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Susceptibility and immune response of Deroceras reticulatum, Milax gagates and Limax pseudoflavus exposed to the slug parasitic nematode Phasmarhabditis hermaphrodita
Journal of
INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 97 (2008) 61–69 www.elsevier.com/locate/yjipa
Susceptibility and immune response of Deroceras reticulatum, Milax gagates and Limax pseudoflavus exposed to the slug parasitic nematode Phasmarhabditis hermaphrodita Robbie G. Rae a
a,b,*
, Jamie F. Robertson b, Michael J. Wilson
b
Max Planck Institute for Developmental Biology, Department of Evolutionary Biology, Spemannstraße 37-39, Tu¨ebingen 72076, Germany b University of Aberdeen, School of Biological Sciences, St. Machar Drive, Aberdeen AB24 3UU, UK Received 4 March 2007; accepted 12 July 2007 Available online 20 July 2007
Abstract We exposed three slug species (Deroceras reticulatum (Mu¨ller), Milax gagates (Draparnaud) and Limax pseudoflavus L.) to the parasitic nematode Phasmarhabditis hermaphrodita Schneider. P. hermaphrodita was able to cause mortality and feeding inhibition to both D. reticulatum and M. gagates but did not negatively affect L. pseudoflavus. On dissection of surviving L. pseudoflavus large numbers of P. hermaphrodita were found encapsulated in the shell of the slug. We found that by increasing shell size, the slug was able to trap invading nematodes, which could be an immune response to P. hermaphrodita invasion. This is the first report of a slug defense mechanism to inhibit P. hermaphrodita. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Phasmarhabditis hermaphrodita; Deroceras reticulatum; Milax gagates; Limax pseudoflavus; Nematodes; Slugs; Parasite; Immunity; Biological control; Shell
1. Introduction Phasmarhabditis hermaphrodita Schneider is a lethal slug parasitic nematode (Wilson et al., 1993; Speiser et al., 2001; Iglesias and Speiser, 2001; Grewal et al., 2003) that has been formulated into a biological control agent (NemaslugÒ) available from Becker Underwood, UK. Infective juveniles are mixed with water and applied using a watering can or spraying equipment. The nematodes search for slugs in soil and respond to slug associated cues such as mucus and faeces (Rae et al., 2006; Hapca et al., 2007). On locating a potential host P. hermaphrodita enters into the shell cavity at the back of the slugs mantle and is thought to release a bacterium (Moraxella osloensis), which kills the slug between 4 and 21 days after infection (Wilson et al., * Corresponding author. Address: Max Planck Institute for Developmental Biology, Department of Evolutionary Biology, Spemannstraße 37-39, Tu¨ebingen 72076, Germany. Fax: +49 (0) 7071 601498. E-mail address: [email protected] (R.G. Rae).
0022-2011/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2007.07.004
1993; Tan and Grewal, 2001). M. osloensis lipopolysaccharide is thought to be responsible for slug death (Tan and Grewal, 2002). Once the slug dies the nematodes grow to hermaphroditic adults and reproduce on the slug cadaver (Wilson et al., 1993; Tan and Grewal, 2001). As the food source depletes, new infective juveniles are produced which then search for potential slug hosts in the soil. Phasmarhabditis hermaphrodita can parasitize and cause mortality to nine slug species including Deroceras reticulatum (Mu¨ller), Deroceras panormitanum (Lessona and Pollonera), Deroceras laeve (Mu¨ller), Arion silvaticus Lohmander, Arion intermedius Normand, Arion distinctus Mabille, Tandonia sowerbyi (Fe´russac), Tandonia budapestensis (Hazay) and Leidyula floridana (Hazay) (Wilson et al., 1993; Grewal et al., 2003; Iglesias and Speiser, 2001). However, some species are not susceptible such as Limax maximus (L.), Arion subfuscus Draparnaud and Arion hortensis Fe´russac (Grewal et al., 2003; Iglesias and Speiser, 2001). For certain other species including Arion lusitanicus Mabille, Arion ater (L.) and the snail Helix aspersa Mu¨ller,
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juveniles are known to be susceptible whereas adults are not (Speiser et al., 2001; Grimm, 2002; Wilson et al., 1993; Glen D.M., unpublished). Similarly, some snail species are susceptible whereas others are not (Wilson et al., 2000). There is no information about the susceptibility of Limax pseudoflavus L. and Milax gagates (Draparnaud) to P. hermaphrodita. L. pseudoflavus is widely distributed in Europe, lives in crevices, under rubbish, in gardens, cellars, outhouses, farmyards and damp basements (Kerney, 1999). It is not generally considered to be a pest species and usually feeds on fungi, decayed plants and rubbish (Janus, 1965). M. gagates is generally distributed in gardens where it is considered to be a pest (Taylor, 1902– 1907) and can be found on farms where it is a pest of rape (Godan, 1983), leaf beet (Iglesias et al., 2001), Chinese cabbage (Vernava´ et al., 2004) and lettuce (Wilson et al., 1995). Relatives of M. gagates and L. pseudoflavus have been tested for susceptibility to P. hermaphrodita. Both relatives of M. gagates (T. sowerbyi and T. budapestensis) are susceptible to P. hermaphrodita (Wilson et al., 1993), therefore, we hypothesize that M. gagates is also susceptible. If M. gagates is susceptible to P. hermaphrodita then nematodes can be recommended as a control measure. In contrast to M. gagates, relatives of L. pseudoflavus such as L. maximus are not susceptible (Grewal et al., 2003). The reasons why some slug species are not susceptible to P. hermaphrodita has received very little attention. Grewal et al. (2003) proposed that non-susceptible gastropods will suspend feeding and contract into a nonfeeding posture to prevent nematodes entering into the shell cavity, but there has been no further research to support this hypothesis. Defense strategies for insects have evolved to stop entomopathogenic nematodes entering the mouth, anus or spiracles. Popillia japonica grubs exhibit grooming behaviour upon nematode contact and evasive behaviour when sensing nematode attack (Gaugler et al., 1994). Some insects such as Phyllophaga hirticola have mechanical barriers such as sieve plates that protect the spiracles from nematode entry (Dowds and Peters, 2002). Frequent defecation may eject nematodes entering via the anus and mandibles may crush any entering nematodes to death (Gaugler and Molloy, 1981; Georgis and Hague, 1981; Cui et al., 1993). Once nematodes have penetrated the insect’s haemocoel, the insect’s non-self response will attempt to entrap the nematode in cellular capsules that are frequently hardened by melanin (Dunphy and Thurston, 1990; Wang et al., 1994). Little is known about the immune responses in slugs and snails (South, 1992). The internal shell of slugs lies immediately beneath the mantle in the shell sac and consists of an oval flattened plate, which tends to be convex above and concave below and generally shows concentric zones of growth (South, 1992). We hypothesize that nematodes entering the mantle area will come into direct contact with
the shell and the shell may have defensive properties that immobilize and encapsulate nematodes, a phenomenon that has never previously been reported for slugs and nematodes. We exposed D. reticulatum, M. gagates and L. pseudoflavus to a range of doses of P. hermaphrodita and recorded mortality and feeding inhibition. At the end of the experiment shells of non-susceptible species were removed and examined for nematode encapsulation. 2. Materials and methods 2.1. Source of invertebrates Slugs were collected from Seaton Park, Aberdeen and Aberdeen University Botanic Gardens and stored in nonairtight plastic boxes lined with cotton wool and fed with fresh lettuce leaves. Any diseased or dead slugs were removed and only healthy slugs were used in experiments. P. hermaphrodita was supplied by Becker Underwood, UK. 2.2. Assessing susceptibility of D. reticulatum, M. gagates and L. pseudoflavus Fifteen Petri dishes (diameter 13.6 cm) were filled with 150 g of Craibstone series soil (loamy sand texture, 73.85% sand, 20.04% silt and 6.11% clay containing 4.25% w/w organic matter). The soil was air dried, sieved to 2 mm and rewetted with 23% water (v/v). Slugs were exposed to P. hermaphrodita at five different doses: 0 (untreated control), 30 per cm 2, 90 per cm 2, 150 per cm 2 and 300 per cm 2. Nematodes were evenly applied over the soil surface using a wash bottle. Ten D. reticulatum (mean weight = 0.24 ± 0.01 g, n = 150), M. gagates (0.58 ± 0.02 g, n = 150) and L. pseudoflavus (0.40 ± 0.02 g, n = 150) were added to separate Petri dishes. Five discs (2.7 cm diameter) of lettuce were added to each dish to examine feeding inhibition. The percentage of each lettuce disc eaten was recorded every three days for 12 days. Fresh discs were added every three days. The dishes were then sealed with ParafilmÒ and placed in an incubator at 16 °C. Mortality was recorded every day for 18 days. Any dead slugs were removed and examined for presence of nematodes. At the end of the experiment surviving slugs were dissected and shells removed, weighed and the number of P. hermaphrodita trapped inside was determined. The experiment was repeated three times with fresh batches of slugs and nematodes. 2.3. Experimental analysis Statistics were performed using MINTAB 12 (Mintab Inc., USA). Significance levels were taken at P 6 0.05. Mortality counts were transformed to square roots and subjected to General Linear Model ANOVA and Tukey’s pairwise comparison. Kruskal–Wallis and Mann–Whitney U tests were used to analyse data from the number of
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Fig. 1. Mean percentage mortality of D. reticulatum (a), M. gagates (b) and L. pseudoflavus (c) exposed to P. hermaphrodita. Bars represent ± one standard error.
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Fig. 2. Mean percentage eaten of lettuce discs by D. reticulatum (a), M. gagates (b) and L. pseudoflavus (c) exposed to P. hermaphrodita. Bars represent ± one standard error.
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P. hermaphrodita entering L. pseudoflavus, the weight of L. pseudoflavus shells and the number of nematodes encapsulated in the shells. The numbers of P. hermaphrodita found within L. pseudoflavus shells were transformed to square roots and the relationship between weight of shells and numbers of entrapped nematodes was analysed using regression analysis. Percentage feeding inhibition data were transformed using arcsine square root transformations and analysed using General Linear Model ANOVA and Tukey’s pairwise comparison. 3. Results 3.1. The effect of P. hermaphrodita on D. reticulatum, M. gagates and L. pseudoflavus Phasmarhabditis hermaphrodita when applied at the highest dose (300 per cm 2) caused significant mortality to D. reticulatum on day 6 (P 6 0.001) (Fig. 1a). On days 12 and 18 P. hermaphrodita applied at 90, 150 and 300 per cm 2 significantly affected the mortality of D. reticulatum (P 6 0.001). Significant mortality to M. gagates was also caused by the highest dose of P. hermaphrodita (300 per cm 2) on day 6 (P 6 0.001) (Fig. 1b). As time of exposure increased, progressively lower doses of P. hermaphrodita effected mortality in this species such that on day 12 P. hermaphrodita (150 per cm 2 and 300 per cm 2) caused significant mortality to slugs (P 6 0.001) and by day 18 P. hermaphrodita caused significant mortality to M. gagates at 90 (P 6 0.05), 150 (P 6 0.001) and 300 per cm 2 (P 6 0.001). Conversely, P. hermaphrodita did not cause significant mortality to L. pseudoflavus and there were no significant differences between mortality of the untreated and nematode-treated L. pseudoflavus at any time point with any dose of P. hermaphrodita (P > 0.05) (Fig. 1c). 3.2. Feeding inhibition of D. reticulatum, M. gagates and L. pseudoflavus exposed to P. hermaphrodita For D. reticulatum, as with mortality, feeding inhibition tended to increase with time of exposure and nematode dose. On day 6 the highest P. hermaphrodita dose (300 per cm 2) caused significant feeding inhibition to D. reticulatum (P 6 0.05) (Fig. 2a) whereas by days 9 and 12 significant feeding inhibition was caused by 90 (P 6 0.05), 150 (P 6 0.05) and 300 per cm 2 (P 6 0.001). The effect of P. hermaphrodita on feeding by M. gagates was less pronounced than that seen for D. reticulatum with no significant feeding inhibition being observed on day 6. However, by days 9 and 12 P. hermaphrodita applied at 150 and 300 per cm 2 caused a significant reduction in feeding by M. gagates (P 6 0.05; P 6 0.001, respectively) (Fig. 2b). As was the case with slug mortality, P. hermaphrodita did not have any effect on the feeding of L. pseudoflavus and there were no differences between the feeding of
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untreated and treated L. pseudoflavus at any time point with any nematode dose (P > 0.05) (Fig. 2c). 3.3. Shell weight and number of P. hermaphrodita penetrating and encapsulated in L. pseudoflavus shells Only shells from L. pseudoflavus were examined as both M. gagates and D. reticulatum were susceptible to P. hermaphrodita and in many cases shells were subsequently shed during parasitism. When examining the shells microscopically to count nematodes encapsulated, it could be seen that movement of nematodes was very much restricted by the newly laid down shell material, and it is possible that L. pseudoflavus encapsulates nematodes as an immune response (Fig. 3). There were significant differences between the numbers of P. hermaphrodita entering into the body of L. pseudoflavus exposed to the five doses (H = 33.61; df = 4; P 6 0.001) (Fig. 4). The number of P. hermaphrodita entering L. pseudoflavus differed significantly from the control at 150 per cm 2 (P 6 0.001) and 300 per cm 2 (P 6 0.001) but not at 30 cm 2 (P > 0.05) and 90 cm 2 (P > 0.05). The weights of shells were significantly different among nematode doses (H = 19.79; df = 4; P 6 0.001) (Fig. 5). Shells extracted from L. pseudoflavus exposed to nematodes (150 per cm 2) were significantly heavier than the control (P 6 0.05) but shells from treated slugs at the other doses (30, 90 and 300 per cm 2) did not differ significantly from the control (P > 0.05). The numbers of P. hermaphrodita found encapsulated inside the shells of L. pseudoflavus differed between doses (H = 37.90; df = 4; P 6 0.001) (Fig. 6). There were significantly more P. hermaphrodita found in the shells of slugs treated with 150 (P 6 0.001) and 300 nematodes per cm 2 (P 6 0.05) compared to the control. There were no differences in the numbers of nematodes found in shells of slugs treated with 30 and 90 P. hermaphrodita per cm 2 (P > 0.05). There was a significant, positive relationship between the weight of shells and the numbers of P. hermaphrodita encapsulated in the shells (control weights were removed) (P 6 0.001; r2 = 0.647) (data not shown). 4. Discussion Phasmarhabditis hermaphrodita caused significant mortality and feeding inhibition to D. reticulatum and M. gagates but had no effect on L. pseudoflavus. This is perhaps not surprising as relatives of M. gagates (T. sowerbyi and T. budapestensis) are susceptible to P. hermaphrodita (Wilson et al., 1993) and relatives of L. pseudoflavus (e.g. L. maximus) are not susceptible (Grewal et al., 2003). Therefore, P. hermaphrodita could be recommended for use in the field against M. gagates if applied at 90, 150 or 300 nematodes per cm 2. Lesions were produced at the point of entry to the shell cavity on the back of the mantle of M. gagates. Usually the
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Fig. 3. Shell of (A) untreated L. pseudoflavus and (B) nematode treated shell with P. hermaphrodita encapsulated inside.
infection resulted in shells being shed due to the build up of nematodes. At this time M. gagates was observed in a coiled position where the head was positioned at the lesion at back of the mantle. It is not known what the slug was doing but it could be possible that it was removing any nematodes present. Grooming behaviour has been recorded in P. japonica grubs to prevent nematode attack (Gaugler et al., 1994). Small lesions were also observed on the anterior mantle and on the posterior part of the tail. All these lesions had nematodes present. It may be possible that as well as mechanical means to enter host slugs
through natural openings, P. hermaphrodita may also use proteases or other enzymes to penetrate the slug’s cuticle and produce the observed lesions. The use of enzymes has been shown for a number of parasitic nematode species to gain access to host systems resulting in tissue disruption including Ascaris suum (Knox and Kennedy, 1988) and Strongyloides ransomi (Dresden et al., 1985). AbuHatab et al. (1995) reported Steinernema glaseri releasing proteolytic enzymes that increased the chances of entering through the gut wall of insect hosts. Interestingly, no lesions were observed on L. pseudoflavus, and it could be possible that this slug species may produce a lectin in its mucus that acts as a protease inhibitor, a phenomenon that has been reported for A. ater (Habets et al., 1979), which is also known to be resistant to P. hermaphrodita. Phasmarhabditis hermaphrodita did not cause mortality or feeding inhibition to L. pseudoflavus. Grewal et al. (2003) speculate that non-susceptible species will stop eating and contract to reduce the chances of nematode penetration; however, we found that L. pseudoflavus did not stop eating and no feeding inhibition was observed for 12 days. Little is known about the immune responses in slugs and snails (South, 1992). We found L. pseudoflavus encapsulated P. hermaphrodita inside the shell underneath the mantle area. There was a positive significant relationship between the weight of shell and the numbers of nematodes encapsulated within the shell. In some slugs the shells extracted contained all life stages of P. hermaphrodita including J2, J3 and adults in large abundance. Either the nematode is able to reproduce when encapsulated in the shell or the nematode can reproduce inside the slug and the resultant nematodes are trapped. This increase in shell weight and encapsulation of P. hermaphrodita may be one reason why this slug species is not susceptible to nematodes. As well as the characteristic swelling of the mantle area, infection of P. hermaphrodita causes a variety of symptoms to slugs such as reduced locomotion (Bailey et al., 2003), feeding inhibition (Glen et al., 1994), a lesion on the back of the mantle (Wilson et al., 1993) and slugs are more likely to be found under refuge traps (Wilson et al., 1994). The reasons behind these symptoms are poorly understood but parasite induced changes have been recorded in a number of molluscs. For example trematode parasites of the snail Hydrobia ulvae cause symptoms including castration (Rothschild, 1936; Lauckner, 1980), changes in crawling behaviour (Honer, 1961; Mouritsen and Jensen, 1994) and gigantism (Wesenberg-Lund, 1934; Rothschild and Rothschild, 1939; Mouritsen and Jensen, 1994; Huxham et al., 1995). The cellular defense of bivalve molluscs is controlled by haemocytes (granulocytes, hyalinocytes and serous cells) and are involved in a number of functions including wound repair, shell repair, nutrient digestion and transport, excretion and internal defense (Cheng, 1981). The bivalve’s internal defense system mainly consists of phagocytosis and encapsulation of foreign material (Cheng, 1981;
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Fig. 4. Mean number of P. hermaphrodita found inside L. pseudoflavus after 18 days. Bars represent ± one standard error.
Fig. 5. Mean weight of shells dissected from L. pseudoflavus after exposure to P. hermaphodita after 18 days. Bars represent ± one standard error.
Fig. 6. Mean number of P. hermaphrodita found encapsulated in L. pseudoflavus shells after 18 days. Bars represent ± one standard error.
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Ratcliffe et al., 1985). For example Montes et al. (1995) demonstrated that protozoa (Perkinsus sp.) were encapsulated by granulocytes in the clam (Tapes semidecussatus). The increase in size and encapsulation of P. hermaphrodita in the internal shell of L. pseudoflavus may be an immune response to invading parasites and may be one of the reasons why L. pseudoflavus is not susceptible to the nematode. This is the first report of a possible defense mechanism against slug parasitic nematodes that may contribute to immunity of slugs. Further research could concentrate on the role of the shell in other non-susceptible species such as L. maximus or in species where the shell is not an oval plate but made of granules such as in A. ater and A. subfuscus. Acknowledgments We are grateful to the Kintail Land Foundation for funding this project and to Becker Underwood for supplying nematodes. References AbuHatab, M., Selven, S., Gaugler, R., 1995. Role of proteases in penetration of insect gut by the entomopathogenic nematode Steinernema glaseri (Nematoda: Steinernematidae). J. Invertebr. Pathol. 66, 125–130. Bailey, S.E.R., Cairns, A., Latham, R., Abdel Kasi, M., Manning, P., 2003. Onset of immobilization in the slug Deroceras reticulatum Mu¨ller parasitized by the nematode Phasmarhabditis hermaphrodita Schneider. In: Slugs and Snails: Agricultural, Veterinary and Environmental Perspectives, British Crop Protection Council (BCPC) Symposium Proceedings, vol. 80, pp. 215–220. Cheng, T.C., 1981. Bivalves. In: Ratcliffe, N.A., Rowley, A.F. (Eds.), Invertebrate Blood Cells. Academic Press, London, pp. 233–300. Cui, L., Gaugler, R., Wang, Y., 1993. Penetration of steinernematid nematodes (Nematoda: Steinernematidae) into Japanese beetle larvae, Popillia japonica (Coleoptera: Scarabaeidae). J. Invertebr. Pathol. 62, 73–78. Dowds, B.C.A., Peters, A., 2002. Virulence mechanisms. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Wallingford, pp. 79–98. Dresden, M.H., Rege, A.A., Murrell, K.D., 1985. Strongyloides ransomi: proteolytic enzymes from larvae. Exp. Parasitol. 59, 257–263. Dunphy, G.B., Thurston, G.S., 1990. Insect immunity. In: Gaugler, R., Kaya, H.K. (Eds.), Entomopathogenic Nematodes in Biological Control. CRC Press, Florida, pp. 301–323. Gaugler, R., Molloy, D., 1981. Instar susceptibility of Simulium vittatum (Diptera: Simuliidae) to the entomogenous nematode Neoaplectana carpocapsae. J. Nematol. 13, 1–5. Gaugler, R., Wang, Y., Campbell, J.F., 1994. Aggressive and evasive behaviours in Popillia japonica (Coleoptera: Scarabaeidae) larvae: defenses against entomopathogenic nematode attack. J. Invertebr. Pathol. 64, 193–199. Georgis, R., Hague, N.G.M., 1981. A neoaplectanid nematode in the larch sawfly Cephalcia lariciphila (Hymenoptera: Pamphiliidae). Ann. Appl. Biol. 99, 171–177. Glen, D.M., Wilson, M.J., Pearce, J.D., Rodgers, P.B., 1994. Discovery and investigation of a novel nematode parasite for biological control of slugs. In: Pests and Diseases, Brighton Crop Protection Council (BCPC) Symposium Proceedings, vol. 2, pp. 617–624. Godan, D., 1983. Pest Slugs and Snails—Biology and Control. SpringerVerlag, Berlin.
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R.G. Rae et al. / Journal of Invertebrate Pathology 97 (2008) 61–69 Taylor, J.W., 1902–1907. Monograph of the Land and Freshwater Mollusca of the British Isles (Testacellidae, Limacidae, Arionidae). Taylor Brothers, UK. Vernava´, M.N., Phillps-Aalten, P.M., Hughes, L.A., Rowcliffe, H., Wiltshire, C.W., Glen, D.M., 2004. Influences of preceding cover crops on slug damage and biological control using Phasmarhabditis hermaphrodita. Ann. Appl. Biol. 145, 279–284. Wesenberg-Lund, C., 1934. Contributions to the development of the Trematoda Digenea. Part 2. The biology of the freshwater cercariae in Danish freshwaters. Kangel. Dan. Vidensk. Biol. Skrift. 5, 1–221. Wang, Y., Gaugler, R., Cui, L., 1994. Variations in immune response of Popillia japonica and Acheta domesticus to Heterorhabditis bacteriophora and Steinernema species. J. Nematol. 26, 11–18.
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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, 503–511. Wilson, M.J., Glen, D.M., George, S.K., Pearce, J.D., Wiltshire, C.W., 1994. Biological control of slugs in winter wheat using the rhabditid nematode Phasmarhabditis hermaphrodita. Ann. Appl. Biol. 125, 377– 390. Wilson, M.J., Glen, D.M., George, S.K., Hughes, L.A., 1995. Biocontrol of slugs in protected lettuce using the rhabditid nematode Phasmarhabditis hermaphrodita. Biocontrol Sci. Technol. 5, 233–242. Wilson, M.J., Hughes, L.A., Hamacher, G.M., Glen, D.M., 2000. Effects of Phasmarhabditis hermaphrodita on non-target molluscs. Pest Manag. Sci. 56, 711–716.
INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 97 (2008) 61–69 www.elsevier.com/locate/yjipa
Susceptibility and immune response of Deroceras reticulatum, Milax gagates and Limax pseudoflavus exposed to the slug parasitic nematode Phasmarhabditis hermaphrodita Robbie G. Rae a
a,b,*
, Jamie F. Robertson b, Michael J. Wilson
b
Max Planck Institute for Developmental Biology, Department of Evolutionary Biology, Spemannstraße 37-39, Tu¨ebingen 72076, Germany b University of Aberdeen, School of Biological Sciences, St. Machar Drive, Aberdeen AB24 3UU, UK Received 4 March 2007; accepted 12 July 2007 Available online 20 July 2007
Abstract We exposed three slug species (Deroceras reticulatum (Mu¨ller), Milax gagates (Draparnaud) and Limax pseudoflavus L.) to the parasitic nematode Phasmarhabditis hermaphrodita Schneider. P. hermaphrodita was able to cause mortality and feeding inhibition to both D. reticulatum and M. gagates but did not negatively affect L. pseudoflavus. On dissection of surviving L. pseudoflavus large numbers of P. hermaphrodita were found encapsulated in the shell of the slug. We found that by increasing shell size, the slug was able to trap invading nematodes, which could be an immune response to P. hermaphrodita invasion. This is the first report of a slug defense mechanism to inhibit P. hermaphrodita. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Phasmarhabditis hermaphrodita; Deroceras reticulatum; Milax gagates; Limax pseudoflavus; Nematodes; Slugs; Parasite; Immunity; Biological control; Shell
1. Introduction Phasmarhabditis hermaphrodita Schneider is a lethal slug parasitic nematode (Wilson et al., 1993; Speiser et al., 2001; Iglesias and Speiser, 2001; Grewal et al., 2003) that has been formulated into a biological control agent (NemaslugÒ) available from Becker Underwood, UK. Infective juveniles are mixed with water and applied using a watering can or spraying equipment. The nematodes search for slugs in soil and respond to slug associated cues such as mucus and faeces (Rae et al., 2006; Hapca et al., 2007). On locating a potential host P. hermaphrodita enters into the shell cavity at the back of the slugs mantle and is thought to release a bacterium (Moraxella osloensis), which kills the slug between 4 and 21 days after infection (Wilson et al., * Corresponding author. Address: Max Planck Institute for Developmental Biology, Department of Evolutionary Biology, Spemannstraße 37-39, Tu¨ebingen 72076, Germany. Fax: +49 (0) 7071 601498. E-mail address: [email protected] (R.G. Rae).
0022-2011/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2007.07.004
1993; Tan and Grewal, 2001). M. osloensis lipopolysaccharide is thought to be responsible for slug death (Tan and Grewal, 2002). Once the slug dies the nematodes grow to hermaphroditic adults and reproduce on the slug cadaver (Wilson et al., 1993; Tan and Grewal, 2001). As the food source depletes, new infective juveniles are produced which then search for potential slug hosts in the soil. Phasmarhabditis hermaphrodita can parasitize and cause mortality to nine slug species including Deroceras reticulatum (Mu¨ller), Deroceras panormitanum (Lessona and Pollonera), Deroceras laeve (Mu¨ller), Arion silvaticus Lohmander, Arion intermedius Normand, Arion distinctus Mabille, Tandonia sowerbyi (Fe´russac), Tandonia budapestensis (Hazay) and Leidyula floridana (Hazay) (Wilson et al., 1993; Grewal et al., 2003; Iglesias and Speiser, 2001). However, some species are not susceptible such as Limax maximus (L.), Arion subfuscus Draparnaud and Arion hortensis Fe´russac (Grewal et al., 2003; Iglesias and Speiser, 2001). For certain other species including Arion lusitanicus Mabille, Arion ater (L.) and the snail Helix aspersa Mu¨ller,
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juveniles are known to be susceptible whereas adults are not (Speiser et al., 2001; Grimm, 2002; Wilson et al., 1993; Glen D.M., unpublished). Similarly, some snail species are susceptible whereas others are not (Wilson et al., 2000). There is no information about the susceptibility of Limax pseudoflavus L. and Milax gagates (Draparnaud) to P. hermaphrodita. L. pseudoflavus is widely distributed in Europe, lives in crevices, under rubbish, in gardens, cellars, outhouses, farmyards and damp basements (Kerney, 1999). It is not generally considered to be a pest species and usually feeds on fungi, decayed plants and rubbish (Janus, 1965). M. gagates is generally distributed in gardens where it is considered to be a pest (Taylor, 1902– 1907) and can be found on farms where it is a pest of rape (Godan, 1983), leaf beet (Iglesias et al., 2001), Chinese cabbage (Vernava´ et al., 2004) and lettuce (Wilson et al., 1995). Relatives of M. gagates and L. pseudoflavus have been tested for susceptibility to P. hermaphrodita. Both relatives of M. gagates (T. sowerbyi and T. budapestensis) are susceptible to P. hermaphrodita (Wilson et al., 1993), therefore, we hypothesize that M. gagates is also susceptible. If M. gagates is susceptible to P. hermaphrodita then nematodes can be recommended as a control measure. In contrast to M. gagates, relatives of L. pseudoflavus such as L. maximus are not susceptible (Grewal et al., 2003). The reasons why some slug species are not susceptible to P. hermaphrodita has received very little attention. Grewal et al. (2003) proposed that non-susceptible gastropods will suspend feeding and contract into a nonfeeding posture to prevent nematodes entering into the shell cavity, but there has been no further research to support this hypothesis. Defense strategies for insects have evolved to stop entomopathogenic nematodes entering the mouth, anus or spiracles. Popillia japonica grubs exhibit grooming behaviour upon nematode contact and evasive behaviour when sensing nematode attack (Gaugler et al., 1994). Some insects such as Phyllophaga hirticola have mechanical barriers such as sieve plates that protect the spiracles from nematode entry (Dowds and Peters, 2002). Frequent defecation may eject nematodes entering via the anus and mandibles may crush any entering nematodes to death (Gaugler and Molloy, 1981; Georgis and Hague, 1981; Cui et al., 1993). Once nematodes have penetrated the insect’s haemocoel, the insect’s non-self response will attempt to entrap the nematode in cellular capsules that are frequently hardened by melanin (Dunphy and Thurston, 1990; Wang et al., 1994). Little is known about the immune responses in slugs and snails (South, 1992). The internal shell of slugs lies immediately beneath the mantle in the shell sac and consists of an oval flattened plate, which tends to be convex above and concave below and generally shows concentric zones of growth (South, 1992). We hypothesize that nematodes entering the mantle area will come into direct contact with
the shell and the shell may have defensive properties that immobilize and encapsulate nematodes, a phenomenon that has never previously been reported for slugs and nematodes. We exposed D. reticulatum, M. gagates and L. pseudoflavus to a range of doses of P. hermaphrodita and recorded mortality and feeding inhibition. At the end of the experiment shells of non-susceptible species were removed and examined for nematode encapsulation. 2. Materials and methods 2.1. Source of invertebrates Slugs were collected from Seaton Park, Aberdeen and Aberdeen University Botanic Gardens and stored in nonairtight plastic boxes lined with cotton wool and fed with fresh lettuce leaves. Any diseased or dead slugs were removed and only healthy slugs were used in experiments. P. hermaphrodita was supplied by Becker Underwood, UK. 2.2. Assessing susceptibility of D. reticulatum, M. gagates and L. pseudoflavus Fifteen Petri dishes (diameter 13.6 cm) were filled with 150 g of Craibstone series soil (loamy sand texture, 73.85% sand, 20.04% silt and 6.11% clay containing 4.25% w/w organic matter). The soil was air dried, sieved to 2 mm and rewetted with 23% water (v/v). Slugs were exposed to P. hermaphrodita at five different doses: 0 (untreated control), 30 per cm 2, 90 per cm 2, 150 per cm 2 and 300 per cm 2. Nematodes were evenly applied over the soil surface using a wash bottle. Ten D. reticulatum (mean weight = 0.24 ± 0.01 g, n = 150), M. gagates (0.58 ± 0.02 g, n = 150) and L. pseudoflavus (0.40 ± 0.02 g, n = 150) were added to separate Petri dishes. Five discs (2.7 cm diameter) of lettuce were added to each dish to examine feeding inhibition. The percentage of each lettuce disc eaten was recorded every three days for 12 days. Fresh discs were added every three days. The dishes were then sealed with ParafilmÒ and placed in an incubator at 16 °C. Mortality was recorded every day for 18 days. Any dead slugs were removed and examined for presence of nematodes. At the end of the experiment surviving slugs were dissected and shells removed, weighed and the number of P. hermaphrodita trapped inside was determined. The experiment was repeated three times with fresh batches of slugs and nematodes. 2.3. Experimental analysis Statistics were performed using MINTAB 12 (Mintab Inc., USA). Significance levels were taken at P 6 0.05. Mortality counts were transformed to square roots and subjected to General Linear Model ANOVA and Tukey’s pairwise comparison. Kruskal–Wallis and Mann–Whitney U tests were used to analyse data from the number of
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Fig. 1. Mean percentage mortality of D. reticulatum (a), M. gagates (b) and L. pseudoflavus (c) exposed to P. hermaphrodita. Bars represent ± one standard error.
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Fig. 2. Mean percentage eaten of lettuce discs by D. reticulatum (a), M. gagates (b) and L. pseudoflavus (c) exposed to P. hermaphrodita. Bars represent ± one standard error.
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P. hermaphrodita entering L. pseudoflavus, the weight of L. pseudoflavus shells and the number of nematodes encapsulated in the shells. The numbers of P. hermaphrodita found within L. pseudoflavus shells were transformed to square roots and the relationship between weight of shells and numbers of entrapped nematodes was analysed using regression analysis. Percentage feeding inhibition data were transformed using arcsine square root transformations and analysed using General Linear Model ANOVA and Tukey’s pairwise comparison. 3. Results 3.1. The effect of P. hermaphrodita on D. reticulatum, M. gagates and L. pseudoflavus Phasmarhabditis hermaphrodita when applied at the highest dose (300 per cm 2) caused significant mortality to D. reticulatum on day 6 (P 6 0.001) (Fig. 1a). On days 12 and 18 P. hermaphrodita applied at 90, 150 and 300 per cm 2 significantly affected the mortality of D. reticulatum (P 6 0.001). Significant mortality to M. gagates was also caused by the highest dose of P. hermaphrodita (300 per cm 2) on day 6 (P 6 0.001) (Fig. 1b). As time of exposure increased, progressively lower doses of P. hermaphrodita effected mortality in this species such that on day 12 P. hermaphrodita (150 per cm 2 and 300 per cm 2) caused significant mortality to slugs (P 6 0.001) and by day 18 P. hermaphrodita caused significant mortality to M. gagates at 90 (P 6 0.05), 150 (P 6 0.001) and 300 per cm 2 (P 6 0.001). Conversely, P. hermaphrodita did not cause significant mortality to L. pseudoflavus and there were no significant differences between mortality of the untreated and nematode-treated L. pseudoflavus at any time point with any dose of P. hermaphrodita (P > 0.05) (Fig. 1c). 3.2. Feeding inhibition of D. reticulatum, M. gagates and L. pseudoflavus exposed to P. hermaphrodita For D. reticulatum, as with mortality, feeding inhibition tended to increase with time of exposure and nematode dose. On day 6 the highest P. hermaphrodita dose (300 per cm 2) caused significant feeding inhibition to D. reticulatum (P 6 0.05) (Fig. 2a) whereas by days 9 and 12 significant feeding inhibition was caused by 90 (P 6 0.05), 150 (P 6 0.05) and 300 per cm 2 (P 6 0.001). The effect of P. hermaphrodita on feeding by M. gagates was less pronounced than that seen for D. reticulatum with no significant feeding inhibition being observed on day 6. However, by days 9 and 12 P. hermaphrodita applied at 150 and 300 per cm 2 caused a significant reduction in feeding by M. gagates (P 6 0.05; P 6 0.001, respectively) (Fig. 2b). As was the case with slug mortality, P. hermaphrodita did not have any effect on the feeding of L. pseudoflavus and there were no differences between the feeding of
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untreated and treated L. pseudoflavus at any time point with any nematode dose (P > 0.05) (Fig. 2c). 3.3. Shell weight and number of P. hermaphrodita penetrating and encapsulated in L. pseudoflavus shells Only shells from L. pseudoflavus were examined as both M. gagates and D. reticulatum were susceptible to P. hermaphrodita and in many cases shells were subsequently shed during parasitism. When examining the shells microscopically to count nematodes encapsulated, it could be seen that movement of nematodes was very much restricted by the newly laid down shell material, and it is possible that L. pseudoflavus encapsulates nematodes as an immune response (Fig. 3). There were significant differences between the numbers of P. hermaphrodita entering into the body of L. pseudoflavus exposed to the five doses (H = 33.61; df = 4; P 6 0.001) (Fig. 4). The number of P. hermaphrodita entering L. pseudoflavus differed significantly from the control at 150 per cm 2 (P 6 0.001) and 300 per cm 2 (P 6 0.001) but not at 30 cm 2 (P > 0.05) and 90 cm 2 (P > 0.05). The weights of shells were significantly different among nematode doses (H = 19.79; df = 4; P 6 0.001) (Fig. 5). Shells extracted from L. pseudoflavus exposed to nematodes (150 per cm 2) were significantly heavier than the control (P 6 0.05) but shells from treated slugs at the other doses (30, 90 and 300 per cm 2) did not differ significantly from the control (P > 0.05). The numbers of P. hermaphrodita found encapsulated inside the shells of L. pseudoflavus differed between doses (H = 37.90; df = 4; P 6 0.001) (Fig. 6). There were significantly more P. hermaphrodita found in the shells of slugs treated with 150 (P 6 0.001) and 300 nematodes per cm 2 (P 6 0.05) compared to the control. There were no differences in the numbers of nematodes found in shells of slugs treated with 30 and 90 P. hermaphrodita per cm 2 (P > 0.05). There was a significant, positive relationship between the weight of shells and the numbers of P. hermaphrodita encapsulated in the shells (control weights were removed) (P 6 0.001; r2 = 0.647) (data not shown). 4. Discussion Phasmarhabditis hermaphrodita caused significant mortality and feeding inhibition to D. reticulatum and M. gagates but had no effect on L. pseudoflavus. This is perhaps not surprising as relatives of M. gagates (T. sowerbyi and T. budapestensis) are susceptible to P. hermaphrodita (Wilson et al., 1993) and relatives of L. pseudoflavus (e.g. L. maximus) are not susceptible (Grewal et al., 2003). Therefore, P. hermaphrodita could be recommended for use in the field against M. gagates if applied at 90, 150 or 300 nematodes per cm 2. Lesions were produced at the point of entry to the shell cavity on the back of the mantle of M. gagates. Usually the
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Fig. 3. Shell of (A) untreated L. pseudoflavus and (B) nematode treated shell with P. hermaphrodita encapsulated inside.
infection resulted in shells being shed due to the build up of nematodes. At this time M. gagates was observed in a coiled position where the head was positioned at the lesion at back of the mantle. It is not known what the slug was doing but it could be possible that it was removing any nematodes present. Grooming behaviour has been recorded in P. japonica grubs to prevent nematode attack (Gaugler et al., 1994). Small lesions were also observed on the anterior mantle and on the posterior part of the tail. All these lesions had nematodes present. It may be possible that as well as mechanical means to enter host slugs
through natural openings, P. hermaphrodita may also use proteases or other enzymes to penetrate the slug’s cuticle and produce the observed lesions. The use of enzymes has been shown for a number of parasitic nematode species to gain access to host systems resulting in tissue disruption including Ascaris suum (Knox and Kennedy, 1988) and Strongyloides ransomi (Dresden et al., 1985). AbuHatab et al. (1995) reported Steinernema glaseri releasing proteolytic enzymes that increased the chances of entering through the gut wall of insect hosts. Interestingly, no lesions were observed on L. pseudoflavus, and it could be possible that this slug species may produce a lectin in its mucus that acts as a protease inhibitor, a phenomenon that has been reported for A. ater (Habets et al., 1979), which is also known to be resistant to P. hermaphrodita. Phasmarhabditis hermaphrodita did not cause mortality or feeding inhibition to L. pseudoflavus. Grewal et al. (2003) speculate that non-susceptible species will stop eating and contract to reduce the chances of nematode penetration; however, we found that L. pseudoflavus did not stop eating and no feeding inhibition was observed for 12 days. Little is known about the immune responses in slugs and snails (South, 1992). We found L. pseudoflavus encapsulated P. hermaphrodita inside the shell underneath the mantle area. There was a positive significant relationship between the weight of shell and the numbers of nematodes encapsulated within the shell. In some slugs the shells extracted contained all life stages of P. hermaphrodita including J2, J3 and adults in large abundance. Either the nematode is able to reproduce when encapsulated in the shell or the nematode can reproduce inside the slug and the resultant nematodes are trapped. This increase in shell weight and encapsulation of P. hermaphrodita may be one reason why this slug species is not susceptible to nematodes. As well as the characteristic swelling of the mantle area, infection of P. hermaphrodita causes a variety of symptoms to slugs such as reduced locomotion (Bailey et al., 2003), feeding inhibition (Glen et al., 1994), a lesion on the back of the mantle (Wilson et al., 1993) and slugs are more likely to be found under refuge traps (Wilson et al., 1994). The reasons behind these symptoms are poorly understood but parasite induced changes have been recorded in a number of molluscs. For example trematode parasites of the snail Hydrobia ulvae cause symptoms including castration (Rothschild, 1936; Lauckner, 1980), changes in crawling behaviour (Honer, 1961; Mouritsen and Jensen, 1994) and gigantism (Wesenberg-Lund, 1934; Rothschild and Rothschild, 1939; Mouritsen and Jensen, 1994; Huxham et al., 1995). The cellular defense of bivalve molluscs is controlled by haemocytes (granulocytes, hyalinocytes and serous cells) and are involved in a number of functions including wound repair, shell repair, nutrient digestion and transport, excretion and internal defense (Cheng, 1981). The bivalve’s internal defense system mainly consists of phagocytosis and encapsulation of foreign material (Cheng, 1981;
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Fig. 4. Mean number of P. hermaphrodita found inside L. pseudoflavus after 18 days. Bars represent ± one standard error.
Fig. 5. Mean weight of shells dissected from L. pseudoflavus after exposure to P. hermaphodita after 18 days. Bars represent ± one standard error.
Fig. 6. Mean number of P. hermaphrodita found encapsulated in L. pseudoflavus shells after 18 days. Bars represent ± one standard error.
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Ratcliffe et al., 1985). For example Montes et al. (1995) demonstrated that protozoa (Perkinsus sp.) were encapsulated by granulocytes in the clam (Tapes semidecussatus). The increase in size and encapsulation of P. hermaphrodita in the internal shell of L. pseudoflavus may be an immune response to invading parasites and may be one of the reasons why L. pseudoflavus is not susceptible to the nematode. This is the first report of a possible defense mechanism against slug parasitic nematodes that may contribute to immunity of slugs. Further research could concentrate on the role of the shell in other non-susceptible species such as L. maximus or in species where the shell is not an oval plate but made of granules such as in A. ater and A. subfuscus. Acknowledgments We are grateful to the Kintail Land Foundation for funding this project and to Becker Underwood for supplying nematodes. References AbuHatab, M., Selven, S., Gaugler, R., 1995. Role of proteases in penetration of insect gut by the entomopathogenic nematode Steinernema glaseri (Nematoda: Steinernematidae). J. Invertebr. Pathol. 66, 125–130. Bailey, S.E.R., Cairns, A., Latham, R., Abdel Kasi, M., Manning, P., 2003. Onset of immobilization in the slug Deroceras reticulatum Mu¨ller parasitized by the nematode Phasmarhabditis hermaphrodita Schneider. In: Slugs and Snails: Agricultural, Veterinary and Environmental Perspectives, British Crop Protection Council (BCPC) Symposium Proceedings, vol. 80, pp. 215–220. Cheng, T.C., 1981. Bivalves. In: Ratcliffe, N.A., Rowley, A.F. (Eds.), Invertebrate Blood Cells. Academic Press, London, pp. 233–300. Cui, L., Gaugler, R., Wang, Y., 1993. Penetration of steinernematid nematodes (Nematoda: Steinernematidae) into Japanese beetle larvae, Popillia japonica (Coleoptera: Scarabaeidae). J. Invertebr. Pathol. 62, 73–78. Dowds, B.C.A., Peters, A., 2002. Virulence mechanisms. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Wallingford, pp. 79–98. Dresden, M.H., Rege, A.A., Murrell, K.D., 1985. Strongyloides ransomi: proteolytic enzymes from larvae. Exp. Parasitol. 59, 257–263. Dunphy, G.B., Thurston, G.S., 1990. Insect immunity. In: Gaugler, R., Kaya, H.K. (Eds.), Entomopathogenic Nematodes in Biological Control. CRC Press, Florida, pp. 301–323. Gaugler, R., Molloy, D., 1981. Instar susceptibility of Simulium vittatum (Diptera: Simuliidae) to the entomogenous nematode Neoaplectana carpocapsae. J. Nematol. 13, 1–5. Gaugler, R., Wang, Y., Campbell, J.F., 1994. Aggressive and evasive behaviours in Popillia japonica (Coleoptera: Scarabaeidae) larvae: defenses against entomopathogenic nematode attack. J. Invertebr. Pathol. 64, 193–199. Georgis, R., Hague, N.G.M., 1981. A neoaplectanid nematode in the larch sawfly Cephalcia lariciphila (Hymenoptera: Pamphiliidae). Ann. Appl. Biol. 99, 171–177. Glen, D.M., Wilson, M.J., Pearce, J.D., Rodgers, P.B., 1994. Discovery and investigation of a novel nematode parasite for biological control of slugs. In: Pests and Diseases, Brighton Crop Protection Council (BCPC) Symposium Proceedings, vol. 2, pp. 617–624. Godan, D., 1983. Pest Slugs and Snails—Biology and Control. SpringerVerlag, Berlin.
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