Fundam. appl. NemalOl., 1992, 15 (2), 159-166.
Migration of Caenorhabditis elegans (Nematoda : Rhabditidae) larvae towards bacteria and the nature of the bacterial stimulus
Parwinder S. GREWAL * and Denis J. WRIGHT ** * Microbiology and Crop Protection Department, Horticulture Research International, Worthing Road, Littlehampton, W. Sussex BN17 6Lp, UK and ** Department of Biology, Imperial Co/lege of Science, Technology and Medicine, Silwood Park, Ascot, Berks SL5 7PY, UK. Accepted for publication 7 March 1991.
Summary - Chemotaxis by Caenorhabditis elegans larvae to several species of bacteria was studied on agar in specially designed Petri plates. Ali the bacteria affected the pattern of nematode migration and the degree of this alteration depended on the species of bacteria. AcinelObaeler ealcoaeelicus var. ani/ralUs, EnterobaCler amnigenus, E. cloaeae, Pseudomonas mallOphilia and Serralia liquefaeiens were more attractive than Eseheriehia coli and Pseudomonas j/uorescens biovar reaelans. Baeillus eereus, B. lhuringiensis and Baeil/us sp. showed least influence on nematode migration. Younger bacterial colonies (24-48 h old) were more attractive than the older ones (96-192 h old). When killed in-situ by chloroform fumes, bacterial colonies remained attractive to the nematode. Methods were developed to study the nature of the bacterial stimulus and the involvement of both diffusible and/or volatile attractants was found.
Résumé - Migration des larves de Caenorhabditis elegans (Nematoda : Rhabditidae) vers les bactéries, et nature du stimulus bactérien - Le chimiotactisme de Caenorhabdi/is elegans vis-à-vis de plusieurs espèces de bactéries a été étudié dans des boîtes de Petri specialement conçues. Toutes les bactéries testées ont un effet sur le mode de migration des nématodes et le degrè de modification dèpend des espèces de bacteries. AeinelObaCler ealeoaeelieus var. ani/ralus, Enlerobaeler amnigenus, E. cloaeae, Pseudomonas mallOphilia et Serralia liquefaciens sont plus attractifs quO Eseheriehia coli et Pseudomonas j/uoreseens biovar reaelans. Baeillus cereus, B. lhuringiensis and Baeillus sp. n'ont qu'une faible influence sur la migration. Les colonies bactériennes jeunes (24-48 h) sont plus attractives que les colonies âgées (96-192 h). Lorsqu'elles sont tuées par des vapeurs de chloroforme, les colonies bactériennes restent attractives pour le nèmatode. Des méthodes sont décrites pour étudier la nature du stimulus bactèrien, et la mise en jeu d'attractifs volatils ou diffusant dans la gélose a été trouvèe. Key-words : Caenorhabdilis, migration, bacteria.
Nematodes respond to a variety of extemal stimuli, natural food source has been studied by several investi e.g. mechanical, light, temperature, electrical and gators (Andrew & Nicholas, 1976; Hosono, 1978; Jans chemical (Croll, 1970; Dusenbery, 1980; Coomans & son & Nordbring-Hertz, 1983). Andrew and Nicholas De Grisse, 1981). Chemotaxis, mediated by movement (1976) found that C. elegans was attracted ta three along chemical gradients, plays an important role in bacterial species on which it could feed and was repelled food-finding, mate-finding and other aspects of nema by one toxic species indicating the importance of attrac tode interactions (Green, 1980; Zuckerman & Jansson, tion ta a suitable food. However, the nature of the 1984). However, none of the natural chemicals bacterial stimulus mediating these interactions was not mediating these responses have been identified. known. The present paper reports on the effects of Chemotaxis by the microbivorous nematode Caenor several species of bacteria on the migration of C. elegans habditis elegans to a variety of known chemicals is weil on agar plates and also presents evidence for bath the established (Ward, 1973; Dusenbery, 1980, 1983). It is volatile and diffusible nature of the bacterial stimulus. attracted to cyclic AMP, Na+, Cl-, OH-, pyridine, Oz, COz (in borate buffer, pH 8.8) and repelled by COz (in MateriaI and methods phosphate buffer, pH 6.0), D-tryptaphan, H+ and high osmotic pressure (Dusenbery, 1983). Chemoractic sig NEMATODE CULTURE nais are received at the amphids and the inner labial C. elegans was isolated from a sample of compost sensillae which are located in the cephalic region of collected from a mushroom farrn near Taunton, Somer nematades (Coomans & De Grisse, 1981; Dusenbery, set, U.K. The nematode was cultured with associated 1983). Although the anatomy of cephalic sense organs of bacteria at 22 oC on 3 % (w/v) nutrient agar (Oxoid Ltd.) C. elegans has been studied in detail (Ward et al., 1975; in Petri plates. In ail the experiments, third and fourth Ware et al., 1975; Wright, 1983) there is no direct stage larvae were selected from 8-10 day old cultures and evidence on the manner in which reception of chemotac surface sterilized with 0.1 %" Thimerosal "(w/v sodium tic signal leads ta an orientated response. ethyl mercurithiosalicylate, Sigma Ltd.) following Gre The attraction of bacterial-feeding nematodes to their wal (1990). The sterile larvae were left in sterile distilled
ISSN 1164-5571/92/02/159 08 $ 2.80/ © Gaulhier-Villars - ORSTOM 159 P. S. Grewal and D. J. Wright water in cavity blocks for 24 h at 22 oC and then used for were placed in the centre of each plate, the drops were attraction studies. then allowed ta evaporate for 5 min on a sterile-air laminar flow cabinet. The number of nematodes which BACTERIAL CULTURES moved out of the central ring towards the bacterial Ten species of bacteria out of the thirteen tested in streak (test side) and the bacteria-free side (control side) This study were isolated from the Taunton strain of C. were recorded periodically. The mean distance travelled elegans (Grewal, 1990). They included; AcinelObaCler by nematodes tawards bacteria after each time interval calcoacelicus var. anilralUs, Bacillus cereus, Bacillus sp., in each plate (replicate) was calculated using the equa EnlerobaCler amnigenus, E. cloacae, Pseudomonas aeru tion; ginosa, P. jluorescens biovar reaclans, P. mallOphilia, Pseudomonas sp. and Serralia liquefaciens. Reference X + {1 l, + 2 ~ + 3 G - (J CI + 2 ~ + 3 cJI J' strains of Bacillus lhuringiensis (HD 1), Escherichia coli where the numbers of nematodes in the respective arcs (ATCC 9001) and Pseudomonas 10laasii (Pt 51) were of the test and in the control quadrant are represented obtained from the HRI culture collection. Ali the bac by 1" ~, Gand CI> ~, C:;, respectively. The numerals 1, 2 teria except P. 10laasii were maintained on 3 % nutrient and 3 represent the distance in centimeters of the agar plates, stored at 4 oC and were subcultured at respective arcs from the centre ta the periphery and Tis monthly intervals. P. 10laasii was maintained on King's total number of nematodes added. Ali experiments were B medium (King el al., 1954). conducted at a room Temperature of 20-21 oC and the QUANTIFICATION OF NEMATODE RESPONSE data were subjected to analysis of variance.
Quadrant plates were prepared according to Andrew RELATIVE ATTRACTlVENESS OF BACTERIAL SPECIES and Nicholas (1976) with concentric rings marked at an Using the above technique, the effects of 24 h-old interval of one cm on the bottom of each Petri plate colonies of thirteen species of bacteria, growing on (8 cm diam., Fig. 1). Nutrient agar was poured into the nutrient agar, on the migratory behaviour of C. elegans plates and a loop-full of bacteria, from 24 h old cultures were studied. Bacteria were randomly divided into four grown in nutrient broth in shaker flasks at 25 oC, was groups; one group contained four species and the others streaked along a quarter of the periphery (Fig. 1). The used three species each. Three replicate plates were plates were then incubated at 25 oC for 24 h. Nematodes prepared for each species of bacteria and three bacteria (60 ± 1.8) contained in 30 III of sterile distilled water free control plates containing streaks of nutrient broth were run simultaneously. The response of nematodes to bacteria were assessed periodically and the data analysed as above.
RESPONSE OF NEMATODES TO DIFFERENT AGED BACTERIAL STREAKS Streaks of different ages of culture (24, 48, 96 and 192 h old) of two attractive bacteria; A calcoacelicus var. anilralUS and S. liquefaciens, grown at 25 oC, were tested for their effects on nematode attraction. One species of bacteria was studied at a time with three replicates of each treatment. The data on the mean distance travelled by nematodes in each plate was recorded periodicaUy and analysed as above.
RESPONSE OF NEMATODES TO DEAD BACTERIA Four species of attractive bacteria inciuding, A calcoa celicus var. anilralUs, E. amnigenus, P. mallophilia, and S. liquefaciens were streak-inoculated, individually, around a quarter of the periphery of quadrant plates. Three plates were used for each species. The plates were Fig. 1. A quadrant plate used to quantify the nematode incubated at 25 oC for 24 h. The bacterial cultures were responsc to the test marerial (dotted area). T = toral number then killed in silu by exposing the plates to drops of of nematodes added; t" t2' t) are the respective arcs in the test chloroform (50 III each) for 30 min in a fume cupboard. quadrant; and Ch C2, c; are the respective arcs in the control To test the effectiveness of chloroform treatrnent, a quadrant. Attraction index is defined as the mean distance loop-full of bacteria from each lawn was inoculared onto travelled by nematodes at any time from the central arc fresh nutrient agar plates and examined for next three towards the periphery (see text for details). days for any growth of the bacteria at 25 oc. The
160 Fundam. appl. Nematol. Migration ofCaenorhabditis elegans
response of C. elegans to the dead bacterial streaks was Diffusible-volatile aUraetants studied (immediately after ki11ing) as described earlier. Agar blocks containing the bacterial diffusates were NATURE OF ATTRACTANTS prepared in similar conditions of incubation. These agar-blocks were then placed on thin pieces of micro Volatile aUraetants scope sEde cover-glass in half the periphery of pre Non-vented quadrant plates were divided into two poured nutrient agar plates. The other half of these unequal portions by fitting aseptic impermeable plastic plates (i.e. the control sides) and also the separate control barriers to the sides and bottom of the plate with plates received agar-blocks from bacteria-free plates. " UHU " glue (Beecham, Brentford, UK) before pour Four species of attractive bacteria (that showed the ing the agar (Fig. 2). The level of the agar was kept a little production of diffusible attractive substance) were stud below the partition wall. Bacteria were streaked in the ied simultaneously with three replications for each partitioned area and incubated at 25 oC for 24 h. Nter species. Relative migration of nematodes towards the the introduction of nematodes in the centre, the quad test or control sides was assessed periodically as de rant plates were sealed with Se110tape and responses of scribed above. nematodes to a11 the thirteen species of bacteria were assessed periodically as described earEer. Results
RELATIVE ATTRACTIVENESS OF BACTERIAL SPECIES Ali the species of bacteria studied altered the normal random movement of the test nematode to a more directed and precise orientation towards bacterial col onies (Fig. 3). The degree of alteration in the pattern of nematode migration varied with the bacterial species. For instance, A. calcoaceticus var. anitralUs) E. amnige nus) E. cloacae) P. maltophilia and S. liquefaciens elicited a significant (P < 0.05) and rapid response of the nematodes when compared with any other bacteria. Bacillus species including B. cere us) B. thuringiensis and Bacillus sp. showed the least effects on the nema tode migration and attracted fewest nematodes. Other bacteria such as E. coli, P. aeruginosa and P. jluorescens biovar reaetans, P. IOlaasii and Pseudomonas sp. pro duced intermediate effects. RESPONSE OF NEMATODES TO DIFFERENT AGED BACTERIAL STREAKS Differentiai response was observed when the nema todes were exposed to different aged bacterial streaks Fig. 2. Modifïed quadrant plate to evaluate the volatile nature (24, 48, 96, 192 h old) of two attractive bacteria (Fig. 4). of the bacterial stimulus. Dotted line in the test quadrant The 24 h old streaks of A. calcoaceticus var. anitratus shows the place of plastic barrier. For other details refer to were most attractive whereas 48 h old streaks of S. Fig. 1. liquefaciens elicited significantly vigorous response. Furthermore, the streaks of A. calcoaceticus var. anitra lUs remained attractive even after 192 h incubation Diffusible aUraetants whereas the same aged lawns of S. liquefaciens were not For detection of diffusible attractants, bacteria were attractive. grown on nutrient agar plates in straight lines at an interval of one cm. Nter 24 h incubation at 25 oC, the RESPONSE OF NEMATODES TO DEAD BACTERIA inter-streak portions were taken out with a sterile scalpel Nematodes were attracted towards dead streaks of ail and placed on half the periphery (test side) of pre the bacteria tested (Fig. 5). Three bacteria namely A. poured nutrient agar quadrant plates. In the other half calcoaceticus var. anitralUs) E. cloacae) and S. liquefa of the periphery (control side), were placed similarly ciens were significantly (P < 0.05) more attractive than sized nutrient agar blocks obtained from the bacteria P. mallOphilia. free plates. Control plates received agar blocks from the NATURE OF ATTRACTANTS bacteria-free plates in both halves of the periphery. Relative migration of nematodes towards both the test Volatile aUraetants and control sides were estimated as described above. Fig. 6 shows the effects of bacteria on the orientation
Vol. 15, nO 2 - 1992 161 P. S. Grewal and D. J. Wrighr
22 l l l l l l l l l 18 0-0 A.ca/coacet/cus* 0-0 E.cloacae ..- .. B.cereus A-A P.aerug/nosa A-A S.lIquefac/ens 14 .. - .. P.fluorescens .-. Control .-. Control
li) ~ 6 0> A-A 6 ___A • ..~.. /A______.A/ -----A ~ A--- ..------. ~-- ~o--o 2 ..--..--..----.. 0 A/ ./ L . 0-_-0=-=:::::::.. / ~ .."""-1 '==.~o~.-· __. ..~•..-;;;;;,-----..=--=11::::::::::::;....------·--. -2 1 1 Il! 1 o 20 40 60 80 100 120 140 o 20 40 60 80 100 120 140 Minutes Minutes Fig. 3. Effects of imact bacterial streaks of differem species on the migration of C. elegans. Data are mean distances (mm) travelled by the third stage larvae towards or away from the bacterial streak. Bars represem LSD (p < 0.05).• A. calcoacelicus var. ani/ralus. A. ca/coacetlcus* S./lquefac/ens of C. elegans on partiùoned quadrant plates when the bacterial streak was separated from the rest of the plate l l l l l l III to avoid any diffusion through the agar. Ali the species 0-024h 0-024h A of baeteria except Bacillus sp. and P. fluorescens biovar A-A48h A-A48h / ..- .. 96h reaetans produced volaùle attraetants which significantly .-.192h /0 :=: ~~~h~,/ .. (P < 0.05) affected the nematode migraùon. B. lhu .-.cont~1 .-. contr ringiensis was more attractive to the nematodes on yro partitioned plates (Fig. 6) than on the plates where // diffusion through the agar was unimpeded (Fig. 3). ~/ Diffusible atlraetants ...... -./. ~~:>< Agar-blocks containing the baeterial diffusates eli -2 cited differential response of the nematodes (Fig. 7). 10 30 50 70 90 10 30 50 70 90 Diffusates of A. calcoaceticus var. anitralUs, E. amnige Minutes Minutes nus, E. cloacae, P. aeruginosa, P. fluorescens biovar reac Fig. 4. Effects of age of bacterial streaks on the migratory tans, P. maltophilia, P. tolaasii, Pseudomonas sp. and S. behaviour of C. elegans. Data are mean distances (mm) tra liquefaciens had significant (P < 0.05) attracùve effects velled by the third stage larvae towards or away from the on the nematodes whereas those of B. cereus, B. thur bacterial streaks. Bars represem LSD (p < 0.05).•A. calcoace ingiensis, Bacillus sp. and E. coli did not elicit any lieus var. ani/rarus. significant (P < 0.05) response. 162 Fundam. appl. NemalOl. Migration ofCaenorhabditis elegans 18 16 CD Ol l l l l l l > A ~ 14 ..- .. A.ca/coacetlcus* [;-[; E.c/oacae U) c: 0-0 P.ma/tophl/la Fig. 5. Effeets ofdead bacteria (killed in-situ) on the migratory III Cl 12 .-. S./Iquefac/ens behaviour of C. elegans. Data are mean distances (mm) tra CIl - .-. Control Qi velled by the third stage larvae towards and away from the ci [;/ bacterial streak. Bars represent LSD (p < 0.05).• A. ealcoaee >- 10 D lieus var. ani/ralUS. "Ql ~ > 8 ~ /·t: Ê 6 E / /1/' Fig. 6. Effects ofdifferent bacteria on the migratory behaviour Ql () of C. eleganswhen the diffusion through the agar was impeded. c: 4 Ol Data are mean distances (mm) travelled by the third stage êii J'~' larvae towards and away from the bacterial streak. Bars repre '5 c: 2 / / ./0 sent LSD (p < 0.05). • A. calcoaeetieus var. ani/ralUs. Ol -;6~ Ql [;--- .. :::E 0 ..~!~./.~.------. - 2 '-5'---2--'-5--4.L5,--.---,J6L5--8~5=---10~5=---1:-:!.2 5 Minutes 22 l l l l 18 l l l l l 0-0 A.ca/coaceticus* ..- .. B.cereus 0-0 E.c/oacae [;-[; S./Iquefac/ens [;-6 P.aeruglnosa 14 0-. ContrOl ..- .. P.ffuorescens .-. ContrOl CD 10 Ol > li! U) 6 c: III 0> CIl Qi 2 ci >- D -2 '-L'__L-_-'-__-'-_-'-__--'-=-_---' u !E œ ~ 22 l l l l 1 l l l l l l Ê 0-0 E.co/i E 18 ..- .. Bacil/us sp. 0-0 B.lhurlng/ensls ~; CIl () t.-[; P.maltophllla c: [;-[; E.amnlgenus sp. h[;~~" Ol 14 '-'Controt ..- .. P.lo/aasll ;:/" êii 1-. Pseudomonas '6 c: Ol 10 .-. Con". CIl 1'1./ :::E 6 ~:/ /0/ 2 /./ /.- -~ .. ...~==-"t----[;__..~;::::::=::::::= .~.--. -2 1 ! o 20 40 60 80 100 120 140 o 20 40 60 80 100 120 140 Minutes Minutes Vol. 15, nO 2 - 1992 163 P. S. Grewal and D. J. Wright 22 l l 0-0 E.cJoaeae 18 ! III l 1----1 il-il P.aerug/nosa 0-0 A.ea/eoaeetleus* ~o------P.Nuoreseens B.eereus 8 A-A A-A 0-0 Control 14 il-il S.I/quetae/ens oJ .-. Coo''O' 6 2 -2 1 ! 1 o 20 40 60 80 100 120 140 o 20 '40 60 80 100 120 140 Minutes Minutes Fig. 7. Effects of diffusates (contained in agar blacks) of different bacterial species on the migralOry behaviour of C. elegans. Data are mean distances (mm) travelled by the third stage larvae IOwards and away from the test blacks. Bars represent LSD (p < 0.05). • A. calcoacelicus var. anilralUs. Diffusible-volatile attraetants Bacillus mycoides and B. subtilis were comparatively less Fig. 8 shows that the agar blocks containing bacterial attractive and B. megatherium was a repellent. The diffusates emitted volatile substances that attracted present results expand this list and had shown that sorne nematodes. The agar blocks containing diffusates of the of the bacteria e.g. A. calcoaceticus var. anitratus, E. bacteria, E. amnigenus, E. cloacae and S. liquefaciens amnigenus, E. cloacae, P. maltophilia and S. liquefaciens attracted significantly (P < 0.05) more nematodes than were even more attractive than E. coli and P. fluorescens A. calcoaceticus var. anitratus. biovar reaetans. The group of bacteria including, E. coli, P. aeruginosa, P. fluorescens biovar reaetans, P. tolaasii Discussion and Pseudomonas sp. elicited an intermediate response while B. cereus, B. thuringiensis and Bacillus sp. were the The present study has shown that the presence of least attractive. No bacteria repelled C. elegans. bacteria affected the pattern of C. elegans migration on This species-specific response of C. elegans to various agar plates and that the degree of alteration depended on bacteria may be due ta different types and/or concen the species of bacteria. Similar observations have been trations of attractants produced by bacteria. Apart reported in the past on C. elegans (Andrew & Nicholas, from inherent differences between baeteria, the growth 1976) and on Neoapleetana carpocapsae (Pye & Burman, conditions also affect the quality of bacterial cel1s 1981). Andrew and Nicholas (1976) reported that (Schiemer, 1982). The choice of static populations of Escherichia coli, Pseudomonas fluorescens and P. aerugi bacteria rather than replenishing cultures and also the nosa were the most attractive bacteria for C. elegans. use of a similar nutrient medium for ail the species of 164 Fundam. appl. NemalOl. Migracion ojCaenorhabditis elegans 22 alkaline environment in their vicinity. Therefore, it is also possible that the attraction is based simply on the Q) Vol. 15, nO 2 . 1992 165 P. S. Grewal and D. J. Wrighl arginine and adenine they aggregated in a dense mass HOSONO, R. (1978). Age dependent changes in the behaviour directly beneath the square. This probably explains the of Caenorhabdilis elegans on attraction to Eseheriehia coli. effects of volatile nature of ammonia on nematode Exp. Gerontol., 13 : 31-36. behaviour. IGNOFFO, C. M. & DROPKIN, V. H. (1977). Deleterious effects of the thennostable toxin of Baeil/us lhuringiensis on species Acknowledgments of soil-inhabting, myceliophagous and plant-parasitic nema todes. Kans. EnlOmol. Soc., 50 : 394-398. We tank Mr. Rodney Edmondson for statistical advice, Dr. J. Paul Hand for discussions, Mr. Paul Jarrett and Mr. Steve JANSSON, H.-B. & NORDBRlNG-HERTZ, B. (1983). The en Lincoln for the provision of reference strains of bacteria. The doparasitic nematophagous fungus Meria coniospora infects financial suppon te PSG provided by the Committee of nematodes specifically at the chemosensory organs. J. gen. 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Bergey's ZUCKERMAN, B. M. & JANSSON, H.-B. (1984). Nematode Mannual of Syslemalie Baeteriology. Vol. 1. Baltimore & chemotaxis and possible mechanisms of host/prey recog London, Williams & Wilkins: 477-484. nition. A. Rev. PhylOpalhol., 22 : 95-113. 166 Fundam. appl. Nerna 101.