Growth and Development of Aphelenchus Avenae Bastian

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. were killed for measurement immediately after being taken from food and at 4-hourly intervals for the next 20 hr. The body shortened and became thinner during the time in water without food, as the following tabulation shows:

Hours after Feeding L.S.D. )'-- (5% level) 12 16 24 28 32 Length (/Lm): 649 659 645 638 602 15·3 Width (/Lm): 28 30 30 28 28 1·2

The nematodes in the samples taken at 28 hr had become motionless for the shedding of the cuticle, which had separated at both the head and tail in the 32-hr sample.

(b) Moulting In the term moulting, usually used to describe the period when the larvae are motionless, I include a period preceding the motionless phase, when the larvae are motile but when there are no visible signs that they are undergoing a moult. In this nematode then, the term moulting as I define it covers about five-sixths of the time spent in a stage. Larvae in the motionless phase of moulting were used as the inoculum in the experiments in this section. They were placed in distilled water to regain motility and then underwent the experimental test. All experiments were repeated at least twice. Groups of about 10 larvae moulting to the fourth stage were placed on food for 6,8, 10, 12, or 14 hr, after which they were placed in water for assessment of moulting. The percentages of larvae which moulted after feeding for the above times were 10, 70,89,100, and 100 respectively. A similar experiment was done with larvae moulting GROWTH AND DEVELOPMENT OF A. AVENAE 415 to the third stage. After feeding for 4,8,12,16, and 24 hr, 0, 80, 100, 100, and 100 %, respectively, of the larvae moulted. The larvae were left in water for a further 3 days after completing the moult to the fourth stage and 33% of those which had fed for 24 hr moulted to become adults. The ability of larvae to undergo two successive moults without feeding in the intermediate stage needed further study. Groups of about 20 larvae moulting to the third stage were fed for 12, 16, 20, 24, or 28 hr before being placed in water for moulting. All larvae moulted to the fourth stage and all which had fed for more than 20 hr moulted again to become adult; 47% of the larvae which had fed for 20 hr moulted to become adults. None of the larvae which had fed for less than 20 hr moulted to the adult stage. As duration of feeding affected moulting, the effect of fasting before feeding was examined. Larvae moulting to the third stage were placed on food for 16 hr. They were then placed in water to undergo the third moult and were left in water to starve for 7 days. Groups of about 20 were then placed on food for 4, 6, 7, 9, 11, or 13 hr before being placed in water for assessment of moulting to the adult stage. The percentages of larvae which moulted after feeding for these times were 0, 0, 5, 14, 53, and 69 respectively. To study removal of the cuticle, larvae moulting to the fourth stage were placed in water for 16 hr at 27°C to regain motility. The number oflarvae in three replicates of 20, which had removed their cuticles completely, was recorded after the following treatments: initially; after 1 hr on agar; after 1 hr on fungus on agar; after 24 hr in water. The percentages of larvae which were completely free of their cuticles after these treatments were 47, 63, 98, and 79 respectively. Observations showed that the shed cuticle broke at any point along its length, mostly with a jagged, transverse tear. Some larvae, on which the cuticle was broken but not completely removed, used their stylets in an attempt to pierce the old cuticle, but this happened only when the loose cuticle opposed movement of the head.

(c) Growth of the Gonad Larvae newly moulted to the fourth stage were placed on food for 4, 8, 12, or 16 hr, after which times the length of the gonad (stained in cotton blue) was measured in samples of about 20 nematodes. Also after each time interval, about 20 larvae were placed in water to determine the number which would moult, and after moulting was complete the gonads were stained and measured. In addition, a sample of 20 larvae which had fed for 12 hr was placed in water for 4 hr and the gonads were stained and measured. Mter feeding for 4 hr, 70% of the larvae moulted when placed in water and after feeding 8 hr or longer, 100% moulted. The gonad remained about the same length after the larvae had fed for 4 or 8 hr but was considerably longer after they had fed for 12 hr; thus growth commenced at about 8 hr and continued to 16 hr (Fig. 4). The amount of growth was less when the larvae were removed from food after 12 hr and placed in water for 4 hr than when they were left on food for 16 hr. After the moult the gonad was longer than at any other time but its length after the moult depended on the length of feeding (Fig. 4). Larvae which had fed for 4 hr had an 416 J. M. FISHER average gonad length of 101 {Lm, and 70% of these were able to moult. After moulting, gonad length in the 70% averaged 216 {Lm, while in the 30% unable to moult, gonad length averaged 90 {Lm, which was not significantly different from 101 {Lm.

·100 Fig. 5 x .160 320r Fig. 4 280 /X 320 x / l24ox/x~XI ~ 0 l2sol /x / 240 / ...s::: 200 It-- go I ~ 200x~ x I -'v 160 I 0 I I/O~O 16+ /0 12°1- I 1 o~ 120 ~ l_~ __ ~ ___ L __ 80L4 _____ ~-1 80 ° - x 12 )16 '------R 12 ~~--~-----t 2u 24 Time (h,) 4 Time (h, Fig. 4.-Changes in length of ~ 120r x gonad of fourth-stage larvae fed I Fig.Fi~ 6 ------r for 4,8,12, and 16 hr and meas i x IOOr ~ ured immediately (0) and after i x_-x 180~ ~ the fourth moult (x). ~ ~ - Length of gonad in specimens I x 0 fed for 12 hr and starved in ]60~/I ______o~ water for 4 hr. Arrow indicates 40 r 0 time after which the majority of the larvae would undergo the ¥--j ---0---~L_ 12I 16! 20 24 20~ 4 8 Time (h,) fourth moult. Least significant differences at the 5% level are indicated by vertical lines.

Fig. 5.-Changes in length of gonad in fasted fourth-stage larvae fed for different times and measured immediately (O) and after the fourth moult (x). Arrow and vertical line as for Figure 4.

Fig. 6.-Changes in length of gonad of third-stage larvae fed for different times and measured immediately (O) and after the third moult ( x). Arrow indicates time after which the majority of larvae would undergo the third moult. Vertical lines as for Figure 4.

A similar experiment was set up, but this time the larvae were fasted in distilled water for 13 days immediately after completing the third moult. They were then placed on food for 4, 8, 12, 16, or 24 hr, after which samples of about 20 were either killed immediately or placed in water to determine the number which would moult. Gonads stained in cotton blue were measured in all samples_ After 4 hr of feeding, 24% of the larvae moulted when placed in water and after 8 and 12 hr of feeding, 75 and 100% respectively of the larvae moulted. In the samples measured immediately after removal from food, gonads were of similar length in the 4.,8·, and 12·hr samples but were longer in the 16· and 24·hr samples (Fig. 5). In the samples taken after moulting, the longer the nematodes had fed the longer were their gonads. GROWTH AND DEVELOPMENT OF A. AVENAE 417

A further experiment was set up using larvae newly moulted to the third stage as the initial inoculum. Samples of about 20 larvae were placed on food for 0, 4, 8, 12, 16, or 24 hr and the gonads stained and measured immediately after removal ofthe nematodes from food. Samples of about 20 were also placed in water after each feeding time and gonads measured when moulting was complete. The percentage moult after feeding for 0,4, 8, and 12 hr was 0,48,91, and 100% respectively. After the larvae had fed for 0, 4, or 8 hr, the gonad was about the same length, but after they had fed for 12 hr, it had lengthened and continued to lengthen thereafter. The gonad was longer after the moult than at any time before the moult, but its length increased with increasing duration offeeding before the moult (Fig. 6). In these experiments there was a critical duration of feeding before which less than 100% of the larvae when placed in water moulted. In these samples, the larvae which did not moult contained gonads whose length was about the same as the similar sample measured immediately after removal from food. The larvae which had moulted had considerably longer gonads.

IV. DISCUSSION The individual sigmoidal steps for growth in length in larval stages were recorded when the life cycle was synchronized by selecting, as the initial inoculum, larvae in the process of moulting. The plateaux in the growth curves coincided with the onset of the motionless period of moulting. However, shedding of the cuticle could occur irrespective of whether changes in length followed the sigmoid pattern or not. Increases in length occurred only while larvae were feeding or after the motionless period of moulting. Qhanges in width were similar to those in length except during the motionless phase of moulting and shortly thereafter, when changes in length and width were opposed. At this time, changes in volume (calculated by regarding the nematodes as cylinders) were related to changes in width and not to changes in length. The increase in width and associated decrease in length, when the head and tail withdrew from the shed cuticle, resulted in an increase in volume. Whether the recorded increase in width is valid is doubtful, as it was impossible to take separate measurements of the new and shed cuticles because at the middle of the body they were pressed against each other tightly, and the small increase in width recorded may simply represent the imposition of the new cuticle; if this were true, the nematode body would have a smaller volume. The reverse changes, which occurred when the larvae regained their motility after moulting, resulted in a decrease in volume (decrease in width, increase in length), so that the larvae had a smaller volume when moulting was complete than when the new cuticle was being formed. Presumably this mechanism permits growth in the following stage without stretching the cuticle unduly. During the early growth of a larva, increase in length was not associated with an increase in width. This could be explained in either of two ways: that the nematode could increase in length more easily than in width, or that the circumference of the nematode immediately after the moult was not circular and increases in width could not be recorded till the circumference became circular. lliustrations of cross-sections (Hechler 1962; Goodey and Hooper 1965) support the latter explanation. Whatever 418 J. M. FISHER the reason, changes in width seem more important for measuring growth than changes in length, as width is more important in determining volume. The demonstration that larvae do not have to increase in size in order to undergo the following moult is of importance in assessing variation in size that might be expected in adults. Once it has become adult the nematode is capable of increasing its length by about 50% of its initial adult length (Fisher 1969). As more than half ofthe growth in length in the fourth stage is not essential for thef6llowing moult, variation in length in the adult stage can be expected to be quite large, as has been shown (Goodey and Hooper 1965). From the information presented here it can be suggested that the longest adult would be at least twice the length of the shortest. The moulting process is not clearly understood. The important point in a stage is that at which sufficient food has been ingested for moulting to occur. On the basis of Rogers' (1962) hypothesis it seems likely that the stimulus for moulting is received at this point but there is no evidence that a stimulus is involved. If a stimulus is necessary to initiate moulting, then it is not ingestion of food as moulting can take place in the absence of feeding. The time spent before this point is variable as it depends on the duration of feeding in the previous stage; in my experiments it varied from 0 to 12 hr. The time after this point appeared relatively constant at about 20 hr and this same time interval occurred when feeding was not necessary to initiate the moult. The latter constant time suggests a constant mechanism-presumably a series of steps which end with the shedding of the old cuticle and its replacement. From this point till the resumption of motility after the shedding of the cuticle comprises about five· sixths of the time spent in a stage. The irregular breaking of the cuticle during final removal suggests that it is removed by tension. This apparently can be accomplished in a number of ways, one of which is the increase in length of the larva after the motionless period; this accounted for the removal of about 50% of the shed cuticles. Pressure against agar helped the removal of the cuticles but when food was present removal was much more rapid, either due to differences in movement associated with the presence of food or use of the stylet. Moulting in plant nematodes is evidently quite different from exsheathment in animal· parasitic nematodes. The size of the larva when shedding the cuticle lies between two extremes which are determined by the amount of food ingested in the constant period of the stage. No food may be eaten and this fixes the lower size limit; or larvae may feed for 20 hr and this determines the upper size limit. Any size between these extremes is possible depending on the amount of food consumed. Care should be exercised in the use of the word "moulting". It has previously been used to describe the motionless stage when the old cuticle is being replaced, but this appears to be only a portion of the total moulting period. For convenience here, moulting has been divided into motile and motionless periods, but this distinction is somewhat artificial as some nematodes do not have a motionless period (Lee 1965). The point at which ingestion of food no longer influences moulting is important also in the elongation of the gonad. In both the third and fourth stages, elongation of the gonad commenced about 4 hr after this time and continued whether the larvae were feeding or not. Food seems unnecessary for the elongation of the gonad, though presence or absence of food did influence the rate of elongation in the fourth stage. GROWTH AND DEVELOPMENT OF A. AVENAE 419

When both third- and fourth-stage larvae were feeding, rates of elongation of the gonads were remarkably different, being much faster in the fourth stage. This suggests that a factor (or factors) which limits elongation is present in the third-stage larvae or a stimulating factor is present in fourth-stage larvae. Whichever is true, then some mechanism exists for determining the stage of the larva. An overall hypothesis for growth of the gonad (which requires supporting evidence) is that elongation is initiated indirectly by the stimulus for moulting and that some factor(s) regulates the rate at which elongation takes place.

V. ACKNOWLEDGMEN'rS Some of this work was done at Rothamsted Experimental Station, Harpenden, England. I thank Dr. F. G. W. Jones and colleagues for use of facilities and for stimulating discussion.

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