256 Journal o f Nematology, Vol. 4, No. 4, October 1972 6.RASKI, D. J. 1949. The life history and morphology of sugarbeet nematode, Heterodera schachtii Schmidt. Phytopathology 40:135-152. 7.SANDSTEDT, R. and M. L. SCHUSTER. 1963. Nematode-induced callus on carrot discs grown in vitro. Phytopathology 53:1309-1312. 8.STEELE, A. E. 1965. The host range of the sugarbeet nematode Heterodera schachtii Schmidt. J. Amer. Soc. Sugar Beet Technol. 13:573-603. 9 . S T E E L E , A. E. 1971. O r i e n t a t i o n and development of Heterodera schachtfi larvae on tomato and sugarbeet roots. 3. Nematol. 3:424-426. t0.STEELE, A. E. 1971. Morphological changes in roots of sugarbeet and tomato infected with Heterodera schachtii Schmidt 1871. J. Amer. Soc. Sugar Beet Technol. (in press). ll.THORNE, G. 1961. Principles of nematology. McGraw-Hill Book Co., New York, N.Y. 553 p. Observations on the Bionomics of the Freshwater Nematode Chromadorina bioculata N. A. C R O L L and A. Z U L L I N I 1 Abstract: The biology and morphology of Chromadorina bioculata is presented. Tile nematode was abundant on the alga Cladophora of one lily pond, but absent from similar habitats in nearby ponds. The setae, caudal glands, high Iocomotory rates and positive photo response have been interpreted in relation to maintenance on, amongst and between algal filaments, suspended over large volumes of water. When placed in tap or distilled water, C. bioculata became inactive and died. The influence of pH buffers, tonicity, temperature and starvation on activity were investigated. C. bioculata survived longer in artificial sea water diluted 10 or 100 times with distilled water, or in NaC1 isotonic with sea water diluted 100 or 1000 times, than in tap or distilled water. No evidence of wide osmotic toleration or osmoregulation was observed. Activity was influenced by temperature, with peak activities occurring at the seasonal normal temperature. These findings are discussed in terms of general hematology and habitat selection. Key Words: osmotic tolerance, activity, behavior, distribution, morphology, setae, caudal glands. T h e C h r o m a d o r i d a e consist mostly of marine species, but a few are adapted to soil a n d f r e s h w a t e r habitats. Chromadorina bioculata (Schultze) occurs in fresh water and is one o f the few nonmarine nematodes with photoreceptors (4, 5). The species has been reported from North, East and Central Europe, including the Balkans. Almost nothing is known o f the biology o f freshwater nematodes, and some observations made between June and September 1971 on the morphology, biology and physiology o f C. bioculata are presented below. MORPHOLOGY: Chromadorina bioculata (Schultze in Carus, 1857), Wieser, 1954 used in Received for p u b l i c a t i o n 4 February 1 9 7 2 . 1Dep artment of Zoology and Applied E n t o m o l o g y , I m p e r i a l College, L o n d o n University, London, England; and Royal Society Fellow and Accademia Nazionale dei Lincei postdoctoral fellow, Laboratoxio di Zoologia dell Universita statale di Milano, Italy, respectively. t h e s e i n v e s t i g a t i o n s h a d t h e following measurements: Females: L = 0.56-0.63 ram; a = 20-31 ; b = 5.2-6.4; c = 5.4-6.2; V = 46-60%. Males: g = 0.49-0.57 ram; a = 21-28; b = 5.5-6.4 ; c = 5.0-6.4. The population corresponded well with the description o f the species made by Andr~ssy (1). In addition, we found that the female had a total o f 130-132 setae, and the male 124-128 setae. These included four cephalic setae, two subdorsal and two subventral, all about 7 # long. In the cervical and oesophageal regions there were four rows of somatic setae also about 7 p long. The setae in these rows became shorter towards the middle of the body. The distribution of setae had the following pattern (based on the examination o f 5 females and 4 males). 6 10 12 7-8 6 20-22 6-7 9:4 d: 4 5 8-9 10 3-4 5 19-22 3-4 The first number represents cephalic setae, Bionomics of Chromadorina bioculata: Croll, Zullini 257 2 _-I0. s Z345|; suPfaoe ~ ~ .~ e 0 2 4 I 8 10 1~ 14 16 11 '" lo 2g !~ 3o ~40" , > so. i bottom ,,oJ FIG. 1-2. I. A. Anterior part of Chromadorina bioculata (Schultze) and B. tail end showing caudal gland secretions. 2. Algae and C. bioeulata showing depth distxibution, and their coincident occurrence. Algae are assessed in relative quantities; C. bioculata data is expressed as the number of specimens of C. bioculata/mg dry weight of alga. the following numbers above the line are subdorsal somatic and below the line subventral somatic setae. In the female, each pair of figures represents the oesophageal, pre-vulval, post-vulval and tail regions, respectively. In the male t h e y r e p r e s e n t the oesophageal, g e n i t o - i n t e s t i n a l and tail regions. Unlike Andr~ssy (1) illustrated in his Fig. 6B, we observed two setae (one on each side) at the base of the caudal gland duct (Fig. 1). DISTRIBUTION OF CHROMADORINA BIOCULATA AND OTHER NEMATODES: We failed to recover C. bioculata from any of the ponds and lakes around the lmperial College Field Station except for one lily pond in which it abounds. The pond is concrete-lined and measures 7 X 15 m, having a depth of about 1 m. Tap water is added monthly to replace water lost by evaporation. Other than the planktonic habitat, from which no C. bioculata were recovered, the pond presents two distinct habitats. For the summer months the vertical walls are covered in algae, chiefly of the genus Cladophora, growing outward in dense mats of up to 2-3 cm. This periphyton also includes Ciliata, Hydra and Rotifera. The pond bottom detritus is largely derived from decaying beech tree litter, and shelters C h i r o n o m i d a l a r v a e , Ciliata, Cladocera, Diatomeae, Gastrotricha, Ologochaeta, Rotifera and many Thecamoebae, together with species of the algal genera Closterium and Pediastrum as well as nematodes. Of the 748 nematode specimens identified in the pond, nine species of nematodes were found, eight in the algae and four in the bottom mud (Table 1). Seventy-seven percent of all nematodes on the algae were C. bioeulata, and on the deeper algae it was virtually the only species present. In contrast, only 4% of the nematodes in the bottom detritus were C. bioeulata. The mean density of C. bioculata was t h r e e individuals/rag dry weight of alga, although up to 9/rag have been found. Males and females were present in equal proportions. C. bioeulata occurred in highest mean densities (7/rag dry wt. alga) 40-60cm below the 258 Journal of Nematology, Vol. 4, No. 4, October 1972 T A B L E 1. N e m a t o d e s collected from the algae on the sides of the p o n d and from the m u d b o t t o m . Habitat Nematode Number Algae on the p o n d sides Chromadorina bioeulata (Schultze) AphelenehoMes sp. Rhabdolaimus terrestris de Man Plectus parvus Bastian Camallanus sp. a Monhystera vulgaris de Man M. paludieola de Man Eudiplogaster sp. 523 108 39 2 2 2 1 1 Mud on the p o n d b o t t o m Monhystera vulgaris 114. paludicola Tobrilus sp. b C. bioculata 34 28 5 3 aLl larva of fish parasite, probably in pond fish. bTobrilus close to T. consimilis and T. aberrans in the Andr~ssy key (1964). (2) 6 Q ~a Q ! I. t ca. ; ,'o ,*s time hours 2"o 2s ;o 2 | lo 14 11 21 24 30 34 temperature °C tooto, ¢1 4 .o. 7o, TER 5 XaCI 21 5 ~t 20 time hours 3e IS 40 dilutions FIG. 3-6. 3. Mean survival of Chromadorina bioculata at various a q u e o u s dilutions o f NaC1 at 20 C. T h e control was in filtered pond water. Each line is the m e a n for 50 individuals. 4. Mean survival of C. bioculata in a range o f aqueous dilutions of artificial seawater at 20 C. The control was in filtered pond water; each line is tile mean for 50 individuals. 5. Comparison o f C. bioculata survival in NaC1 and seawater o f equal osmotic values (derived from Fig. 3 and 4). Ordinate axis is percent survival × time, abscissal axis is a q u e o u s dilution. 6. L o c o m o t o r y rates o f C. bioculata over a range o f temperatures. Each point represents a single observation;line fitted by eye. Bionomics of Chromadorina bioculata: Croll, Zullini 259 surface, and showed a second peak density at the surface (Fig. 2). LOCOMOTORY HABITS O F C. BIOCULATA: Adult C. bioculata are capable of very rapid movements, reaching a maximum of 6.5 undulations/sec at 20-22 C (Fig. 6), and swim freely by their own efforts. However, no individuals were found in samples of pond water any distance from the algae; instead they moved in and around the algal filaments. Typically they moved over the surface of the filaments in rapid, but highly coordinated bursts of movements following rectilinear or helical paths. On the filaments the body "flowed" so that there was minimum lateral displacement and each part of the body followed the previous position (8). As it flowed over the alga the body appeared to be attached, and did not fall off when on the under side (as one might have anticipated). The setae may be secretory (7), but also provide considerable sensory input regarding posture and nature of the substratum. When n o t actively changing location, movement is dominated by the caudal gland and the slightly hooked tail, the body pivoting from a subterminal position. Individuals spend considerable periods attached to the substratum by caudal gland secretions. This is a viscous, mucoid elastic, often copious secretion which we were unable to stain using Alcian blue for acid mucopolysaccharide and oil red O and Sudan Black A for lipids (10). Occasionally adults were seen attached to the substrate or to algae by means of the four cephalic setae. In this position they may have been feeding, since their intestinal lumens frequently contained green particles, but none was ever seen engulfing food. ACTIVITY AND SURVIVAL LIMITS OF C. BIOCULATA: When removed from the pond and kept in the laboratory on algae in pond water or in filtered pond water (pH 8.0-8.2), C. bioculata survived for several days. If placed in tap water (pH 7.6-8.1) or distilled water (pH 4.8-6.0), they quickly became inactive and died. Because of the survival in filtered pond water, it seemed unlikely that they were starving in the tap and distilled water, but r a t h e r were highly susceptible to some physicochemical conditions. Preliminary to any physiological laboratory studies, some factors likely to influence survival were examined. Osmon'c and ionic conditions. Five adults in each experiment were placed in 0.I ml of NaC1 in m i c r o t i t e r p l a t e s using a range of concentrations calculated in osmotic pressures equivalent to sea water, and each concentration was tested with 50 individuals. At aqueous dilutions of 10 "1 , 10 .4 and 10 "s NaC1 of the solution isosmotic to sea water, with which we started, all specimens were irreversible when inactivated within 2 hr, but they survived 20-30 hr at dilutions between 10 .2 and 10 .3 NaCI at 20 C (-+ 2). The controls in filtered pond water survived over 50 hr (Fig. 3). When repeated using artificial sea water (9), survival was longer than in NaC1 of equal osmotic pressures (Fig. 4). The area subtended by each hyperbola in Fig. 4 corresponding to the different dilutions was calculated at each dilution. This value was plotted as the ordinate (percent survival × time) against the dilution values expressed as a logarithmic scale of the abscissa (Fig. 5). This shows a maximum survival in seawater between 10 "l and 10 -3 in NaC1 dilutions. In undiluted seawater, C. bioeulata did not move after 38 sec (+ 10), and in NaC1 solutions of equal osmosity, they died after 36 sec (+ 10), these differences being statistically insignificant. Analysis of filtered pond water established a mineral salt equivalent, equal to 0.16%, corresponding to a sea water dilution of lO-2.s. pH buffers. C. bioeulata were placed in a range of buffers, but the results were irregular, suggesting that the phosphate and biphosphate ions were having an influence additional to that of the pH. Temperature. The water temperature of the pond between June and September fluctuated from 15 to 29 C. Preliminary tests in constant temperature rooms showed marked differences in locomotory rates at 12 and 20 C, with very small variance compared to the means. Single specimens were placed in 2 ml of filtered pond water in solid watch glasses, and, using ice and hot water in the medium around the watch glasses, they were taken from 0 to 35 C. We measured locomotory rates at each temperature for 30-40 sec by counting the anterior undulations per second. Highest speeds were reached between 16 and 26 C, the rate dropping sharply above and below this region (Fig. 6). By measuring displacements over a distance of 1 cm it may be estimated that an individual, under ideal conditions and with continuous activity, could travel 1.2 m/hr. 260 Journal o f Nematology, Vol. 4, No. 4, October 1972 DISCUSSION Tonicity of the fluids of nematodes from various habitats has been shown to be isotonic with the surrounding medium (3, 6). The ability to regulate water balance has been measured in some (3, 6). The marine species Deontostoma californicum has an osmotic pressure equivalent to 0.6 M NaCI (6). The osmotic pressure in many nematode-parasites o f vertebrates is equivalent to 0.2 M NaCI, and free-living soil and plant parasites about 0.15 M NaC1 (3). Survival o f C. bioculata was longest between 5.20 mM and 0.52 mM NaC1; this may be t h e lowest reported isotonicity for a nematode so far. This emphasizes the isotonic r a n g e f o r nematodes o f about 1:50. C. bioculata appeared to be unable to regulate sufficiently to survive in conditions o f osmotic or ionic stress. Greater survival in artificial seawater dilutions rather than in equivalent osmotic values of NaC1 may have resulted from NaC1 toxicity, t h r o u g h an i m p r o v e d physiological condition being maintained in the more 'balanced' artificial seawater. Temperature data is liable to acclimatization and seasonal and regional factors, but for the p e r i o d observed, this population's greatest activity o c c u r r e d at t h e m e a n d a i l y temperatures o f the season. Winter activities are to be investigated in a comparable manner. T h e s e activities are extremely high when c o m p a r e d with the data of Wallace and Doncaster (11). Distribution o f C. b i o c u l a t a w a s discontinuous, as it dominated one pond but was n o t f o u n d in similar nearby ponds containing adequate algae. Within the pond it was found on the sides in areas o f greatest algal density. This species is photopositive in visible light (4) with paired anterior photoreceptors (5). It may be deduced that such a response leads the nematodes to areas of algal growth. Their absence in the b o t t o m mud may be due to an absence o f food, lower oxygen tensions or the presence o f species potentially predatory to nematodes. The caudal gland and somatic setae have been observed in the behavior o f C. bioculata; they are probably associated with attachment and with the considerable agility and sensory c o o r d i n a t i o n required by a species living suspended or precariously attached to algal filaments over a deep water habitat. The high activity rates may also be related to the need to maintain or regain their station in an aqueous h a b i t a t . S u c h h i g h activity rates, while doubtless permitting entry to an area o f algal growth, could not be maintained, for it would interfere with feeding and mating and would consume large quantities o f energy. LITERATURE CITED 1. ANDRASSY, 1. 1960. Nematoden aus dem Periphyton der Landungsmolen der Donau zwischen Budapest und Moh~cs. Ann. Univ. Sci. Budapest Sect. Biol. 3:3-21. 2. ANDRASSY, 1. 1964. Ein Versuchsschliissel zur Bestimmung der Trobrilus Arten (Nematoda). Ann. Univ. Sci. Budapest Sect. Biol. 7:3-18. 3. ARTHUR, E. J. and R. C. SANDBORN. 1969. Osmotic and ionic regulation in nematodes, p. 429-464. In M. Florkin and B. I. Scheer [ed.]. Chemical zoology, 3rd edit. Academic Press, New York and London. 4. CROLL, N. A. 1966. The phototactic responses and spectral sensitivity of Chromadorina viridis (Nematoda, Chromadorida) with a note on the nature of the paired pigment spots. Nematologica 12:610-614. 5. CROLL, N. A., 1. L. RIDING and J. M. SMITH. 1972. A nematode photoreceptor. Comp. Biochem. Physiol. 41 B. (in press). 6. CROLL, N. A. and D. R. VIGLIERCHIO. 1969. Osmoregulation and the uptake of ions in a marine nematode. Proc. Helminthol. Soc. Wash. 36 : 1-9. 7. GERLACH, S. A. 1953. Die bioz~notische Gliederung der Nematoden fauna an den Deutschen Kusten. Z. Morphol. Tiere 41:411-512. 8. GRAY, SIR JAMES and H. W. LISSMAN. 1964. The locomotion of nematodes. J. Exp. Biol. 41:135-154. 9. HALE, L. J. 1958. Biological laboratory data. Methuen, London. 10. PEARSE, A. E. 1960. Histochemistry theoretical and applied. Churchill, Ltd., London. 11. WALLACE, H. R. and C. C. DONCASTER. 1964. A comparative study of the movement of some microphagous, plant parasitic, and animal parasitic nematodes. Parasitology 54:313-326.