cycling (Yeates, 1979;Griffiths, 1994). They have unique foraging strategies adaptedto cope with an impressive array of environmental conditions(Dusenbery, 1987) and can travel large (m) distances throughthe soil profile (Dusenbery, 1987), distributing otherwise rela-tively immobile populations of bacteria and viruses.

Wallace (1958) was one of the first scientists to presentevidence of the relations between nematode motility, soil poresizes and soil water. We now know that nematodes can adoptforaging strategies in response to chemical stimuli (Robinson,1995; Younget al., 1996), and are attracted to roots via solubleand gaseous attractants produced by the root or the attendantrhizosphere microorganisms (Dusenbery, 1987; Grewal &Wright, 1992), a process commonly known as chemotaxis.There is a reasonable body of literature identifying soil moistureparameters having effects on nematode reproduction and move-ment (Evans, 1969; Griffithset al., 1995).

Yet the effect of soil structure does not stop at definingavailable pore pathways for nematode movement. Soil structureacts to slow diffusion by placing solid particles in the diffusivepathway, or forcing the diffusing molecules to move throughwater-films or water-filled pores. In the case of a gaseous

Correspondence: I. M. Young. E-mail: imy@scri.sari.ac.ukReceived 21 August 1997; accepted 13 October 1997

© 1998 Blackwell Science Ltd 237

attractant such as CO2, a gas implicated in the attraction ofnematodes to roots (Dusenbery, 1987), a characteristic diffusionrate would be approximately one centimetre per day in water,compared with one metre per day in air. In soil there is acomplex interaction of attractants (gaseous and water soluble),soil structure and nematode movement, acting over a widerange of scales. This relation has been investigated recently(Andersonet al., 1997a,b). However, this work concentratedon the analysis of single nematodes in two dimensions, wherethe effect of moisture regimes was not investigated. The aimof the work presented in this paper is to examine the influenceof three-dimensional structured systems, equilibrated at a rangeof matric potentials, on nematode motility, in the presence andabsence of an attractant.

Materials and methods

Coarse (1.00–1.25 mm) or fine (0.125–0.70 mm) acid-washedsand fractions were packed into plastic tubes (20 mm diameterand 40 mm long), and saturated with sterile deionized waterfor 24 h. The sand columns were then equilibrated to a rangeof matric potentials (– 0.5, – 2 or – 4 kPa) on a tension table.A preliminary experiment, relating nematode population growthto matric potential, was carried out to identify an optimal soilmatric potential range. Five replicate sand columns, for eachsand size, were used in all experiments. The bacteriumEscher-ichia coli was maintained on nutrient agar (NA, Oxoid) andprepared for experiments by growing a batch culture in Luriabroth (Oxoid) at 37°C, a loopful of which was spread uniformly

238 I. M. Younget al.

across an NA plate and incubated for a further 24 h at20°C. The bacterial-feeding nematodesCaenorhabditis elegans(Rhabditida) were cultured monoxenically with the bacteriumE. coli on 3 NA at 20°C, and prepared for experiments bywashing in sterile 5% NaCl. A plug of agar, 20 mm diameterand 3 mm deep, was cut from anE. coli plate and placed atthe bottom of the sand column, and the base of the columnwas sealed. TheE. coli growing on the agar was intended toact as an attractant source for the nematodes (Andersonet al., 1997a). Columns withoutE. coli received a plug ofuninoculated NA. Twenty-four hours after the agar plug hadbeen added to the columns a population ofµ 60 juveniles (J2)C. elegansnematodes were placed at the opposite end of thesand column. The sand columns were then sealed to preventevaporation, stored horizontally, and incubated at 20°C. Col-umns were sampled 18 h after nematode addition for both sandsizes, and 18, 24, 39 and 48 h after nematode addition forthe fine sand. Nematode numbers were sampled from foursuccessive 10-mm sections (A, B, C, D) from each tube.Nematodes had been placed in Section A and the plug of agar(with or without E. coli) was placed at the base of Section D.Nematodes in each section were separated from the sand grainsby repeated shaking and decanting with distilled water, andcounted using a microscope. As percentage distribution is usedany effects of population mortality or recruitment are ignored.

Analysis of variance was used to assess treatment effectson the nematode percentages in each section of the tube.Residual plots indicated that a transformation of the percentagedata was not necessary.

Diffusivities of the replicated fine sand cores, equilibratedat – 0.5 and – 4 kPa, were measured using the method of Ballet al. (1981), and standard errors calculated.

Results

The results of nematode movement in relation to sand size,matric potential and sampling time are shown in Figures 1–3.The general observation is that the presence ofE. coli actedas an effective source of attractant toC. elegansnematodes.A significantly (P , 0.001) larger percentage of nematodeswas found in Section D (i.e. next to the agar plug), in thepresence ofE. coli, than in the control, withoutE. coli, forboth sand size ranges and at all times (Figures 1–3). In thefine sand treatment, the percentage of nematodes remaining inSection A, where they were added, increased as matric potentialdecreased (P , 0.01), as shown in Figure 1. This trend wasmore pronounced in the presence ofE. coli. In Section C, inthe presence ofE. coli in both sand sizes, nematodes weresignificantly fewer (P , 0.01) than in the control. For fine andcoarse sand, equilibrated at – 0.5 kPa, 12 and 1% of the totalnematode population were found in Section D in the absenceof E. coli, respectively (Figures 1 and 2). No significant effectsof sand size on the distribution of nematodes were observed.

Figure 3 shows that, over time, there was a significant

© 1998 Blackwell Science Ltd,European Journal of Soil Science, 49, 237–241

decrease (P , 0.01) in the percentage ofC. elegansin SectionA in the presence ofE. coli. This was mirrored by a significantincrease (P , 0.001) in Section D. In the control, noC. elegansnematodes were found, at any time, in Section D. In SectionB, significantly fewer (P , 0.01) nematodes were found in thepresence ofE. coli than in the control.

The relative diffusivities, with associated standard errors, ofthe fine sand columns at – 4 and – 0.5 kPa were 0.200 (6 0.004)and 0.119 (6 0.010), respectively.

Discussion

The chemotactic response used by nematodes, in the presenceof an attractant, is the main mechanism by which they acquireinformation about the surrounding environment (Dusenbery,1992). We have shown that the physical habitat, and relatedmoisture conditions, play an important role in the rate at whichnematodes sense and respond to sources of attractants, in thiscaseE. coli. The optimum environment for nematodes foragingin soil relies on a compromise between the hydraulic connectiv-ity of pores and the diffusive characteristics of the porepathways. Whilst all nematodes are aquatic, requiring water-films to survive and move in, all gaseous attractants diffusevery slowly in water. The speed of diffusion will increase onlywhere significant mixing or flow occurs within the water-films.In this investigation particle size had no significant effect onnematode movement, presumably because within the matricranges examined there were adequate hydraulic connectionswithin the sand columns to permit, but not alter, nematodemovement within respective sand sizes.

In this investigation the relative diffusivities in fine sandreinforce the extent to which structure and moisture interactto control the diffusion of attractants: the relative diffusivityat – 0.5 kPa is 45% slower than that at – 4 kPa. This is reflectedin the final proportion of nematodes observed in the sectionnearest the attractant source: at – 0.5 kPaµ 20% comparedwith µ 10% at – 4 kPa. The diffusion measurement probablyreveals little about the hydraulic connectivity, which is partlydefined by thin water-films around sand particles, and notnecessarily by water-filled pore throats, which can have a largeimpact on diffusion rates. Thus the similarity between thedecreases in diffusivities and the increases in nematode percent-ages is coincidental.

The presence of an attractant has been shown significantlyto alter the foraging strategy of the nematodes from a relativelyrandom to a more directed movement, towards the attractantsource (Andersonet al., 1997a). In the present work, for bothsand sizes, it is clear that there were connected water pathwaysfrom Sections A to D at all matric potentials examined. Inboth controls, at the highest matric potential, a significantpercentage of nematodes reached Section D: over 10% for finesand andµ 1% for coarse sand. This may be due to simplerandom foraging strategies which, as described in our earlierwork (Young et al., 1996), allow nematodes to forage some

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Figure 1 The movement ofC. elegansnematodes through fine sand, in the presence and absence ofE. coli, at different soil matric potentialsover 18 h. Standard errors of the means are indicated. Nematodes were placed in Section A at the start of the experiment.

Figure 2 The movement ofC. elegansnematodes through coarse sand, in the presence and absence ofE. coli, at different soil matric potentialsover 18 h. Standard errors of the means are indicated. Nematodes were placed in Section A at the start of the experiment.

© 1998 Blackwell Science Ltd,European Journal of Soil Science, 49, 237–241

240 I. M. Younget al.

Figure 3 The movement ofC. elegansnematodes through fine sand, in the presence or absence ofE. coli, over time, equilibrated at – 2 kPa.Standard errors of the means are indicated. Nematodes were placed in Section A at the start of the experiment.

distance from the main population in search of food not solelyin response to a chemical gradient. An alternative reason maybe linked to the method of adding the nematodes to the sandcores. After matric equilibration, a small aliquot of watercontaining the nematodes was added to the sand cores. Duringthe experimental period there would have been some redistribu-tion of moisture, and thus nematodes, within the sand profile.However, no nematodes were found in Section D in the controltime-series, at any sampling time, suggesting that redistributionof nematodes by our method of application was not significant.The time-course of nematode movement (Figure 3) shows asignificant decrease in Section A and a corresponding increasein Section D in the presence of an attractant.

An interesting observation from all experiments is therelatively large number of nematodes that remain in the sectionfurthest from the attractant population (Section A). In the finesand structures between 50 and 70% remain, and there is adirect negative relation between nematode numbers and matricpotential, presumably due to the decreasing probability thatthe nematodes will find a water connection to Section D. Evenafter 48 h more than 30% of nematodes remain in SectionA (Figure 3). The question is why? There are four mainpossibilities.1 First, the nematodes remaining in Section A may havebeen damaged during transfer into the sand cores and thus beless motile. We examined a subset of nematodes in Section Afor viability, and all were active. Therefore, we rule out damageto the nematodes as being significant.

© 1998 Blackwell Science Ltd,European Journal of Soil Science, 49, 237–241

2 Secondly, prior to transfer into the sand cores the nematodeswere grown on a population ofE. coli. Young et al. (1996)note thatC. elegansnematodes, placed on a Petri dish in thepresence ofE. coli, without first being surface-sterilized, donot initially sense the presence ofE. coli attractant, as theirreceptors are awash with the attractant and so are insensitiveto chemical gradients. A similar response is observed usinginsects which, prior to exposure to a possible attractant in alaboratory, are generally preconditioned to ensure that theinsects have no ‘memory’ of the attractant and do not becomedesensitized (Szentesi & Jermy, 1990). Although the nematodesused in this investigation were surface-washed, this would nothave removedE. coli from their gut. TheC. elegansmaysense theseE. coli when they defaecate in the experimentalsystem, some distance from theE. coli acting as the experi-mental source of the attractant.3 Thirdly, whilst a chemotaxis response is a directed orienta-tion towards an attractant source, an undirected response mayalso occur (Croll, 1970). This is termed ‘kinesis’ and canmanifest itself in nematodes by either an increase in theirspeed or frequency of activity (orthokinesis), or an increase intheir rate of turning (klinokinesis). Although Andersonet al.(1997a) did observe differences inC. elegansturning anglesin the presence of an attractant, this was within an overallchemotaxis response. So, some of the nematodes remaining inSection A may be responding to the attractant, but throughkinesis and not taxis, and thus exhibit no directional movementaway from Section A.

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4 Finally, a variable response by a microbial community, inthis case nematodes, to a stimulus may be an effective strategy,in that the presence of an attractant does not necessarily meanthe presence of good quality substrate. Thus, such a responseto a potential food source may in the long run increase theoptions available to the microbial community and increasetheir chances of survival. Additionally, the nematodes may befeeding at different rates (perhaps differing between develop-mental stage), and over time more nematodes will respond tothe food source, which to some extent is what is observed inFigure 3.

What is clear is that the structure and ambient moistureconditions play a vital role in determining the rate of responseof a biological population to food sources in the soil profile,from the point of view of both the movement of nematodes,at the microbial scale, and diffusion of attractants, at themolecular scale. The physical structure acts as habitat, transportconduit and water reservoir for all organisms in soil, and thusplays a pivotal role in the biological functioning of the soilecosystem.

Acknowledgements

We thank Dr Nick Birch and Mr Daan Kiezebrink for usefuldiscussions on this research, and the Scottish AgriculturalCollege, Bush Estate, for determining the diffusivities. Thisresearch was funded by the Scottish Office Agriculture, Envir-onment and Fisheries Department.

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