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Movement of the parasitic nematode Phasmarhabditis hermaphrodita in the presence of mucus from the host slug Deroceras reticulatum
Biological Control 41 (2007) 223–229 www.elsevier.com/locate/ybcon
Movement of the parasitic nematode Phasmarhabditis hermaphrodita in the presence of mucus from the host slug Deroceras reticulatum Simona Hapca a, John Crawford a, Robert Rae b, Michael Wilson b, Iain Young b
a,*
a SIMBIOS, University of Abertay Dundee, Bell Street, Dundee DD1 1HG, UK School of Biological Sciences, University of Aberdeen, St. Machar Drive, Aberdeen, AB24 3UU, UK
Received 19 September 2006; accepted 12 January 2007 Available online 20 January 2007
Abstract Phasmarhabditis hermaphrodita is a parasitic nematode capable of killing several species of slugs including Deroceras reticulatum, the most widespread slug pest in the world. This nematode can control slug infestations in a wide range of crops such as wheat, lettuce and strawberries. Optimization of this biocontrol agent depends on a proper understanding of the interaction between the host and parasite. In this paper, we investigate the response of P. hermaphrodita to the presence of slug mucus on plates of agar. We define an attraction index and find that the nematodes are significantly attracted by filter paper impregnated with slug mucus compared to paper impregnated with water. Second, nematode trails were recorded on a homogeneous layer of technical agar, with or without the presence of the slug mucus. Mucus was applied in two treatments comprising localization on a piece of filter paper and a uniform distribution across the plate. The different mucus treatments induced significantly different effects on the speed of nematode movement and the distribution of the turning angles, as well as the fractal dimension of nematode foraging trail. These results are consistent with the hypothesis that the nematodes exhibit both a chemotactic and chemokinetic response to a signal emanating from slug mucus. 2007 Elsevier Inc. All rights reserved. Keywords: Phasmarhabditis hermaphrodita; Deroceras reticulatum; Chemotaxis; Chemokinesis; Attraction index; Nematode movement; Turning angle distribution; Fractal dimension
1. Introduction Phasmarhabditis hermaphrodita (Schneider) is a parasite that directly infects slug pests of agricultural and horticultural crops. These nematodes form non-feeding infective juveniles (dauer larvae) that are associated with pathogenic bacteria, such as Moraxella osloensis Bøvre Henriksen, which they carry within their intestines. The larvae move through the soil to find a host. Once they have located a host they penetrate through the dorsal integumental pouch and canal where they release the symbiotic bacterium, which is presumed to kill the host. The nematodes develop and reproduce within the host’s shell cavity eventually producing dauer larvae once the food source is depleted. These *
Corresponding author. Fax: +44 (0) 1382 308117. E-mail address: [email protected] (I. Young).
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dauer larvae finally leave the cadaver and search for new hosts (Wilson et al., 1993; Tan and Grewel, 2001). The nematode’s capacity to effectively control slug damage relies, therefore, on its ability to locate the slug within a defined time period. Like most organisms, nematodes have the capacity to locate resources using physical or chemical gradients released from a source (Dusenbery, 1992). In the case of chemical stimuli, the reaction might be a change in the directed locomotion of the nematode toward or away from the chemical stimulus (chemotaxis), or a change in speed or frequency of locomotion, and/or the frequency and magnitude of turning angle, that is not oriented with respect to the stimulus (chemokinesis) (Dusenbery, 1980). At present little is known about the sensory ecology of P. hermaphrodita despite the fact that a better understanding of how the nematode reacts to slug hosts may enable us to more effectively utilize it as a natural biocontrol agent.
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As a first step to achieve such understanding, we tested the hypothesis that P. hermaphrodita responds to chemical cues elicited by the host slug Deroceras reticulatum. In addition, we characterized the nature of this response.
nematodes zone1
2. Materials and methods
zone2
T
C
2.1. Organism used Dauer larvae of P. hermaphrodita were obtained from Becker Underwood (Littlehampton, U.K.) in a formulated product (Nemaslug) consisting of partially dehydrated dauer larvae and clay particles. Four hours prior to use, the product was mixed with cold water and nematodes were separated from the formulated material using a Baermann funnel. Adults D. reticulatum (about 2 cm long) were collected from grassland sites in Scotland, around the University of Abertay Dundee, and were kept in the dark at 4 C and fed with slices of carrots until used. In all treatments, the experimental arena comprised a 4.5-cm diameter Petri dish containing 1.2% technical agar (Oxoid Ltd., Hampshire, U.K.). 2.2. Is P. hermaphrodita attracted by D. reticulatum mucus? 2.2.1. Experimental treatments Each plate was divided into quadrants and a 1.5 cm diameter centered circle was drawn on the back of the plate. Two pieces of filter paper (7 · 7 mm) were placed 30 mm apart at opposite quadrants of the plate, on the surface close to the boundary. One was impregnated with water to serve as a control and the other with slug mucus. Mucus was swabbed from single slugs that were held carefully with a pair of tweezers while the filter paper was gently rubbed over the slug until the whole surface of filter paper was fully impregnated with mucus. Control plates consisted of filter paper impregnated with water versus an untreated square of filter paper. All plates were sealed with parafilm and kept at 19 C. To determine how the degree of nematode attraction to slug mucus depended on the duration that the plates were exposed to slime prior to nematode inoculation, two different time intervals were chosen. Based on preliminary investigations, the nematodes were added to the plates approximately 4 and 12 h after the filter paper was applied. A 5-ll drop of water containing 30 ± 5 active nematodes was pipetted into the center of each replicate plate. Initially, the surface tension of the water retained the nematodes within the center of the dish. Once the water drop evaporated (typically after 5 min), the nematodes were free to disperse in the Petri dish. To measure how fast the nematodes responded to the presence of the slug mucus, the nematodes were left to move around the Petri dish for either 1 or 2 h. Consequently, five experimental treatments were investigated: (i) nematodes on the plates for 1 h with untreated filter paper applied 4 h before the nematodes were added (‘‘control’’); (ii) nematodes on the test plates
test
control
Fig. 1. Schematic diagram of the experimental design used to compute the attraction index.
for 1 h with treated filter paper applied 4 h before the nematodes were added (‘‘4 h/1 h’’); (iii) as (ii) with the treated filter paper applied 12 h prior to nematodes inoculation (‘‘12 h/1 h’’); (iv) nematode on the test plates for 2 h with treated filter paper applied 4 h before the nematodes were added (‘‘4 h/2 h’’); (v) as (iv) with the treated filter paper applied 12 h prior to nematode inoculation (‘‘12 h/2 h’’). Twenty replicates were prepared for each treatment. The sampling periods and test assays were based on many preliminary studies of slug mucus and nematode interaction. At the end of this time period the nematodes present on the control and test filter paper strips denoted in Fig. 1 by zone (1) were counted under a microscope after immersing and fragmenting the filter paper in water. The number of nematode presented in the neighborhood of these filter papers (the corresponding quadrant outside the centered circle), zone (2) was also monitored. 2.2.2. Attraction index An ‘‘attraction index’’ was calculated as follows: AI ¼
aðN T ;1 N C;1 Þ þ bðN T ;2 N C;2 Þ aðN T ;1 þ N C;1 Þ þ bðN T ;2 þ N C;2 Þ
with
a ¼ 2 and b ¼ 1; where AI represents the attraction index; NT,1 the number of nematode in and underneath the test filter paper (zone 1), and NT,2 the number of nematodes in its neighborhood (zone 2); NC,1 the number of nematode in and underneath the control filter paper (zone 1), and NC,2 the number of nematodes in its neighborhood (zone 2) (Fig. 1). This attraction index is a generalization of a previously used index (Thurston et al., 1994). The coefficients a and b serve as differential weights for the different sections of the plate based on how far the nematode is from the food source. If b = 0, only the nematodes situated on the filter paper are counted and the index is exactly that used by Thurston et al. (1994). The choice of a = 2 and b = 1 above means that nematodes in both zones are considered and zone 1 carries twice the weight of zone 2 in the attraction index. The generalized formula above is consistent with the definition of an attraction index for which the value ranges from 1.0 to 1.0. Values close to 0 indicate that the test
S. Hapca et al. / Biological Control 41 (2007) 223–229
compound had no effect on nematode attraction, positive values indicate attraction to, and negative values indicate repellence by the test compound. Statistical significance among the different AI values corresponding to the five experimental treatments was determined by one-way ANOVA and post hoc multiple comparisons were made by using Turkey’s test (Montgomery, 1991; Zar, 1999). 2.3. Does D. reticultum mucus produce a chemotactic or chemokinetic responses in P. hermaphrodita? 2.3.1. Experimental treatments Two experimental treatments were used to examine the activity of nematodes in the presence of slug mucus. In the first, two pieces of filter paper of 7 · 7 mm were placed 30 mm apart at opposite quadrants of the plate, on the surface close to the boundary (mucus on filter paper treatment). One was impregnated with water to serve as a control and the other one with slug mucus. In the second treatment, slug mucus was applied uniformly to treatment plates by gently swabbing the slug across the Petri dish (mucus uniformly applied treatment); untreated agar plates were included as controls. All plates were sealed with parafilm and kept at 19 C for 4 h. Typically three nematodes were picked from water, using a mounted eyelash and placed in the center of the plate, about 5 mm apart, to avoid any direct interaction. Their movement was monitored for 15 min. Preliminary investigations on untreated agar plates revealed that P. hermaphrodita remain active, having a constant speed, for approximately 1 h. In total, the movement of 25 nematodes was recorded for each treatment. 2.3.2. Image capture and analysis An Axio MRc Zeiss camera mounted on a Leica MZ16 microscope connected to a computer, monitored nematode movement with an image field size of 30 · 40 mm. Movement was recorded at 8-s intervals over a period of 15 min. Images were processed using Axio Vision 3.1 software, then analyzed by an image analysis software incorporating a tracking algorithm (Image-Pro Plus v.5, Media Cybernetics Inc., U.S.A.), in order to obtain the x and y coordinates at each time point. MatLab v.6.1 was used to plot the final digitized nematode trail and to compute the distance traveled between frames, the value of the turning angle and the fractal dimension of nematode trail. The speed of the nematode movement was determined by calculating the average distance between successive positions, traveled in 8-s time interval. An 8-s interval was chosen on the basis that in this time the nematodes travel an average distance of 1 mm. This is approximately equal to the body length. Shorter timescales would have introduced artifactual error due to changes in the body shape during movement, and longer timescale would have reduced the amount of data available for analysis. The statistical significance of differences in nematode speed among treatments was determined by t-tests (Zar, 1999).
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The turning angle distribution was generated by computing the angle between successive line segments on the trail. The angular range chosen was from p to p radians, and all angles were computed with respect to the previous angle. The negative values correspond to the right turns and the positive values to the left turns. v2 goodness of fit test (Zar, 1999) was used to determine significant differences in turning angle distributions corresponding to different treatments. We also studied the fractal dimension of the nematode trails to give a quantitative measure that characterize the degree to which the nematode trails fill the 2-D space of the agar plates (Anderson et al., 1997). One of the most common methods for calculating the fractal dimension of an object is the box counting method (Falconer, 1990). This method consists in counting the number of boxes of length lb needed to completely cover the nematode trail. If we denote this number by Nb (lb), then the fractal dimension Db is given by: Db ¼ lim
ld !0
log10 ðN b ðlb ÞÞ . log10 ðlb Þ
In practice Db
corresponds to the slope of the plot log10 (Nb (lb)) versus log10 (lb). The lowest dimension we would expect is 1 (the dimension of a straight line) which indicates a linear trail. Where Db is close to 2 (the dimension of brownian motion), the trail will be more rugged and tortuous, and given sufficient time the nematode will visit all points on the agar plate. To compare the fractal dimension, the t-test for means (Zar, 1999) was used. 3. Results 3.1. General observations In the absence of any attractant, the nematode trails displayed frequently two types of movement (Fig. 2): looping (spiraling around an imaginary point) and reversal (rapidly alternating forward and backward movements) (Anderson et al., 1997). Second, there was significant individual variation in the population, from nematodes that move slowly and in reversals, to nematodes that move fast making large and smooth loops. This variation in nematode population was also observed even in the presence of the attractant, either in the form of the localized slug mucus, or uniformly distributed on the whole surface. Such variation in populations resulted in relatively large statistical variances. 3.2. Attraction response The attraction index (AI) values corresponding to the five experimental treatments (Table 1) were significantly different (ANOVA, F = 174.4; df = 4, 95; P < 0.001). Phasmarhabditis hermaphrodita was significantly attracted by D. reticulatum mucus (q = 9.73, df = 95; k = 5; P < 0.001) in all experimental treatments compared to the control. Further, when the nematode were allowed to move on the Petri dish for 1 h, the attraction response was significantly greater in the plates where the nematodes were
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Fig. 2. Digitized nematode movement. (a) Control (technical agar with no potential attractant); (b) Treatment 1 (technical agar with slug mucus localized on a strip of filter paper on the left); (c) Treatment 2 (technical agar with slug mucus uniformly applied on the surface of the plate); (+ denotes the starting position).
Table 1 Values of the attraction index (AI) ranging between 1.0 and 1.0 (negative values for repellence and positive values for attraction) together with the standard error for n = 20 replicates Treatmentsa
AI ± SE
Control 4 h/1 h 12 h/1 h 4 h/2 h 12 h/2 h
0.02 ± 0.09 0.43 ± 0.12 0.24 ± 0.09 0.73 ± 0.09 0.67 ± 0.10
a Numbers relate to the time the amount of time the treatment was applied prior to the amount of time the nematode was exposed to the treatment. See text for full details.
added 4 h after the slime paper than in the cases where the nematodes were added after 12 h (q = 8.28, df = 95; k = 5; P < 0.001). But, when the nematode were allowed to move on the Petri dish for 2 h, the difference between the attraction index corresponding to treatments (4 h/2 h) and (12 h/ 2 h) was not significant (q = 2.63, df = 95, k = 5; P = 0.37). On the other hand, comparing treatments ‘‘(4 h/ 1 h) + (12 h/1 h)’’ with ‘‘(4 h/2 h) + (12 h,2 h)’’ a significantly greater response (q = 11.02; df = 4, 95; P < 0.001) was obtained when the nematodes were left to explore the Petri dish for 2 h with respect to 1 h. In all tests, 1 h after applying the nematodes to the agar surface, at least 50% of the nematodes had moved out of the central circle, and at least 20% were in the two response quadrants.
the filter paper near the boundary of the plate, the distribution became more peaked around value 0 of the turning angle, corresponding to a predominantly straight-line movement with an increase in the proportion of forward directed movements. A nematode in the presence of localized mucus typically moved in the same direction more often following the attractant gradient emanating from the slug mucus. When the mucus was uniformly applied on the whole surface, the distribution of the turning angle became more uniform with a reduction in the frequency of values around 0 and an increase in frequencies around p and p, compared to the control. This distribution is associated with a random behavior; with forward directed movement being replaced by an increase in reversal movements. The nematode trails in plates with uniformly distributed mucus had a significantly greater fractal dimension FD = 1.252 (t = 2.22, df = 48, P < 0.05), compared to the control FD = 1.192, as a consequence of the more random foraging behavior, which leads to a dense and rugged trail. When the mucus was localized near the boundary, on the piece of filter paper, the movement becomes smoother, with a consequent fractal dimension FD = 1.129 significantly smaller (t = 2.24, df = 48, P < 0.05) compared to the control.
4. Discussion 3.3. Chemotactic and chemokinesis response Typical examples of nematode trails produced are presented in Fig. 2, for the control plate as well as for the test plates with slime on it either localized on the piece of filter paper, or uniformly disposed on the surface of the agar. Nematode movement was significantly faster in plates in which slime was impregnated to filter paper, speed average = 0.113 mm/s (t = 2.10; df = 48; P < 0.05), and significantly slower in the plates with a uniform layer of slime, speed average = 0.042 mm/s (t = 5.42; df = 48; P < 0.05), compared to the control speed average = 0.084 mm/s. Furthermore, the turning angle distributions corresponding to both treatments were significantly different (P < 0.001) compared to the control. When mucus was localized on
Our study has clearly shown that the presence of the D. reticulatum slug mucus significantly affected the behavior of P. hermaphrodita on agar plates. Related investigations have been reported in the past on entomopathogenic nematodes (Lewis et al., 1992, 1993), and similar effects have been recorded for Caenorhabditis elegans (Maupas migration) to several species of bacteria (Rodger et al., 2004; Grewal and Wright, 1992; Andrew and Nicholas, 1976; Ward, 1973). Recently, Rae et al., 2006 have studied P. hermaphrodita attraction to different slug cues and reported an aggregation of nematodes after 24 h, under a point source of slug cues situated about 30 mm away from the inoculation point. Our contribution consists of a rigorous quantitative analysis of nematode behavior in the presence of the
S. Hapca et al. / Biological Control 41 (2007) 223–229
slug mucus subject to different spatial and temporal treatments. Moreover, quantitative trail analysis has never previously been applied to slug parasitic nematodes. A first and very important finding is that the degree of impact on their behavior depends on the duration that the plates were exposed to mucus, prior to the inoculation with nematodes. Longer exposure decreases the effect. Preliminary investigations have also shown that the attraction response was smaller for a shorter 1 h exposure compared to 4 h. Whilst, it is beyond the scope of this work to identify the signaling compounds emanating from the slug mucus, our results suggest that the compound is in an aqueous phase. A typical signaling compound in air (e.g., CO2) will cross a 10 cm Petri dish in a timescale of the order of 1 min (dependent, only weakly, on the square root of the molecular weight) (Lyman et al., 1982). In the aqueous phase, it will cross the same dish in a timescale of the order of 10 h. Our results show that gradients are becoming established and declining over the latter timescales, consistent with aqueous-phase diffusion. All of the reported results, are consistent with a reaction-diffusion scenario, in which, the nematodes are dispersing on the agar plate along the chemical gradient diffusing from the slug slime (Fig. 4). In our assay, 4 h was the most efficient interval between slime application and nematode inoculation (AI = 0.43). From the preliminary results, 1 h seems to be an insufficient timescale for the mucus to diffuse through the agar in order to be sensed by the nematode chemoreceptor. Thus, initially the nematode can go in both directions with equal probability. However, after 12 h, the attraction index was significantly lower (AI = 0.24, P < 0.001) with respect to 4 h, suggesting that the gradient of chemical signal from the mucus had declined to be close to zero. When the nematodes were left to move on the plate for 2 h, the responses corresponding to 4 and 12 h mucus exposure converge (AI = 0.73 and AI = 0.67, respectively, P = 0.37). In both situations, the high value of the attraction index signifies that 2 h was sufficient time for most of the nematodes to migrate in the test quadrant.
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Fig. 4. Comparison of the gradient profile of a diffusible attractant a, released by localized slug mucus, corresponding to three interval time 1, 4 and 12 time units. The formula of the attractant a, is given by the solution of normal diffusion equation with an instantaneous point source (Crank, 1975).
In previous publications, this index is termed the ‘‘chemotactic index’’ (Thurston et al., 1994), whilst others term it the ‘‘attraction index’’ (Andrew and Nicholas, 1976; Grewal and Wright, 1992; Rodger et al., 2003). It is important to note that it is possible to have AI close to 1 even although there is no attraction or chemotaxis simply because chemokinetic response exists. In order to differentiate between these responses, a more detailed analysis of the nematode trail is required. The fact that the attraction index depends significantly (P < 0.001) on the diffusion of the attractant suggests that it is unlikely that the nematodes encountered the cue by chance, implying that there is chemotaxis behavior. Therefore, to further interpret the attraction data recorded in the first experiments we did, an individual based analysis of nematode behavior in the presence of slug mucus. Firstly, a large and significant increase in speed was found when the nematode was placed at some distance from the attraction source. The sugges-
0.3 control
frequency
0.25
treatment 1
0.2
treatment 2
0.15 0.1 0.05 0 -3
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
3
radians Fig. 3. Comparison of the effect of the three experimental treatments (including the control) on the turning angle distribution of nematode trail.
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tion is that orthokinesis, that is a kinesis in which forward speed is changed (Dusenbery, 1980) is operating at this scale. Second, when the nematodes were placed in direct contact with the mucus their speed significantly decreased compared to that of a control. Croll (1975) reported that C. elegans move much more slowly in a lawn of bacteria than on bare agar. Our analyzes of turning angle distributions clearly show that nematodes follow a straighter foraging trail in response to a signal emanating from a localized food source (Fig. 3). Conversely, when the signal is coming from everywhere, as the mucus is uniformly applied on the Petri dish, then nematode movement becomes less directed; consequently, the turning angle distribution tends to be more uniform in the interval [p, p]. This statement is consistent with the results obtained from the fractal dimension analysis where a higher value corresponding to a better exploitation of each potential food source area, was found in this case. When the nematode is challenged by the presence of a localized potential food source a lower fractal dimension, corresponding to a relatively linear trail, occurs. This is in agreement with the result obtained for C. elegans trails when searching for bacterial food (Anderson et al., 1997). Foraging strategies of P. hermaphrodita, and of nematodes, in general, are complex (Young et al., 1996) and depend on the diffusion of the attractant released by the food source (Grewal and Wright, 1992). There is evidence that random movement persists in the presence of directed movement (Robinson and Heald, 1993; Schroeder and Beavers, 1987; Anderson et al., 1997). It has also been observed that, in order to maintain a certain direction over large distances, some aquatic organism adopt a constant rotation around an axis parallel to their direction of locomotion (Jennings, 1904; Schaeffer, 1928; Foster and Smyth, 1980). In our study, the results suggest that where no food is sensed, P. hermaphrodita adopts a random exploration with looping and reversals that optimizes their opportunities of locating substrate. This behavior changes in the presence of an attractant gradient to a faster (chemokinesis) and more direct movement toward the stimulus (chemotaxis). Therefore, after a period of time there is a high probability that they will find the potential host. The next question is, what happens when this event takes place? When the nematodes were directly challenged by the slug mucus, spatially distributed in the domain of action, their speed was slower (chemokinesis) and the trail was denser and more localized, compared with nematodes in the absence of an attractant. Even in the presence of an attraction agent, we still observe looping and reversals. This suggests that such movement is an additional requirement for exploring in a complex spatial environment where nematodes may otherwise become trapped in pores where a signal exists but movement in the direction of the gradient is obstructed. The analysis we have developed presents a detailed picture of the exploration and exploitation
responses of nematodes to a range of environmental perturbations. Acknowledgments We thank K. Macmillan (University of Aberdeen) for his help and advice with respect to the experimental techniques and H. Staines (Scottish Informatics Mathematics Biology and Statistics Centre) for statistical advice. We are also grateful to the referees for their constructive comments. This research was funded by the Biotechnology and Biological Sciences Research Council (Grant No.: BBSB01065). References Anderson, A.R.A., Sleeman, B.A., Young, I.M., Griffiths, B.S., 1997. Nematode movement along a chemical gradient in a structurally heterogeneous environment. 1. Experiment. Fundam. Appl. Nematol. 20, 165–172. Andrew, P.A., Nicholas, W.L., 1976. Effect of bacteria on dispersal of Caenorhabditis elegans (Rhabditidae). Nematologica 22, 451–461. Crank, J., 1975. The mathematics of diffusion, second ed. Oxford University Press, Oxford, UK. Croll, N.A., 1975. Componenets and patterns in the behaviour of nematode Caenorhabditis elegans. J. Zool. 176, 159–176. Dusenbery, D.B., 1980. Behaviour of free-living nematodes. In: Zuckerman, B.M. (Ed.), . Nematodes as Biological Models, vol. 1. Academic Press, New York, pp. 127–158. Dusenbery, D.B., 1992. Sensory Ecology. W.H. Freeman, New York. Falconer, K., 1990. Fractal Geometry, Mathematical Foundations and Applications. John Wiley and Sons, Chichester, UK. Foster, K.W., Smyth, R.D., 1980. Light antennas in phototactic algae. Microbiol. Rev. 44, 572–630. Grewal, P.S., Wright, D.J., 1992. Migration of Caenorhabditis elegans (Nematoda: Rhabditidae) larvae towards bacteria and the nature of bacteria stimulus. Fund. Appl. Nematol. 15, 159–166. Jennings, H.S., 1904. Contributions to the study of the behaviour of lower organisms. Carnegie Institute, Washington DC. Lewis, E.E., Gaugler, R., Harrison, R., 1992. Entomopathogenic nematode host finding: response to host cues by cruise and ambush foragers. Parasitology 105, 309–315. Lewis, E.E., Gaugler, R., Harrison, R., 1993. Response of cruiser and ambusher entomopathogenic nematodes (Steinernematidae) to host volatile cues. Can. J. Zool. 71, 761–769. Lyman, W.J., Reehl, W.J., Rosenblatt, D.H., 1982. Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds. McGraw-Hill, New York. Montgomery, D.G., 1991. Design and Analysis of Experiments. John Wiley and Sons, New York. Rae, R.G., Roberson, J.F., Wilson, M.J., 2006. The chemotactic response of Phasmarhabditis hermaphrodia (Nematoda: Rhabditida) to cues of Deroceras reticulatum (Mollusca: Gastropoda). Nematology 8, 197–200. Robinson, A.F., Heald, C.M., 1993. Movement of Rotylenchulus reniformis through sand and agar in response to temperature, and some observations on vertical descent. Nematologica 39, 92–103. Rodger, S., Bengough, A.G., Griffiths, B.S., Stubbs, V., Young, I.M., 2003. Does the presence of detached root border cells of Zea mays alter the activity of pathogenic nematode Meloidogyne incognita. Am. Phytopathol. Soc. 93, 1111–1114. Rodger, S., Griffiths, B.S., McNicol, J.W., Wheatley, R.W., Young, I.M., 2004. The impact of bacterial diet on the migration and the navigation of Caenorhabditis elegans. Microbiol Ecol. 48, 358–365.
S. Hapca et al. / Biological Control 41 (2007) 223–229 Schaeffer, A.A., 1928. Spiral movement in man. J. Morphol. 45, 293–398. Schroeder, W.J., Beavers, J.B., 1987. Movement of the entomogeneous nematodes of the families Heterorhabditidae and Steinernematidae in soil. J. Nematol. 19, 257–259. Tan, L., Grewel, P.S., 2001. Infection behaviour of the rhabdtid nematode Phasmarhabditis hermaphrodita to the grey garden slug Deroceras reticulatum. J. Parasitol. 87, 1349–1354. Thurston, G.S., Yule, W.N., Dunphy, G.B., 1994. Explanations for low susceptibility of Leptinotarsa decemlineata to Steinernema carpocapsae. Biol. Control 4, 53–58.
229
Ward, S., 1973. Chemotaxis by the nematode Caernorhabditis elegans: identification of attractants and analysis of the response by the use of mutants. Proc. Natl. Acad. Sci. USA 70, 817–821. Wilson, M.J., Glen, D.M., George, S.K., 1993. The Rhabditid nematode Phasmarhabditis hermaphrodita as a potential biological control agent for slugs. Biocontrol Sci. Technol. 3, 513–521. Young, I.M., Griffiths, B.S., Robertson, W.M., 1996. Continuous foraging by bacterial feeding nematodes. Nematologica 42, 378–382. Zar, J.H., 1999. Biostatistical Analysis, Fourth ed. Prentice Hall, Upper Saddle River, NJ.
Movement of the parasitic nematode Phasmarhabditis hermaphrodita in the presence of mucus from the host slug Deroceras reticulatum Simona Hapca a, John Crawford a, Robert Rae b, Michael Wilson b, Iain Young b
a,*
a SIMBIOS, University of Abertay Dundee, Bell Street, Dundee DD1 1HG, UK School of Biological Sciences, University of Aberdeen, St. Machar Drive, Aberdeen, AB24 3UU, UK
Received 19 September 2006; accepted 12 January 2007 Available online 20 January 2007
Abstract Phasmarhabditis hermaphrodita is a parasitic nematode capable of killing several species of slugs including Deroceras reticulatum, the most widespread slug pest in the world. This nematode can control slug infestations in a wide range of crops such as wheat, lettuce and strawberries. Optimization of this biocontrol agent depends on a proper understanding of the interaction between the host and parasite. In this paper, we investigate the response of P. hermaphrodita to the presence of slug mucus on plates of agar. We define an attraction index and find that the nematodes are significantly attracted by filter paper impregnated with slug mucus compared to paper impregnated with water. Second, nematode trails were recorded on a homogeneous layer of technical agar, with or without the presence of the slug mucus. Mucus was applied in two treatments comprising localization on a piece of filter paper and a uniform distribution across the plate. The different mucus treatments induced significantly different effects on the speed of nematode movement and the distribution of the turning angles, as well as the fractal dimension of nematode foraging trail. These results are consistent with the hypothesis that the nematodes exhibit both a chemotactic and chemokinetic response to a signal emanating from slug mucus. 2007 Elsevier Inc. All rights reserved. Keywords: Phasmarhabditis hermaphrodita; Deroceras reticulatum; Chemotaxis; Chemokinesis; Attraction index; Nematode movement; Turning angle distribution; Fractal dimension
1. Introduction Phasmarhabditis hermaphrodita (Schneider) is a parasite that directly infects slug pests of agricultural and horticultural crops. These nematodes form non-feeding infective juveniles (dauer larvae) that are associated with pathogenic bacteria, such as Moraxella osloensis Bøvre Henriksen, which they carry within their intestines. The larvae move through the soil to find a host. Once they have located a host they penetrate through the dorsal integumental pouch and canal where they release the symbiotic bacterium, which is presumed to kill the host. The nematodes develop and reproduce within the host’s shell cavity eventually producing dauer larvae once the food source is depleted. These *
Corresponding author. Fax: +44 (0) 1382 308117. E-mail address: [email protected] (I. Young).
1049-9644/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2007.01.005
dauer larvae finally leave the cadaver and search for new hosts (Wilson et al., 1993; Tan and Grewel, 2001). The nematode’s capacity to effectively control slug damage relies, therefore, on its ability to locate the slug within a defined time period. Like most organisms, nematodes have the capacity to locate resources using physical or chemical gradients released from a source (Dusenbery, 1992). In the case of chemical stimuli, the reaction might be a change in the directed locomotion of the nematode toward or away from the chemical stimulus (chemotaxis), or a change in speed or frequency of locomotion, and/or the frequency and magnitude of turning angle, that is not oriented with respect to the stimulus (chemokinesis) (Dusenbery, 1980). At present little is known about the sensory ecology of P. hermaphrodita despite the fact that a better understanding of how the nematode reacts to slug hosts may enable us to more effectively utilize it as a natural biocontrol agent.
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As a first step to achieve such understanding, we tested the hypothesis that P. hermaphrodita responds to chemical cues elicited by the host slug Deroceras reticulatum. In addition, we characterized the nature of this response.
nematodes zone1
2. Materials and methods
zone2
T
C
2.1. Organism used Dauer larvae of P. hermaphrodita were obtained from Becker Underwood (Littlehampton, U.K.) in a formulated product (Nemaslug) consisting of partially dehydrated dauer larvae and clay particles. Four hours prior to use, the product was mixed with cold water and nematodes were separated from the formulated material using a Baermann funnel. Adults D. reticulatum (about 2 cm long) were collected from grassland sites in Scotland, around the University of Abertay Dundee, and were kept in the dark at 4 C and fed with slices of carrots until used. In all treatments, the experimental arena comprised a 4.5-cm diameter Petri dish containing 1.2% technical agar (Oxoid Ltd., Hampshire, U.K.). 2.2. Is P. hermaphrodita attracted by D. reticulatum mucus? 2.2.1. Experimental treatments Each plate was divided into quadrants and a 1.5 cm diameter centered circle was drawn on the back of the plate. Two pieces of filter paper (7 · 7 mm) were placed 30 mm apart at opposite quadrants of the plate, on the surface close to the boundary. One was impregnated with water to serve as a control and the other with slug mucus. Mucus was swabbed from single slugs that were held carefully with a pair of tweezers while the filter paper was gently rubbed over the slug until the whole surface of filter paper was fully impregnated with mucus. Control plates consisted of filter paper impregnated with water versus an untreated square of filter paper. All plates were sealed with parafilm and kept at 19 C. To determine how the degree of nematode attraction to slug mucus depended on the duration that the plates were exposed to slime prior to nematode inoculation, two different time intervals were chosen. Based on preliminary investigations, the nematodes were added to the plates approximately 4 and 12 h after the filter paper was applied. A 5-ll drop of water containing 30 ± 5 active nematodes was pipetted into the center of each replicate plate. Initially, the surface tension of the water retained the nematodes within the center of the dish. Once the water drop evaporated (typically after 5 min), the nematodes were free to disperse in the Petri dish. To measure how fast the nematodes responded to the presence of the slug mucus, the nematodes were left to move around the Petri dish for either 1 or 2 h. Consequently, five experimental treatments were investigated: (i) nematodes on the plates for 1 h with untreated filter paper applied 4 h before the nematodes were added (‘‘control’’); (ii) nematodes on the test plates
test
control
Fig. 1. Schematic diagram of the experimental design used to compute the attraction index.
for 1 h with treated filter paper applied 4 h before the nematodes were added (‘‘4 h/1 h’’); (iii) as (ii) with the treated filter paper applied 12 h prior to nematodes inoculation (‘‘12 h/1 h’’); (iv) nematode on the test plates for 2 h with treated filter paper applied 4 h before the nematodes were added (‘‘4 h/2 h’’); (v) as (iv) with the treated filter paper applied 12 h prior to nematode inoculation (‘‘12 h/2 h’’). Twenty replicates were prepared for each treatment. The sampling periods and test assays were based on many preliminary studies of slug mucus and nematode interaction. At the end of this time period the nematodes present on the control and test filter paper strips denoted in Fig. 1 by zone (1) were counted under a microscope after immersing and fragmenting the filter paper in water. The number of nematode presented in the neighborhood of these filter papers (the corresponding quadrant outside the centered circle), zone (2) was also monitored. 2.2.2. Attraction index An ‘‘attraction index’’ was calculated as follows: AI ¼
aðN T ;1 N C;1 Þ þ bðN T ;2 N C;2 Þ aðN T ;1 þ N C;1 Þ þ bðN T ;2 þ N C;2 Þ
with
a ¼ 2 and b ¼ 1; where AI represents the attraction index; NT,1 the number of nematode in and underneath the test filter paper (zone 1), and NT,2 the number of nematodes in its neighborhood (zone 2); NC,1 the number of nematode in and underneath the control filter paper (zone 1), and NC,2 the number of nematodes in its neighborhood (zone 2) (Fig. 1). This attraction index is a generalization of a previously used index (Thurston et al., 1994). The coefficients a and b serve as differential weights for the different sections of the plate based on how far the nematode is from the food source. If b = 0, only the nematodes situated on the filter paper are counted and the index is exactly that used by Thurston et al. (1994). The choice of a = 2 and b = 1 above means that nematodes in both zones are considered and zone 1 carries twice the weight of zone 2 in the attraction index. The generalized formula above is consistent with the definition of an attraction index for which the value ranges from 1.0 to 1.0. Values close to 0 indicate that the test
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compound had no effect on nematode attraction, positive values indicate attraction to, and negative values indicate repellence by the test compound. Statistical significance among the different AI values corresponding to the five experimental treatments was determined by one-way ANOVA and post hoc multiple comparisons were made by using Turkey’s test (Montgomery, 1991; Zar, 1999). 2.3. Does D. reticultum mucus produce a chemotactic or chemokinetic responses in P. hermaphrodita? 2.3.1. Experimental treatments Two experimental treatments were used to examine the activity of nematodes in the presence of slug mucus. In the first, two pieces of filter paper of 7 · 7 mm were placed 30 mm apart at opposite quadrants of the plate, on the surface close to the boundary (mucus on filter paper treatment). One was impregnated with water to serve as a control and the other one with slug mucus. In the second treatment, slug mucus was applied uniformly to treatment plates by gently swabbing the slug across the Petri dish (mucus uniformly applied treatment); untreated agar plates were included as controls. All plates were sealed with parafilm and kept at 19 C for 4 h. Typically three nematodes were picked from water, using a mounted eyelash and placed in the center of the plate, about 5 mm apart, to avoid any direct interaction. Their movement was monitored for 15 min. Preliminary investigations on untreated agar plates revealed that P. hermaphrodita remain active, having a constant speed, for approximately 1 h. In total, the movement of 25 nematodes was recorded for each treatment. 2.3.2. Image capture and analysis An Axio MRc Zeiss camera mounted on a Leica MZ16 microscope connected to a computer, monitored nematode movement with an image field size of 30 · 40 mm. Movement was recorded at 8-s intervals over a period of 15 min. Images were processed using Axio Vision 3.1 software, then analyzed by an image analysis software incorporating a tracking algorithm (Image-Pro Plus v.5, Media Cybernetics Inc., U.S.A.), in order to obtain the x and y coordinates at each time point. MatLab v.6.1 was used to plot the final digitized nematode trail and to compute the distance traveled between frames, the value of the turning angle and the fractal dimension of nematode trail. The speed of the nematode movement was determined by calculating the average distance between successive positions, traveled in 8-s time interval. An 8-s interval was chosen on the basis that in this time the nematodes travel an average distance of 1 mm. This is approximately equal to the body length. Shorter timescales would have introduced artifactual error due to changes in the body shape during movement, and longer timescale would have reduced the amount of data available for analysis. The statistical significance of differences in nematode speed among treatments was determined by t-tests (Zar, 1999).
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The turning angle distribution was generated by computing the angle between successive line segments on the trail. The angular range chosen was from p to p radians, and all angles were computed with respect to the previous angle. The negative values correspond to the right turns and the positive values to the left turns. v2 goodness of fit test (Zar, 1999) was used to determine significant differences in turning angle distributions corresponding to different treatments. We also studied the fractal dimension of the nematode trails to give a quantitative measure that characterize the degree to which the nematode trails fill the 2-D space of the agar plates (Anderson et al., 1997). One of the most common methods for calculating the fractal dimension of an object is the box counting method (Falconer, 1990). This method consists in counting the number of boxes of length lb needed to completely cover the nematode trail. If we denote this number by Nb (lb), then the fractal dimension Db is given by: Db ¼ lim
ld !0
log10 ðN b ðlb ÞÞ . log10 ðlb Þ
In practice Db
corresponds to the slope of the plot log10 (Nb (lb)) versus log10 (lb). The lowest dimension we would expect is 1 (the dimension of a straight line) which indicates a linear trail. Where Db is close to 2 (the dimension of brownian motion), the trail will be more rugged and tortuous, and given sufficient time the nematode will visit all points on the agar plate. To compare the fractal dimension, the t-test for means (Zar, 1999) was used. 3. Results 3.1. General observations In the absence of any attractant, the nematode trails displayed frequently two types of movement (Fig. 2): looping (spiraling around an imaginary point) and reversal (rapidly alternating forward and backward movements) (Anderson et al., 1997). Second, there was significant individual variation in the population, from nematodes that move slowly and in reversals, to nematodes that move fast making large and smooth loops. This variation in nematode population was also observed even in the presence of the attractant, either in the form of the localized slug mucus, or uniformly distributed on the whole surface. Such variation in populations resulted in relatively large statistical variances. 3.2. Attraction response The attraction index (AI) values corresponding to the five experimental treatments (Table 1) were significantly different (ANOVA, F = 174.4; df = 4, 95; P < 0.001). Phasmarhabditis hermaphrodita was significantly attracted by D. reticulatum mucus (q = 9.73, df = 95; k = 5; P < 0.001) in all experimental treatments compared to the control. Further, when the nematode were allowed to move on the Petri dish for 1 h, the attraction response was significantly greater in the plates where the nematodes were
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Fig. 2. Digitized nematode movement. (a) Control (technical agar with no potential attractant); (b) Treatment 1 (technical agar with slug mucus localized on a strip of filter paper on the left); (c) Treatment 2 (technical agar with slug mucus uniformly applied on the surface of the plate); (+ denotes the starting position).
Table 1 Values of the attraction index (AI) ranging between 1.0 and 1.0 (negative values for repellence and positive values for attraction) together with the standard error for n = 20 replicates Treatmentsa
AI ± SE
Control 4 h/1 h 12 h/1 h 4 h/2 h 12 h/2 h
0.02 ± 0.09 0.43 ± 0.12 0.24 ± 0.09 0.73 ± 0.09 0.67 ± 0.10
a Numbers relate to the time the amount of time the treatment was applied prior to the amount of time the nematode was exposed to the treatment. See text for full details.
added 4 h after the slime paper than in the cases where the nematodes were added after 12 h (q = 8.28, df = 95; k = 5; P < 0.001). But, when the nematode were allowed to move on the Petri dish for 2 h, the difference between the attraction index corresponding to treatments (4 h/2 h) and (12 h/ 2 h) was not significant (q = 2.63, df = 95, k = 5; P = 0.37). On the other hand, comparing treatments ‘‘(4 h/ 1 h) + (12 h/1 h)’’ with ‘‘(4 h/2 h) + (12 h,2 h)’’ a significantly greater response (q = 11.02; df = 4, 95; P < 0.001) was obtained when the nematodes were left to explore the Petri dish for 2 h with respect to 1 h. In all tests, 1 h after applying the nematodes to the agar surface, at least 50% of the nematodes had moved out of the central circle, and at least 20% were in the two response quadrants.
the filter paper near the boundary of the plate, the distribution became more peaked around value 0 of the turning angle, corresponding to a predominantly straight-line movement with an increase in the proportion of forward directed movements. A nematode in the presence of localized mucus typically moved in the same direction more often following the attractant gradient emanating from the slug mucus. When the mucus was uniformly applied on the whole surface, the distribution of the turning angle became more uniform with a reduction in the frequency of values around 0 and an increase in frequencies around p and p, compared to the control. This distribution is associated with a random behavior; with forward directed movement being replaced by an increase in reversal movements. The nematode trails in plates with uniformly distributed mucus had a significantly greater fractal dimension FD = 1.252 (t = 2.22, df = 48, P < 0.05), compared to the control FD = 1.192, as a consequence of the more random foraging behavior, which leads to a dense and rugged trail. When the mucus was localized near the boundary, on the piece of filter paper, the movement becomes smoother, with a consequent fractal dimension FD = 1.129 significantly smaller (t = 2.24, df = 48, P < 0.05) compared to the control.
4. Discussion 3.3. Chemotactic and chemokinesis response Typical examples of nematode trails produced are presented in Fig. 2, for the control plate as well as for the test plates with slime on it either localized on the piece of filter paper, or uniformly disposed on the surface of the agar. Nematode movement was significantly faster in plates in which slime was impregnated to filter paper, speed average = 0.113 mm/s (t = 2.10; df = 48; P < 0.05), and significantly slower in the plates with a uniform layer of slime, speed average = 0.042 mm/s (t = 5.42; df = 48; P < 0.05), compared to the control speed average = 0.084 mm/s. Furthermore, the turning angle distributions corresponding to both treatments were significantly different (P < 0.001) compared to the control. When mucus was localized on
Our study has clearly shown that the presence of the D. reticulatum slug mucus significantly affected the behavior of P. hermaphrodita on agar plates. Related investigations have been reported in the past on entomopathogenic nematodes (Lewis et al., 1992, 1993), and similar effects have been recorded for Caenorhabditis elegans (Maupas migration) to several species of bacteria (Rodger et al., 2004; Grewal and Wright, 1992; Andrew and Nicholas, 1976; Ward, 1973). Recently, Rae et al., 2006 have studied P. hermaphrodita attraction to different slug cues and reported an aggregation of nematodes after 24 h, under a point source of slug cues situated about 30 mm away from the inoculation point. Our contribution consists of a rigorous quantitative analysis of nematode behavior in the presence of the
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slug mucus subject to different spatial and temporal treatments. Moreover, quantitative trail analysis has never previously been applied to slug parasitic nematodes. A first and very important finding is that the degree of impact on their behavior depends on the duration that the plates were exposed to mucus, prior to the inoculation with nematodes. Longer exposure decreases the effect. Preliminary investigations have also shown that the attraction response was smaller for a shorter 1 h exposure compared to 4 h. Whilst, it is beyond the scope of this work to identify the signaling compounds emanating from the slug mucus, our results suggest that the compound is in an aqueous phase. A typical signaling compound in air (e.g., CO2) will cross a 10 cm Petri dish in a timescale of the order of 1 min (dependent, only weakly, on the square root of the molecular weight) (Lyman et al., 1982). In the aqueous phase, it will cross the same dish in a timescale of the order of 10 h. Our results show that gradients are becoming established and declining over the latter timescales, consistent with aqueous-phase diffusion. All of the reported results, are consistent with a reaction-diffusion scenario, in which, the nematodes are dispersing on the agar plate along the chemical gradient diffusing from the slug slime (Fig. 4). In our assay, 4 h was the most efficient interval between slime application and nematode inoculation (AI = 0.43). From the preliminary results, 1 h seems to be an insufficient timescale for the mucus to diffuse through the agar in order to be sensed by the nematode chemoreceptor. Thus, initially the nematode can go in both directions with equal probability. However, after 12 h, the attraction index was significantly lower (AI = 0.24, P < 0.001) with respect to 4 h, suggesting that the gradient of chemical signal from the mucus had declined to be close to zero. When the nematodes were left to move on the plate for 2 h, the responses corresponding to 4 and 12 h mucus exposure converge (AI = 0.73 and AI = 0.67, respectively, P = 0.37). In both situations, the high value of the attraction index signifies that 2 h was sufficient time for most of the nematodes to migrate in the test quadrant.
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Fig. 4. Comparison of the gradient profile of a diffusible attractant a, released by localized slug mucus, corresponding to three interval time 1, 4 and 12 time units. The formula of the attractant a, is given by the solution of normal diffusion equation with an instantaneous point source (Crank, 1975).
In previous publications, this index is termed the ‘‘chemotactic index’’ (Thurston et al., 1994), whilst others term it the ‘‘attraction index’’ (Andrew and Nicholas, 1976; Grewal and Wright, 1992; Rodger et al., 2003). It is important to note that it is possible to have AI close to 1 even although there is no attraction or chemotaxis simply because chemokinetic response exists. In order to differentiate between these responses, a more detailed analysis of the nematode trail is required. The fact that the attraction index depends significantly (P < 0.001) on the diffusion of the attractant suggests that it is unlikely that the nematodes encountered the cue by chance, implying that there is chemotaxis behavior. Therefore, to further interpret the attraction data recorded in the first experiments we did, an individual based analysis of nematode behavior in the presence of slug mucus. Firstly, a large and significant increase in speed was found when the nematode was placed at some distance from the attraction source. The sugges-
0.3 control
frequency
0.25
treatment 1
0.2
treatment 2
0.15 0.1 0.05 0 -3
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
3
radians Fig. 3. Comparison of the effect of the three experimental treatments (including the control) on the turning angle distribution of nematode trail.
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tion is that orthokinesis, that is a kinesis in which forward speed is changed (Dusenbery, 1980) is operating at this scale. Second, when the nematodes were placed in direct contact with the mucus their speed significantly decreased compared to that of a control. Croll (1975) reported that C. elegans move much more slowly in a lawn of bacteria than on bare agar. Our analyzes of turning angle distributions clearly show that nematodes follow a straighter foraging trail in response to a signal emanating from a localized food source (Fig. 3). Conversely, when the signal is coming from everywhere, as the mucus is uniformly applied on the Petri dish, then nematode movement becomes less directed; consequently, the turning angle distribution tends to be more uniform in the interval [p, p]. This statement is consistent with the results obtained from the fractal dimension analysis where a higher value corresponding to a better exploitation of each potential food source area, was found in this case. When the nematode is challenged by the presence of a localized potential food source a lower fractal dimension, corresponding to a relatively linear trail, occurs. This is in agreement with the result obtained for C. elegans trails when searching for bacterial food (Anderson et al., 1997). Foraging strategies of P. hermaphrodita, and of nematodes, in general, are complex (Young et al., 1996) and depend on the diffusion of the attractant released by the food source (Grewal and Wright, 1992). There is evidence that random movement persists in the presence of directed movement (Robinson and Heald, 1993; Schroeder and Beavers, 1987; Anderson et al., 1997). It has also been observed that, in order to maintain a certain direction over large distances, some aquatic organism adopt a constant rotation around an axis parallel to their direction of locomotion (Jennings, 1904; Schaeffer, 1928; Foster and Smyth, 1980). In our study, the results suggest that where no food is sensed, P. hermaphrodita adopts a random exploration with looping and reversals that optimizes their opportunities of locating substrate. This behavior changes in the presence of an attractant gradient to a faster (chemokinesis) and more direct movement toward the stimulus (chemotaxis). Therefore, after a period of time there is a high probability that they will find the potential host. The next question is, what happens when this event takes place? When the nematodes were directly challenged by the slug mucus, spatially distributed in the domain of action, their speed was slower (chemokinesis) and the trail was denser and more localized, compared with nematodes in the absence of an attractant. Even in the presence of an attraction agent, we still observe looping and reversals. This suggests that such movement is an additional requirement for exploring in a complex spatial environment where nematodes may otherwise become trapped in pores where a signal exists but movement in the direction of the gradient is obstructed. The analysis we have developed presents a detailed picture of the exploration and exploitation
responses of nematodes to a range of environmental perturbations. Acknowledgments We thank K. Macmillan (University of Aberdeen) for his help and advice with respect to the experimental techniques and H. Staines (Scottish Informatics Mathematics Biology and Statistics Centre) for statistical advice. We are also grateful to the referees for their constructive comments. This research was funded by the Biotechnology and Biological Sciences Research Council (Grant No.: BBSB01065). References Anderson, A.R.A., Sleeman, B.A., Young, I.M., Griffiths, B.S., 1997. Nematode movement along a chemical gradient in a structurally heterogeneous environment. 1. Experiment. Fundam. Appl. Nematol. 20, 165–172. Andrew, P.A., Nicholas, W.L., 1976. Effect of bacteria on dispersal of Caenorhabditis elegans (Rhabditidae). Nematologica 22, 451–461. Crank, J., 1975. The mathematics of diffusion, second ed. Oxford University Press, Oxford, UK. Croll, N.A., 1975. Componenets and patterns in the behaviour of nematode Caenorhabditis elegans. J. Zool. 176, 159–176. Dusenbery, D.B., 1980. Behaviour of free-living nematodes. In: Zuckerman, B.M. (Ed.), . Nematodes as Biological Models, vol. 1. Academic Press, New York, pp. 127–158. Dusenbery, D.B., 1992. Sensory Ecology. W.H. Freeman, New York. Falconer, K., 1990. Fractal Geometry, Mathematical Foundations and Applications. John Wiley and Sons, Chichester, UK. Foster, K.W., Smyth, R.D., 1980. Light antennas in phototactic algae. Microbiol. Rev. 44, 572–630. Grewal, P.S., Wright, D.J., 1992. Migration of Caenorhabditis elegans (Nematoda: Rhabditidae) larvae towards bacteria and the nature of bacteria stimulus. Fund. Appl. Nematol. 15, 159–166. Jennings, H.S., 1904. Contributions to the study of the behaviour of lower organisms. Carnegie Institute, Washington DC. Lewis, E.E., Gaugler, R., Harrison, R., 1992. Entomopathogenic nematode host finding: response to host cues by cruise and ambush foragers. Parasitology 105, 309–315. Lewis, E.E., Gaugler, R., Harrison, R., 1993. Response of cruiser and ambusher entomopathogenic nematodes (Steinernematidae) to host volatile cues. Can. J. Zool. 71, 761–769. Lyman, W.J., Reehl, W.J., Rosenblatt, D.H., 1982. Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds. McGraw-Hill, New York. Montgomery, D.G., 1991. Design and Analysis of Experiments. John Wiley and Sons, New York. Rae, R.G., Roberson, J.F., Wilson, M.J., 2006. The chemotactic response of Phasmarhabditis hermaphrodia (Nematoda: Rhabditida) to cues of Deroceras reticulatum (Mollusca: Gastropoda). Nematology 8, 197–200. Robinson, A.F., Heald, C.M., 1993. Movement of Rotylenchulus reniformis through sand and agar in response to temperature, and some observations on vertical descent. Nematologica 39, 92–103. Rodger, S., Bengough, A.G., Griffiths, B.S., Stubbs, V., Young, I.M., 2003. Does the presence of detached root border cells of Zea mays alter the activity of pathogenic nematode Meloidogyne incognita. Am. Phytopathol. Soc. 93, 1111–1114. Rodger, S., Griffiths, B.S., McNicol, J.W., Wheatley, R.W., Young, I.M., 2004. The impact of bacterial diet on the migration and the navigation of Caenorhabditis elegans. Microbiol Ecol. 48, 358–365.
S. Hapca et al. / Biological Control 41 (2007) 223–229 Schaeffer, A.A., 1928. Spiral movement in man. J. Morphol. 45, 293–398. Schroeder, W.J., Beavers, J.B., 1987. Movement of the entomogeneous nematodes of the families Heterorhabditidae and Steinernematidae in soil. J. Nematol. 19, 257–259. Tan, L., Grewel, P.S., 2001. Infection behaviour of the rhabdtid nematode Phasmarhabditis hermaphrodita to the grey garden slug Deroceras reticulatum. J. Parasitol. 87, 1349–1354. Thurston, G.S., Yule, W.N., Dunphy, G.B., 1994. Explanations for low susceptibility of Leptinotarsa decemlineata to Steinernema carpocapsae. Biol. Control 4, 53–58.
229
Ward, S., 1973. Chemotaxis by the nematode Caernorhabditis elegans: identification of attractants and analysis of the response by the use of mutants. Proc. Natl. Acad. Sci. USA 70, 817–821. Wilson, M.J., Glen, D.M., George, S.K., 1993. The Rhabditid nematode Phasmarhabditis hermaphrodita as a potential biological control agent for slugs. Biocontrol Sci. Technol. 3, 513–521. Young, I.M., Griffiths, B.S., Robertson, W.M., 1996. Continuous foraging by bacterial feeding nematodes. Nematologica 42, 378–382. Zar, J.H., 1999. Biostatistical Analysis, Fourth ed. Prentice Hall, Upper Saddle River, NJ.