Journal of Nematology 21(2):284-291. 1989. © The Society of Nematologists 1989. Predaceous Behavior and Life History of Odontopharynx Iongicaudata (Diplogasterida) 1

J. J. CHITAMBAR AND E. MAE NOFFSINGER 2

Abstract: The behavior of a California isolate of the predaceous nematode, Odontopharynx longi- caudata de Man, was studied in water agar culture. When feeding on an Acrobeloides sp. the predator completed its life cycle in 13 to 14 days at 25 C. Optimum temperature for reproduction of the predator was 25 C, few individuals survived at 10 C, and 30 C was lethal. Males were necessary for reproduction, and at 25 C the sex ratio was about 1:1. All postembryonic stages were voracious feeders. A single female predator consumed 30 individuals of another Acrobeloides sp. in 1.5 days. Juveniles must feed in order to complete their development. Three modes of feeding were observed depending on the prey selected. A high degree of prey selectivity occurred; 6 of 17 nematode prey species were readily consumed by the predator, but there was little or no feeding on the remaining 11 species. Predation percentage varied with prey species. Consumption of Anguina pacificae and the two Acrobeloides spp. was almost 100%, consumption of A. amsinckiae, Pratylenchus vulnus, and Trichodorus sp. was 70-78%. Difference in final predator population densities was obtained after feeding on the two species of Acrobeloides. Final predator population densities increased linearly with increasing inoculum levels of the first Acrobeloides sp. Key words: Acrobeloides, Diplogasterida, life history, Odontopharynx longicaudata, predator, prey selectivity, reproduction, temperature.

In 1985 Odontopharynx longicaudata de flee-living and plant-parasitic nematodes Man was recovered from soil around the that were extracted with the predator from roots of Poa annua L. (annual bluegrass) the original soil sample. Predator popula- growing on a golf course in San Francisco, tions were subsequently increased and California (1). Emended descriptions of the maintained on an unidentified Acrobeloides adults and morphology of the juvenile sp. (UCD No. 1) in 1.5% water agar dishes stages have been reported separately (2). incubated at 25 C. De Man (3) suggested that the two adult O. Two species of Acrobeloides in culture longicaudata found in a rotting hyacinth were originally obtained from the Depart- bulb were actually predators of the other ment of Nematology, University of Cali- free-living nematodes extracted from the fornia, Riverside. Both species and their bulb, but its biology has remained largely unidentified bacterial food source were unknown. The objective of our study was cultured on oatmeal agar. No special tech- to determine the life cycle and examine the niques were needed to maintain the bac- predaceous behavior of O. longicaudam. terial cultures because sufficient numbers of bacteria were transferred from culture MATERIALS AND METHODS to culture on the bodies of the nematodes. Odontopharynx longicaudata was first cul- Identification of the two Acrobeloides species tured on a 1.5% water agar dish containing is pending; hereafter, the two will be re- ferred to as UCD Acrobeloides No. 1 (close Received for publication 8 April 1988. to A. labiatus Ivanova) and UCD Acrobe- This study was supported in part by National Science Foundation Grant No. BSR 8213566. Mention of a trade loides No. 2 (close to A. nanus de Man). name does not indicate an endorsement of that product. Life cycle: Newly hatched second-stage 2 Postdoctoral Researcher and Senior Museum Scientist, Department of Nematology, University of California, Davis, juveniles (J2) of O. longicaudata were ob- CA 95616. Present address of first author: State of California, tained from eggs deposited by females on Department of Food and Agriculture, Analysis and Identi- fication, 1220 N Street, P.O. Box 942871, Sacramento, CA water agar culture dishes. Each dish had 94271-0001. been inoculated with four females and one The authors thank L. R. Costello, Farm Advisor, Univer- sity of California Cooperative Extension, San Mateo-San male. Five J2 were transferred to each of Francisco Counties, for assistance with field collection; D. W. 60 35-mm-d petri dishes containing water Freckman, Department of Nematology, U.C. Riverside, for the two Acrobeloides species in culture; and B. A. Jaffee and agar. Approximately 100 UCD Acrobeloides H. Ferris for their encouragement. No. 1 were transferred to each dish in one 284 Odontopharynx longicaudata Behavior: Chitambar, Noffsinger 285 or two drops of sterilized distilled water. F. & M. growing in Santa Clara County, The dishes were sealed with Parafilm and California. incubated at 25 C. Nematodes were ex- Prey inocula varied because of limited tracted from the agar dishes during a pe- availability of some species. Treatments in- riod of 11 days. Extractions were done cluded inoculations with 1) five O. longi- three times daily at 1000, 1400, and 1800 caudata (four females and one male) and a hours. To extract the nematodes, the agar single prey species per 35-mm-d water agar in each dish was quartered and then blend- dish, 2) five O. longieaudata and no prey per ed for 5-8 seconds. All stages of O. longi- dish, and 3) no O. longicaudata and a single caudata were handpicked from the suspen- prey species per dish. Each treatment for sion, processed to glycerin (7), and mounted each prey species was replical/ed four times for microscope examination. This experi- and incubated at 25 C for 2-23 days de- ment was repeated once. pending on the prey. The 23-day trial pe- Reproduction without males was tested riod allowed 1.5 life cycles of the predator. by transferring a single J4 female to a water Shorter trials were necessary with the plant- agar dish containing 1,000 UCD Acrobe- parasitic nematodes because most survived loides No. 1. Four replicates of this treat- poorly in the absence of a host. Plant-para- ment were incubated at 25 C for 25 days, sitic nematode prey were surface sterilized and observed daily for egg production. with 133 ppm ethoxyethylmercurychlo- Prey selectivity trials: The following 17 ride and 200 ppm dihydrostreptomycin nematode species representing four orders sulfate. were tested as prey for O. longicaudata. 1) Dishes containing both predator and prey Order Tylenchida, suborder Tylenchina: were examined daily for predation. At the Anguina pacificae Cid del Prado Vera & end of the experiment, percentage of pre- Maggenti, A. amsinckiae (Steiner & Scott) dation by O. longicaudata was calculated Thorne, Criconemella xenoplax (Raski) Luc from the final prey population densities & Raski, J2 ofMeloidogyne incognita (Kofoid with and without the predator. To insure & White) Chitwood, M. javanica (Treub) accuracy, the final nematode populations Chitwood, and M. hapta Chitwood, Praty- were counted before and after extraction lenchus vulnus Allen & Jensen, and Tylen- from diced agar pieces placed in modified chulus semipenetrans Cobb. 2) Order Tylen- Baermann funnels in a mist chamber for 2 chida, suborder Aphelenchina: Aphelenchus days. avenae Bastian, Aphelenchoides fragariae Effects of UCD Acrobeloides No. 1 and 2 (Ritzema Bos) Christie, and Seinura sp. 3) on final predator population were deter- Order Rhabditida: UCD Acrobeloides No. 1 mined statistically by analysis of variance. and 2, and Rhabiditis sp. 4) Order Dory- Because of differences in inoculum density ]aimida: Trichodorus sp. and Xiphinema in- and trial duration, statistical analyses of data dex Thorne & Allen. 5) Order Mononchi- from trials with the other test prey were da: Mononchus sp. not possible; therefore, the analyses made Most of the species tested were obtained were mostly qualitative. from laboratory and greenhouse cultures Effect of temperature on final population maintained in the Department of Nema- densities of O. longicaudata: One thousand tology, University of California, Davis. UCD Acrobeloides No. 1 and five O. longi- Seinura sp. and UCD Acrobeloides No. 1 and caudata adults (four females and one male) 2 were obtained from the Department of were transferred in 2 or 3 drops of steril- Nematology, University of California, Riv- ized distilled water to water agar in 35- erside. Anguina pacificae was collected from mm-d dishes in the following treatments: green stem galls on Poa annua L. growing 1) prey species and no predator, 2) pred- on the same golf course and habitat as 0. ator and no prey species, and 3) predator longicaudata, and A. amsinckiae came from and prey species. Treatments were repli- stem and seed galls on Amsinckia intermedia cated four times and incubated at 10, 15,

, L ...... 286 Journal of Nematology, Volume 21, No. 2, April 1989

TABLE 1. Time required for the development of and incubated at 25 C. After 36 hours, the the postembryonic stages of O. longicaudata at 25 C. nematodes were extracted by blending the agar for 8 seconds in 10 ml water. All post- Stage Day Hours from hatch embryonic nematode stages were counted. Eggs laid 0 Population percentage of UCD Acrobeloides First molt'i" 1-2 J2 3 0-1 No. 2 consumed by a single predator was Second molt 4 24-25 calculated. Data were subjected to analysis J3 5 48-49 of variance. Third molt 6 75-77 Voucher specimens of O. longicaudata are J4 7 96-97 Fourth molt 8 120-124 on deposit in the University of California Adults 9 144-145 Davis Nematode Collection (UCDNC). Eggs laid 13-14 240-265 RESULTS t First molt occurs within egg. Life cycle: Odontopharynx longicaudata completed its life cycle (egg to egg) in 13 20, 25, and 30 C. After 23 days, the nema- to 14 days at 25 C (Table 1). The first molt todes were extracted as in the prey selec- occurred within the egg, and postem- tivity trials. Prey and predators were bryonic stages developed at intervals of 2 counted, and data were subjected to anal- days between molts. Different juvenile ysis of variance to determine differences in stages were distinguished by i) gonad de- final nematode population densities at dif- velopment and length of reflex, 2) total ferent temperatures. body length, 3) esophagus length, and 4) Effect of inoeuIum levels ofUCD Acrobeloides distance of nerve ring and excretory pore No. 1 on reproduction of O. longicaudata: UCD from the anterior end. Sexual differences Acrobeloides No. I was transferred to water were first distinguishable in the J4 by the agar in 35-mm-d dishes at rates of 10, 20, development of the gonad and length of 50, and 100 nematodes per dish; four fe- its reflex. males and one male 0. longicaudata were Males were necessary for reproduction; immediately added to each dish. Control the sex ratio was about 1:1 at 25 C. In tests dishes contained either the prey or pred- where a single J4 female was cultured in ator species alone. Treatments were rep- the absence of males, the nematode de- licated five times and incubated at 25 C for veloped to the adult stage but failed to pro- 15 days. Nematodes were extracted and duce eggs in 25 days. Mating, first observed counted as in the prey selectivity trials, and 10 days after hatching from eggs, began data were subjected to regression analysis. with coiling of the posterior part of the Effect of activity space on predaceous ability male body around the female. Often the of O. longicauclata: Activity spaces of 480 female moved forward and backward until cm s, 240 cm 3, and 120 cm 3 were obtained the male was coiled around the vulvar re- in 35-mm-d dishes containing water agar gion. Spicules of O. longicaudata are un- by cutting and removing none, one-half, equal in size, with the left spicule 31-40 and three-fourths of the agar. UCD Acro- um long and the right spicule 5-7/zm long. beloides No. 2 were extracted from a stock The left spicule was extruded and opened culture plate as described in the prey se- the vulva for the introduction of sperm lectivity trials. Large adults were extracted into the uterus. Two female postuterine by passing the nematode suspension sacs both serve as seminal receptacles. One through a 43-~m-pore sieve. Eighty-five or two additional males often were found UCD Acrobeloides No. 2 adults and one non- nearby in the agar or coiled around the gravid female O. longicaudata were trans- anterior part of the female. The nema- ferred in a water drop to each dish. Control todes did not feed during mating. Gravid dishes contained only UCD Acrobeloides No. females were present 2 days after mating 2. Treatments were replicated four times or 12 days after hatching from eggs. In- Odontopharynx longicaudata Behavior: Chitambar, Noffsinger 287

FIG. 1. Light micrographs of Odontopha~Tnx longicaudata female. A) Feeding on UCD Acrobelo~des No. 1. B) Stomatal region during feeding. trauterine eggs had three or four cells. Fe- of the muscular anterior part of the esoph- males tended to lay more eggs inside the agus, then rupturing the cuticle with the agar medium than on its surface. large dorsal tooth (Fig. 1A, B). Depending In order to develop to mature adults, the largely upon the size of the prey, feeding juveniles must feed. Of 30 newly hatched was achieved by one of three modes: 1) J2, two survived 40 days without prey but piercing the prey's body, while maintain- developed only to the J3 stage. Survival ing continuous contact, and ingesting the and development to J3 by these two juve- body contents; 2) piercing and rupturing niles may have been due to their ingestion the prey, without maintaining continuous of the decomposed contents of the other contact, then ingesting the oozing body dead juveniles. contents; or 3) ingestion of the entire body Feeding behavior: The feeding apparatus of the prey. The predator showed no pref- of O. longicaudata is composed of a large erence for a particular feeding site on the open stoma containing one large dorsal prey's body; however, when it ingested the tooth and 12 smaller teeth. The anterior entire body, the prey was attacked near the part of the stoma is globose shaped, and tail. the posterior one-third is funnel shaped. Time required to consume a prey was 1- The large dorsal tooth is located about mid- 30 minutes for all postembryonic stages of stoma and is movable in a dorsoventral the predator. The voracious feeding be- plane within the stoma. The 12 smaller havior of 0. longicaudata was evident when teeth are nonmovable; six are positioned a newly hatched J2 completely ingested a opposite the large tooth and the other six UCD Acrobeloides No. 2 juvenile and im- are in the posterior region of the stoma. mediately severed another prey into two Odontopharynx longicaudata moved slowly pieces and consumed half of that body. through the agar and made contact with Cannibalism was observed only twice, each the prey by probing (pressing) its lip region time in a 4-5-week-old starved culture against the body. This probing action oc- where an adult female ingested aJ2. With- curred once or twice before the prey was out prey, males usually died within a few attacked, but once selected for feeding, the days, but the females survived for a much prey was attacked quickly. If the prey was longer time. Cultures without prey lasted not selected for feeding, the predator about 4 weeks before the predator popu- moved away, often without probing. lation began to decrease. The basic feeding action after probing Prey selectivity: Odontopharynx longicau- involved drawing part of the prey's cuticle data was very selective in the nematode and body into the stoma by pumping action species upon which it preyed (Table 2). Of 288 Journal of Nematology, Volume 21, No. 2, April 1989

TABL~ 2. Predation by Odontopharynx longicaudatat on selected nematode species.

Prey inoculum Final predator Nematode prey species Period (days) (no./dish) Predation (%) population:~

UCD Acrobeloides No. 1 23 100 99.7 80 a UCD Acrobeloides No. 2 23 100 99.9 132 b Anguina amsinckiae 11 100 78.0 50 a. pacificae Females 2 20 100.0 5 J2 2 20 100.0 5 Aphelenchoides fraga riae 10 40 0.0 4 Aphelenchus avenae 23 1O0 0.0 3 Criconemella xenoplax 23 50 0.0 2 Meloidogyn e hapla J 2 23 100 O. 0 0 M. incognita J2 23 100 0.0 0 M. javanica J2 23 100 0.0 1 Mononchus sp.§ 2 38 1 Prat3'lenchus vulnus 2 20 70.0 5 Rhabditis sp.§ 23 100 2 Seinura sp. 10 20 0.0 1 Trichodorus sp. 11 20 71.0 6 Tylenchulus semipenetrans 23 1O0 0.0 0 Xiphinema index 5 30 0.0 0 t Four females and one male O. Iongicaudata were added per trial. Means of four replicates per treatment. Means followed by different letters are significantly different (P = 0.01) according to Duncan's multiple-range test. Remaining nonlettered means were not statistically analyzed. § Predation observed but not quantified.

17 nematode species tested, six were readi- in either percentage of predation or final ly consumed. High prey selectivity was rep- control populations. Final population den- resented by differences in prey population sities in controls of predator and prey were consumed. Consumption of Trichodorus sp., 40. [ongicaudata, 7,635 UCD Acrobeloides Pratylenchus vulnus, and Anguina amsinckiae No. 1, and 5,863 UCD Acrobeloides No. 2. wag relatively high (70-78%), whereas it Effect of temperature: Odontopharynx lon- was almost 100% for UCD Acrobeloides No. gicaudata did not survive at 30 C with or 1, UCD Acrobeloides No. 2, and Anguina without the prey UCD Acrobeloides No. 1 pacificae. In only 2 days, Anguina pacificae (Table 3). The prey not only survived at was completely eliminated by the predator. 30 C, the population levels increased in Predation percentage of either the Monon- numbers equivalent to the final control. chus sp. or the Rhabditis sp. could not be In treatments with prey, final population determined. Odontopharynx longicaudata and densities were significantly greater at 25 C the Mononchus sp. were observed feeding than at the other temperatures (P = 0.01). on each other. Only two or three Rhabditis Few predators survived at 10 C, but their sp. were attacked, and their bodies were numbers increased significantly at 15 and only partly consumed. Sometimes feeding 20 C. Prey populations decreased as the on Rhabditis sp. was unsuccessful because number of predators increased. Even of the rapid movement of the prey after though there was no difference in the num- being attacked by O. longicaudata. bers of prey recovered at 15, 20, and 25 The final population density of O. Ion- C, their populations were significantly low- gicaudata was significantly greater when the er at 20 and 25 C than at 10 C. predator fed on UCD Acrobeloides No. 2, Predator population densities decreased than when it fed on UCD Acrobeloides No. in the absence of prey, and there were no 1 (P = 0.01). There were no significant significant differences among population differences, however, between these trials densities at the different temperatures. At Odontopharynx longicaudata Behavior: Chitambar, Noffsinger 289

TABLE 3. Effects of temperature on reproduction (final population densities) of the predator, Odontopharynx longicaudata, and prey, UCD Acrobeloides No. I.

Final population densitiest Treatment 10 C 15 C 20 C 25 C 30 C

Prey (alone) 810 a 1,540 a 1,655 a 4,600 b 5,600 b Predator (alone) 2 a 1 a 1 a 1 a 0 a Combined Prey 645 a 263 ab 27 b 53 b 6,105 a Predator 2 a 78 b 81 b 165 c 0 a "~ Numbers are means of four replications. Analyses were done on square root transformed values. Means in horizontal rows followed by the same letter are not significantly different (P = 0.01) according to Duncan's multiple-range test. the termination of the experiment, no level, final prey population density was re- males of O. longicaudata were recovered at duced to ¼7th (129 nematodes) of the con- any temperature. trol prey population density; at the 100 Prey population densities increased with inoculum level, it was reduced to ~4th (106 temperature in the absence of the preda- nematodes) of the control prey population tor. Final population densities at 25 and 30 density. C were significantly greater than at the Effect of activity space on predaceous ability: other temperatures (P = 0.01). At 15 and After 36 hours, the predator had con- 20 C, prey population densities were sim- sumed about the same number of UCD ilar and were significantly greater than Acrobeloides No. 2 regardless of activity populations at 10 C (P = 0.05). space. During the 36 hours, O. longicaudata Effect of prey inoculum levels: Final popu- consumed 35% (30 nematodes) of the prey lation densities of the predator increased population (data not shown). linearly with increasing initial density of the prey, explaining 65% of the variation DISCUSSION and describing the relationship (Fig. 2). The The present study is the first investiga- predator eliminated the prey at the 10 and tion of the biology of O. longicaudata since 20 inoculum levels. At the 50 inoculum its description by de Man in 1912 (3). Re-

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• ..... ,,...... I0 ~ 40 50 60 70 (30 100 POPULATION DENSITY OF THE PREY FIG. 2. Relationship between final population densities of the predator, Odontopharynx longicaudata, and initial population densities of the prey, UCD Acrobeloides No. 1. 290 Journal of Nematology, Volume 21, No. 2, April 1989 ports on the behavior of other predaceous ser reported that prey density can affect diplogasterids are limited to the genera predation by affecting the number of con- Mononchoides (9) and Butlerius (5,6). Studies tacts (4). Predation in Mononchoides potohi- of the quantitative behavior of certain kus is also directly related to prey density species of Mononchoides are the most com- (9). One generation of O. longicaudata is monly reported. able to eliminate populations of UCD Ac- Odontopharynx longicaudata, an obligate robeloides No. 1 at initial nematode levels predator, is very prey selective. The ability of 10 and 20. At initial nematode levels of of O. longicaudata to eliminate populations 50 and above, the predator reduces prey ofAnguina pacificae is of particular interest population progressively as the number of because it suggests that a target prey-pred- predator-prey encounters increase. ator relationship existed in the field habitat Attraction between predaceous nema- from which both species were collected. In todes and prey has been suggested by some our study, endoparasitic plant nematodes workers but has not been proven. Preda- were attacked more readily than the ec- tion by O. longicaudata may occur by chance toparasitic ones. This is generally true of encounter with its prey, but the possible predaceous nematodes (4); however, not presence of an attractant needs investiga- all endoparasitic nematodes are susceptible tion. High numbers of predators were ob- to attack by O. longicaudata, as was dem- served near concentrations of UCD Acro- onstrated with MeloidogyneJ2 and Aphelen- beloides No. 1 contained in an agar wedge choidesfragariae. The ability to attack and within 1 hour of transferring the wedge to feed on the ectoparasitic Trichodorus sp. and a water agar dish. Pheromone activity is the predator Mononchus sp. indicates a more suggested by the frequent presence of two vigorous feeding behavior than that re- or more males near each female during ported for the predaceous diplogasterid copulation. Butlerius sp. which did not feed on T. spar- At 25 C, O. longicaudata has a rather short sus Szczygiel and adults of M. aquaticus life cycle of 13 to 14 days. All of the post- Coetzee (5). This may be due, in part, to embryonic life stages are voracious feed- the elaborate stomatal apparatus of O. lon- ers, and a single female is capable of con- gicaudata and (or) the presence or absence suming 30 Acrobeloides No. 2 in 1.5 days. of specific chemical stimuli. Among the other diplogasterid predators Prey selectivity has been reported in But- reported, Butlerius sp. has a shorter life lerius sp.; however, all of the endoparasitic cycle (8.5-9 days at 25 C), will eat 12 or prey tested were susceptible to the preda- 13 PanagreUus sp. in 1 day, and frequently tor, namely, Pratylenchus sp., Meloidogyne is cannibalistic (5). Mononchoides potohikus, naasi Franklin, and A. fragariae (8,9). Mo- however, is reported to have consumed nonchoides potohikus Yeates, is not a prey- 180-380 Mesorhabditis littoralis (now Bur- selective species (9). silla littoralis (Yeates) Andrfissy) in 24 hours, Differences in final predator population and only one case of cannibalism was ob- densities in trials with UCD Acrobeloides No. served (9). 1 and 2 indicate that different prey species may affect the rate of reproduction of the LITERATURE CITED predator. The difference in reproduction was observed even when percentage of pre- 1. Chitambar, J. J., and E. M. Noffsinger. 1986, dation on those prey species was similar. Life cycle and morphology of a predaceous nematode, Odontopharynx sp. (Diplogasterida: Odontopharyngi- Increase in final populations of Odonto- dae). Journal of Nematology 18:636 (Abstr.). pharynx was directly related to prey den- 2. Chitambar, J.J., and E. Mae Noffsinger. 1989. sity. As the inoculum levels increased, the Taxonomy and postembryonic stages of the nema- tode predator Odontophar3~xlongicaudata de Man, 1912 number of prey per unit area of predator (Diplogasterida).Journal of Nematology 21 : 189-201. increased, thereby increasing the number 3. De Man,J. G. 1912. Odontopharynx longicaudata of contacts between predator and prey. Es- n. g. n. sp. Eine neue Form yon Anguilluliden. Zoo- Odontopharynx longicaudata Behavior: Chitambar, Noffsinger 291 logische Jahrbficher Abteilung f/Jr Systematik, Oko- dae) with notes on its culture. Nematologica 25:203- logie und Geographie der Tiere 33:637-642. 214. 4. Esser, R. P., and E. K. Sobers. 1964. Natural 7. Seinhorst, J. W. 1959. A rapid method for the enemies of nematodes. Proceedings of Soil and Crop transfer of nematodes from fixative to anhydrous Science Society of Florida 24:326-353. glycerin. Nematologica 4:67-69. 5. Grootaert, P., A.Jaques, and R. W. Small. 1977. 8. Small, R. W., and P. Grootaert. 1983. Obser- Prey selection in Butlerius sp. (Rhabditida: Diplogas- vations on the predation abilities of some soil dwelling teridae). Mededelingen van de Faculteit Land- predatory nematodes. Nematologica 29:109-118. bouwwetenschappen RijksuniversiteitGent 42:1559- 9. Yeates, G. W. 1969. Predation by Mononchoides 1563. potohikus (Nematoda: Diplogasteridae) in laboratory 6. Grootaert, P., and A.Jaques. 1979. Description culture. Nematologica 15:1-9. of Butlerius degrissei n. sp. (Nematoda: Diplogasteri-