GROWTH AND DEVELOPMENT OF APHELENCHUS AVENAE BASTIAN By J. M. FrsHER* [Manu8cript received October 6, 1969] Summary Increases in length and breadth of larvae of A. avenae followed a roughly sigmoid pattern with plateaux representing the motionless phases of moulting. During these phases length decreased slightly, resulting in withdrawal of the head and tail from the old cuticle, and increased again as motility was resumed after the moult. It was not always necessary for larvae to feed (and hence grow) before moulting would take place. The crucial time in a stage was the point when moulting was able to occur without further feeding. The time before this was variable and depended on the previous feeding of the larvae. The time spent in a stage after this was constant. Growth of the gonad was related to this point and occurred without feeding, though more rapidly if the larva had fed. Size of adults depended partly on the amount of feeding which had occurred between the time when moulting was first able to occur and the onset of the motionless phase. 1. INTRODUCTION Growth and development of plant-parasitic nematodes have received little attention, the information available mainly concerning saccate forms such as Meloidogyne spp. (Bird 1959). On the fusiform forms there is even less information; growth curves for length of Ditylenchus dipsaci (Blake 1962) suggest a sigmoid curve as the nematodes develop through the fourth larval stage to adults, but this curve is considered to be composed of individual sigmoid steps representing growth of each larval stage (Lee 1965). There is little evidence for such steps. The periods of larval growth are separated by periods of no growth when the nematodes are moulting. Why nematodes have to moult has never been satisfactorily explained. The usual explanation is that growth stretches the cuticle, and that it would burst if the nematode did not shed it to allow for further increase in size. This hypothesis also implies that moulting is initiated when the nematode becomes too large for its cuticle. There is little information on moulting, except that relating to the last part, i.e. exsheathment (Rogers and Sommerville 1968), which is under endocrine control in some animal-parasitic nematodes (Davey and Kan 1968). This information has led to the development of an hypothesis for moulting-stimulus, elaboration of materials (under neurosecretory control), ecdysis (Rogers 1962)-and this has been partly confirmed in the fourth· stage larvae of the plant parasite, Paratylenchus nanus (Fisher 1966). Most experiments on moulting have been done with larvae terminating a survival stage and there is no evidence that the hypothesis can be applied to free­ living nematodes in which moults are separated by regular periods of feeding. * Department of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, Private Bag No. I, Glen Osmond, S.A. 5064. Aust. J. biol. Sci., 1970, 23, 411-19 412 J. M. FISHER Final shedding of the cuticle of plant-parasitic nematodes has not received much attention. Abrasion against soil particles is assumed to be responsible (Wallace 1963) but there is little evidence for this, nor for the circumferential line of weakness in the oesophageal region of the cuticle of some animal-parasitic nematodes (Rogers 1962). Finally, little is known about growth of gonads in nematodes. Each larval stage can usually be recognized by the length ofthe gonad (YukselI960; Hirschmann 1962) and nuclear changes in the gonads occur during shedding of the cuticle (Hirschmann and Triantaphyllou 1967), but there is no understanding of the physiological mechanisms. In this paper, growth and development of Aphelenchu8 avenae Bastian, 1865, a free-living, parthenogenetic nematode, which feeds both on fungi (Townshend 1964) and higher plants (Ohin and Estey 1966) are studied, especially increase in size, the relation of feeding to the onset of moulting, and the relation of onset of moulting to development of the gonad. II. MATERIALS AND METHODS A. avenae was obtained from moist soil beside Brownhill Creek, S.A., and cultured on Rhizoctonia 80lani Kuhn, strain 48 (Flentje, Stretton, and Hawn 1963) grown on one-tenth or one-twentieth dilution of Czapek-Dox agar (Ainsworth and Bisby 1954) plus yeast extract (except where otherwise stated) to which streptomycin (100 fLgJml) had been added. Nematodes in eggs and in individual stages of moulting were hand-picked for the various experiments. When required, they were added to 1-day·old cultures of the fungus, incubated at 25 or 27°C, and hand­ picked from these cultures at the appropriate time. Nematodes were measured after being in fixative (formalin-alcohol 4 : 10 vJv) for at least 24 hr. III. RESULTS (a) Growth of A. avenae Newly hatched larvae were placed on the fungus on tap water agar. About 15 were measured initially and at different times thereafter. The growth curves for length, width, and stylet length (Fig. 1) were roughly sigmoidal but their shapes give little indication of the moulting periods. Three moults occurred but the variation in moulting time of individual larvae obscured all but the final plateau in the curves. The stylet increased in length until the nematodes became adult. Nematodes moulting to the fourth stage were hand-picked for inoculum. Samples of about 14 larvae were killed initially and others after having been placed in distilled water for 4 hr. Further samples which had been placed on food initially were killed after 8 hr and at 4-hourly intervals thereafter. The growth curves for both length and width were sigmoidal (Fig. 2) with plateaux representing the motionless stage of moulting. Until the time when the nematodes became motionless, length increased continuously, even in the absence of food during the first 4 hr when the larvae were regaining their motility after the previous moult. Width decreased during this initial 4 hr and so increases in width were delayed compared to increases in length. Stylets showed no increases in length. Hand-picked larvae moulting to the third stage were left in water till they became motile and were then transferred to food. Samples of about 10 larvae were measured after 0,4, 8, 12, 16, and 24 hr. A second experiment was set up and samples GROWTH AND DEVELOPMENT OF A. AVENAE 413 of about 20 larvae were measured after 16, 24, 28, and 32 hr. The growth curves for both length and width were sigmoidal (Fig. 3); the initial decrease in width was not recorded. 18gg~ Fig. 1 _0-0----° 700 Fig. 2 800~ oP 0 600 0/0 __ 0 700C /0,0/ f -°- 600 Length 0 I 0/ /x_x l30 40 E x Leogth ./ x ----x ~ 500 t~ /e! /_x-X-1 ~ 400 0 X X/X 30 20 500f-\ /0/ /x-- - 1 ~ 1 ~ ...c 300 0.......-0 X X/WIdth E - I / ./ :i. .§" 0l /'x __ x __x/W;dth 25 ~' 20 .......l5' 0° /0 x_x.x 1;...... 200~ /- ~ - 20 ~ ~.x Stylet length 16 ~ StylellcngLh a-c,-6--"--d 15[ "--"--"-,,.--~ 12 D.-i:>.-""--- JIG ,/ 10 8~-=~,~L_~_L_L__.L...._J L--'---' o 20 40 60 80 IOU 120 140 160 180 200 12 16 20 24 28 32 36 Time (hr) Time (hr) Fig. I.-Ohanges in body length, body width, and stylet length as newly hatched larvae develop to adults. Fig. 2.-0hanges in body length, body width, and stylet length of fourth-stage larvae. Larvae about to cease moving (indicated by change in mode of movement) before shedding the cuticle of the third stage were placed on a thin layer of agar on a microscope slide and. covered with a coverslip. As soon as they became motionless, length and width were measured, and were remeasured at 4-hourly intervals till the nematodes became motile again. When the cuticle had been shed at both the head and tail, measurements of the length of the shed and new cuticles were recorded separately. /0----0 450 I ./ ...... "'... _-x -,25 0'" x ... - Length;' // 24 Fig. 3.-0hanges in body length and E ./° 1 1 I / 23 body width of third -stage larvae. Least 20 400 0/ / ''''/r 22 E significant differences in these -£ en f /0/1 ./ e!x I r/:! 21 20 measurements at the 5% level are ~ 1 20 -£ indicated for the first (---) and .....l 350 /0 ________ X~h 1/ 19 ~ second (- -- -) experiments. aI /x I x/ 18 x--x 117 3001 _ ........l- __~) 116 o 12 16 20 21 28 32 Time (hr) For the final measurement, after the larvae had become motile, they were anaesthetized in CO2• As the cuticle was being released, it remained about the same length but the body ofthe nematode shortened (Table 1), parting from the old cuticle at the head and tail. At the same time body width increased a little. With time, the old cuticle also became shorter. When the nematodes became motile after the moult, the body became elongated and thinner. The experiment was repeated with larvae moulting to the adult stage; similar changes were noted (Table 1). 414 J. M. FISHER If, after feeding for a while, larvae are removed from food and placed in water, they will later shed their cuticles without further feeding. Larvae moulting to the fourth stage were picked out, placed in water to regain their motility, and then placed on food for 12 hr, after which they were replaced in water. Samples of about 20 larvae TABLE 1 CHANGES IN BODY LENGTH AND WIDTH AS NEMATODES SHED THE THIRD AND FOURTH CUTICLES S rofers to shed cuticle, N to new cuticle Time after Length of Outicle (/Lm) Width of Outicle (/Lm) Onset of J'-- J'------., Motionless \ Third Fourth Third Fourth Phase (hr) 1 522 746 27 35 4 520(S), 517(N) 752(S), 743(N) 27 36 8 507(S), 497(N) 749(S),736(N) 28 36 12 567* 738(S), 726(N) 23* 37 16 753* 33* * Nematodes regained motility after completing moult.