Journal of Nematology 20(2):327-329. 1988. © The Society of Nematologists 1988. Cephalobus litorali . Biology and Tolerance to Desiccation

M. SAEED, S. A. KHAN, V. A. SAEED, AND H. A. KHAN1

Abstract: Cephalobus litoralis (Akhtar, 1962) Andr~ssy, 1984 reproduced parthenogenetically and completed its life cycle in 72-90 hours. Each female deposited 200-300 eggs. The nematodes showed synchronized movements in the rhythms of the anterior parts of the body. The nematodes were coiled when dried in culture medium or in slowly evaporating water droplets on the tops of culture plates, but in pellets they assumed irregular postures. Nematodes in pellets stored at high humidity could be reactivated after storage for 28 days. Key words: behavior, Cephalobus litoralis, coiling, desiccation tolerance, life cycle, parthenogenesis, synchronized movement.

Cephalobus litoralis (Akhtar) Andrfissy, by a modified Baermann funnel method. 1984 was described in 1962 by Akhtar (1) A single gravid female was placed in a cav- as Paracephalobus litoralis based on only two ity slide containing water. After oviposi- female specimens collected from soil tion, a single egg was transferred to a petri around the roots of sugarcane (Saccharum dish containing finely blended pea meal o~cinarum L.) in the agricultural farms of paste (PMP) and water. All experiments the Pakistan Council of Scientific and In- were conducted with nematodes from sin- dustrial Research (PCSIR), Lahore, Paki- gle egg progeny raised in this manner. stan. It had not been reported elsewhere For mass rearing, nematodes were cul- until the authors found it in Karachi. In tured in 14-cm-d petri dishes containing 1967 Andrfissy (3) transferred Eucephalo- pea meal paste. The dishes were placed bus diversipapiUatus (Altherr) Goodey to under a bell jar lined with black paper to Paracephalobus; however, Anderson and eliminate light. Nematodes were harvested Hooper (2) did not agree with the change. after 10 days. The contents of petri dishes In 1984 Andrfissy (4) synonymized Para- were filtered to remove the liquid and were cephalobus with Cephalobus. extracted from the substrate on a Baer- We collected C. litoralis in 1984 from soil mann funnel fitted with transparent nylon around the roots of doob grass (Cynodon tubing. Nematodes were visible as whitish dactylon (L.) Pers.) from a lawn at Aga Khan turbidity in the nylon tubing. They were Medical University Hospital, Stadium Road, collected frequently to avoid damage from Karachi, and identified it as Paracephalobus oxygen shortage (anoxia). Water was added litoralis. In the same year, however, An- to the funnel periodically. Millions of dr~ssy (4) transferred it to the genus Cepha- nematodes were collected in this manner. lobus as C. litoralis. This paper reports the Mode of reproduction: A gravid female was mode of reproduction, life-cycle duration, placed in a cavity slide. After oviposition, desiccation tolerance, and behavior of C. an egg was transferred to another cavity litoralis. slide and washed several times with dis- tilled water. The slide was enclosed in a MATERIALS AND METHODS petri dish and was viewed periodically Production of inoculum and mass rearing: through an inverted microscope. When the Nematodes were extracted from the soil egg hatched, a small amount of PMP was placed beside the newly emerged juvenile Received for publication 18 December 1986. and development was observed until the z Nematology Laboratory, Division of Applied Biology and juvenile became a female and laid eggs. Marine Resources, Pakistan Council of Scientific and Indus- trial Research (PCSIR), Rahe Saleem, Karachi-B9, Pakistan. Water was added as needed. Observations The authors thank Dr. I. Andrfissy for identification of of the development of individuals were re- nematode to species and Drs. G. Fassuliotis and M. S. Jai- rajpuri for reviewing the manuscript during their visit to the peated 10 times at a temperature of 28 + PCSIR. 5 C. The elapsed time between egg hatch- 327 328 Journal of Nematology, Volume 20, No. 2, April 1988

TABLE 1. Reactivation of, and water uptake by, RESULTS AND DISCUSSION dehydrated Cephalobus litoralis Mode of reproduction: Cephalobus litoralis Rate of water reproduced parthenogenetically and com- Days stored after Percent uptake pleted its life cycle in 3-4 days (72-90 dehydration reactivation'[" (rain) hours) at 28 _+ 5 C. Each female deposited 4 99 1 200-300 eggs which soon began embryo- 8 99 I genic development. 12 84 1-2 16 76 2-3 Desiccation: Dehydrated nematodes in 20 59 3-4 pellets were initially irregularly shrunken, 24 45 3-4 twisted, and apparently lifeless. Upon re- 28 42 4-5 hydration, the nematodes separated from t Mean of l 0 replicates. each other and became turgid in 1-5 min- utes. Most of the reactivated nematodes were either juveniles or young females, but ing and oviposition by the emerged indi- some were gravid females. As many as 11 vidual was considered the length of life juveniles were observed moving about in- cycle. Increase in number of nematodes side the body of a female ("endotokia"). was considered proof of parthenogenesis. Nematodes were noted to survive in pellets Desiccation: Nematodes were placed in for 28 days (Table 1). Within this period, watch glasses, and excess water was aspi- the nematodes stored longer resumed ac- rated with a fine-tipped dropper. Remain- tivity more slowly upon rehydration. ing moisture was removed by passing hot Behavior." C. litoralis showed synchro- air from a hair dryer over the watch glasses nized movement in the rhythms of the an- while simultaneously moving the nema- terior part of the body in the medium (pea tode mass with a small spatula to form ag- meal paste,), When the culture medium be- gregates called pellets (5). The pellets were came dry, the nematodes migrated to water dried further by rolling them on absorbent droplets on the top of the petri dish. As paper till they ceased to leave a moist spot. these droplets evaporated, the nematodes To slow down the rate of water loss, the aggregated and about 90% of them be- dried pellets were coated with paraffin oil came coiled while the others were twisted by rolling them in a petri dish containing irregularly. Coiled nematodes were also a thin layer of oil. Excess oil was removed observed in the slowly dried culture me- by rolling the pellets again on absorbent dium. When placed in slowly evaporating papers. The pellets, each weighing 60-100 water in petri dishes, the nematodes ag- rag, were placed in a humidity chamber gregated in spindle-shaped masses as the containing a glycerine-water solution water evaporated. (R.H. = 94-98%) for 4-28 days. Every 4 Cephalobus litoralis survived water stress days a pellet was removed from the cham- and withstood unfavorable environmental ber and rehydrated. The pellet was ob- conditions. Coiling under water stress has served in a petri dish with a stereoscopic been observed in several anhydrobiotic microscope while drops of water were nematodes including both parasitic and poured over it. The time required for the free-living forms (6). Saeed and Roessner worms to regain their natural posture, tur- (8) studied the behavior of some plant-as- gidity, and movement was recorded. sociated nematodes in paraffin oil and not- Behavior: Culture plates containing ed a marked reduction in body surface area. nematodes were observed with an inverted Coiling and transverse and longitudinal microscope. The plates were stored under folding of the body cuticle is generally an a bell jar, and the nematodes were ob- indication of anhydrobiotic capability in served periodically for a period of 4 weeks nematodes. C. litoralis seems to have these at 28 + 5 C. characteristics, but further studies will be Biology of Cephalobus litoralis: Saeed et al. 329 needed to ascertain whether this nematode neotype for Cephalobus persegnis Bastian, 1865, re- is merely desiccation tolerant or has also description of the species, and observations on vari- ability in taxonomic characters. Canadian Journal of the capability of surviving anhydrobioti- Zoology 48:457-469. cally. 3. Andrfissy, I. 1967. Die Unterfamilie Cephalo- Since Cephalobus litoralis matures rapidly binae (Nematoda: Cephalobidae) und Ihre Arten. Acta Zoological Academy of Sciences, Hungary 11:1-37. and has a short life cycle, it appears to be 4. Andrfissy, I. 1984. Klasse nematoda (Ordnung ideal for the study of nematode morpho- Monhysterida, Desmoscolecida, Araeolaimida, Chro- genesis. It is also inexpensive to maintain madorida, Rhabditida). Stuttgart, Federal Republic as it feeds on bacteria which can be easily of Germany: Gustav Fischer Verlag. P. 509. (In Ger- man.) grown on a medium as simple as pea meal, 5. Crowe, J. H., and K. A. A. Madin. 1975. An- and it seems to be quite malleable to a va- hydrobiosis in nematodes: Evaporative water loss and riety of laboratory purposes. Therefore, survival. Journal of Experimental Zoology 193:323- although it has not been widely reported 333. 6. Freckman, D. W. 1978. Ecology of anhydro- geographically, it meets all the criteria (7) biotic soil nematodes. Pp. 345-357 in J. H. Crowe for a suitable eucaryote model for aging and J. s. Cleggs, eds. Dry biological systems. New research and genetic manipulation. York: Academic Press. 7. Johnson, T. E. 1984. Analysis of biological basis LITERATURE CITED of aging in the nematode, with special emphasis on Caenorhabditis elegans. P. 195 in D. H. Mitchell and 1. Akhter, S.A. 1962. Paracephalobus(Nematoda: T. E. Johnson, eds. Invertebrate models in aging re- Cephalobidae) a new genus of soil-inhabiting nema- search, Boca Raton, FL: CRC Press. todes. Proceedings of the Helminthological Society 8. Saeed, M., and J. Roessner. 1984. Anhydro- of Washington 29:207-210. biosis in five species of plant associated nematodes. 2. Anderson, R. V., and D. J. Hooper. 1970. A Journal of Nematology 16:119-124.