differential reduction of vital dyes in certain oligochetes

DIFFERENTIAL REDUCTION O F VITAL DYES IN CERTAIN OLIGOCHETES

C. M. CHILD AND OLIN RULON Zm7ogbal Laboratory, The UlcZvers-ity of Chicago

Reduction of vital dyes in decreased oxygen provides a method which makes possible the direct demonstration of physiological gradients or diff erentials in living intact individuals of many species during development and later life. The present paper is concerned with results obtained by this method in Tubifex tubifex, with some data on other microdrilous forms and earthworms.

METHODS

Several preparations of methylene blue were used with no appreciable difference in results except that in a given concentration by weight of some preparations, for example, a Coleman and Bell methylene blue ‘for bacilli’ the solution was deeper in color and stained more rapidly than some other preparations (e.g., Coleman and Bell methylene blue ‘vital’). Concentrations from 1/5000 to 1/200,000 were used and the period of staining before sealing varied from a minute or less to several hours. In some cases the leucobase of methylene blue prepared with HCl and Na2S20, (Child, ’34 b) wa.s used but because of its toxicity proved less satisfactory than aque- ous solutions of the oxidized dye. With sufEicient staining methylene blue is toxic for all species used. Evidences of toxicity are decrease in motility, apparent stiffness appear-

‘Thin investigation waa aided in part by funds from a grant by the Rockefeller Foundation for biological research at The University of Chicago,

427

428 C. M. CHILD AND OLIN RULON

ing first in the more susceptible anterior and posterior regions, moniliform outline of the body, particularly in the posterior region, resulting from strong intersegmental con- striction, retardation or absence of reduction and finally disintegration, beginning in the more susceptible regions and gradually extending over the whole body.

Janus green was found more satisfactory than methylene blue because the differentials appeared more clearly with the change of color from blue-green to red with partial reduction of the dye than with the mere loss of color of methylene blue, but the differentials shown by the two dyes are the same. Since J a m s green is more toxic than methylene blue, lower concentrations, 1/50,000 to 1/500,000, were used with short staining periods. I n some experiments several animals were brought into Janus green 1/250,000 or 1/500,000 and sealed at once in a small volume of the solution. With these concentrations the amount of dye in the solution was so small that only slight staining occurred before reduction. The usual procedure, however, was 1 to 3 minutes staining in Janus green 1/50,000 followed by washing and sealing in water.

For reduction one to several animals with a small amount of fluid were placed in a shallow depression slide or in a small cell formed by a ring of melted vaseline, covered with exclu- sion of air bubbles and sealed, either by running melted vaseline around the cover glass or by pressing it down upon the vaseline ring. With decrease in oxygen content in the sealed preparation, reduction of the dye in the tissues occurs. The continuous movements of motile animals provide for equal distribution of oxygen in the sealed preparation, but when motility is decreased or eliminated by anesthetics or otherwise body regions which are bent or coiled on each other may reduce more rapidly than other parts because oxygen decrease occurs more rapidly about and in the greater volume of tissue than elsewhere. Repeated observations of reduction in individuals sealed singly shows, however, beyond ques- tion the relation of such local irregularities to position of

DIFFERENTIAL REDUCTION IN OLIGOCHETES 429

parts of the body in relation to each other. Moreover, the body of the non-motile animal can usually be arranged before sealing so that such local irregularities are avoided. Also in sufficiently large cells position of animals can be changed by gravity.

Time between sealing and occurrence of reduction varies of course with number and size of animals, volume of fluid and depth of staining. For example, when three or four individuals of Tubifex, 25 to 35 mm. in length, are sealed in a shallow depression slide in 2 to 3 drops of fluid, reduction of methylene blue may become evident in 3 to 4 minutes and be complete or almost complete in 10 minutes, provided no parts are so deeply stained that reduction is retarded by injury. With a single animal in a larger volume of fluid reduction is of course less rapid. With three or four similar individuals sealed after staining 1 to 2 minutes in Janus green 1/50,000 the red color of the partially reduced dye begins to be visible in 15 to 20 minutes, but complete reduction may require an hour or more. With continued decrease of oxygen reduction of Janus green to the colorless form usually occurs.

On opening the sealed preparation after reduction of methylene blue the\ blue color of the oxidized dye reappears in a few seconds and, since the animals are sealed in dye solution, the stain may be deeper than before reduction, but with the lower concentrations and rapid reduction repetition of reduction on the same animals with the same result is possible until the stain becomes toxic. Janus green reduced to the red form does not become blue-green again on opening, but after reduction to the colorless form return to red may occur on exposure to air.

In working with different species the most satisfactory range of concentration and staining period must be determined for each. With methylene blue the range is in general wider than with Janus green. In Tubifex a considerable number of segments from the posterior region anteriorly is usually injured by 3 minutes staining with Janus green

THE JOWRXAL OP EXPERIMENTAL zoIJLoaY, VOL. 74, NO. 3


1/50,000. Reduction after 1 minute staining with this concentration usually occurs without injury.

By sealing a number of animals in a relatively small volume of methylene blue in low concentration it is often possible to obtain visible staining only in the parts which reduce less rapidly. In such preparations oxygen decrease occurs so rapidly that the more rapidly reducing regions reduce the dye as rapidly as it enters. Because of rapid staining and slower reduction such differentials are less readily obtained with Janus green.

The attempt is made to indicate stages of differential reduction by curves (figs. 1 to 15). These curves, however, are not based on quantitative data but are merely representations of stages of reduction as they appear to the eye. In all figures the anterior end is at the left, posterior end at the right. The horizontal base line indicates no visible reduction and the height of the curve above this line the degree of reduction. Parts of curves drawn in broken line indicate regions injured by the dye, as shown by loss of motility or moniliform outline. Regions of more extreme injury with separation of segments and beginning disintegration are indicated by short cross lines.

The differentials described and figured below concern primarily the body wall. Animals remaining in the dyes long enough for staining of internal organs throughout the body usually show considerable injury of the more susceptible regions of the body wall. It is possible, however, to stain the segmental ganglia of the younger segments without visible injury of the body wall. Ganglia of other regions can also be stained slightly without visible injury of the body wall of the segment concerned. After such staining rate of reduction of the ganglia in relation to that of the body wall can be observed, but because of the impossibility of providing for equal distribution of oxygen inside the body the significance of differences between external and internal parts is somewhat uncertain.


DIFFERENTIAL REDUCTION IN TUBIFEX TUBIFEX

Motile, alzirnals. Staining of the body wall is at first almost or quite uniform throughout, but differentials in depth of staining gradually appear. The prostolhium becomes more deeply stained than other parts and depth of staining decreases posteriorly from it over a few segments. The clitellum, when present also becomes more deeply stained than adjoining segments and the posterior growing region stains deeper than middle regions but less deeply than the prostomium and anterior segments and depth of stain decreases anteriorly from the growing region over a large number of segments. Stages of reduction in the body wall of animals without clitellum are represented in figures 1 to 5. Reduction first becomes evident anteriorly in the prostomium and about the same time, or in Janus green 5 to 10 minutes later, occa- sionally slightly earlier, in the posterior growing region immediately anterior to the anal segment (fig. 1). From the prostomium it progresses posteriorly to the second segment and to following segments in a steep short gradient extending over only a few segments (figs. 2 to 5). From the posterior growing region it progresses anteriorly much more rapidly in a long, much less steep gradient which finally extends over the posterior two-thirds or more of the body length (figs. 2 to 5). The sharp difference in rate of reduction in most animals between the anal segment and the region immediately anterior to it is of particular interest (figs. 1 to 4). The original anal segment is an old segment and the region anterior to it is either a growing region giving rise to new segments, or in full-grown animals, a much younger segment. The difference in reduction rate indicates clearly a marked physiological difference between the two regions. Regen- erating posterior ends consisting of small developing segments are also rather sharply marked off from older se-gnents anterior to them by more rapid reduction.

In animals with developing or developed clitellum this reduces almost as rapidly as the prostomiurn and posterior region and usually with a slight gradient from anterior to

432 C. M. CHILD AND OWN RULON

posterior end (fig. 6, C). Small, apparently physiologically young animals reduce in general more rapidly than large.

After staining 1 minute with Jams green 1/50,000 reduction usually occurs without visible injury but 2 to 3 minutes staining usually produces injury indicated by moniliform outline and decrease or loss of motility, beginning at the posterior growing region and progressing anteriorly over a

3 c 5

a

F i g s . l t o 6 Stages of differential reduction of vital dyes in body wall of actively motile individuals of Tubifex tubifex not appreciably injured by the dyes. Horizontal base line indicates no visible reduction, height of curve, degree of reduction; anterior end left, posterior, right. Figures 1 to 5, animals without clitellum; figure 6, animals with clitellum, C.


variable number of segments, but reduction may still occur in the injured' region, or may be retarded in the body wall and still occur in internal organs, so far as they are stained. Injury and retardation of reduction begin to appear at the anterior end a t about the same time and progress slowly posteriorly as staining continues. With still longer staining before reduction disintegration and death progress rapidly from the posterior growing region anteriorly in a long gradient with slight differential and slowly from the prostomium posteriorly in a short steep gradient, and reduction is much

Figs. 7 to 9 Differential reduction indicated as in figures 1 to 6 after injury of anterior and posterior regions by the dyes; broken lines represent regions visibly injured, as indicated by decrease or lorn of motility and moniliform outline; short cross lines, disintegrating regions. Figure 7, early, figure 8, late stage in animals without clitellum; figure 9, late stage in animals with clitellum, C.

retarded or does not occur at all as death approaches or occurs. The posterior region is more susceptible than the anterior region and disintegrates earlier, though usually somewhat less deeply stained. Methylene blue is much less toxic than Janus green, but with sufficient staining similar differentials of injury and disintegration appear.

Reduction gradients after injury and retardation of reduction by the dye has begun are indicated in figure 7 early, and figure 8 later stage in animals without clitellum. In figure 7 reduction is retarded in prostomiurn and posterior


region, but posterior and anterior to the retarded regions the usual reduction differentials appear. In later stages of reduction the middle uninjured regions may show more rapid reduction than any other parts (fig. 8), not because their rate is altered, but because the rates of other parts are decreased. Reduction in a deeply stained animal with retardation in clitellum (C) as well as anteriorly and posteriorly is indicated in figure 9.

To sum up, reduction gradients in the normal animal, staining gradients with sufficient staining, injury and disintegration gradients, are all the same as regards regions concerned and directions, but since the regions which normally reduce most rapidly are also injured first with continued staining, differential injury may result in more or less complete reversal of the reduction gradients.

Besides the longitudinal differentials, a very distinct ventrodorsal differential appears at all levels from the oral region posteriorly in all animals observed. Reduction occurs first in the midventral body wall and progresses laterally and dorsally. In segments of the posterior fourth more or less 1/50,000 Janus green usually reaches the ventral nerve cord with 1 to 2 minutes staining and as reduction occurs it can be seen that at each level in this region reduction in the segmental ganglion precedes reduction in the ventral body wall of the same segment. In the middle body region the ganglia become appreciably stained by Janus green 1/50,000 only after 10 to 15 minutes in the solution and with this staining period the posterior region is much injured or killed over a fourth or more of the body length and the prostomium is also usually injured. The segmental ganglia are less readily seen in the middle region, but when they are visible it appears that they and the inner surface of the body wall directly ventral to each ganglion show the first evidences of color change at about the same time, or the ganglion slightly earlier than the body wall, and the body wall reauces from the inner surface outward. This course of reduction in the body wall is perhaps in part the result of local oxygen decrease by the

DIFFERENTIAL REDUCTION IR OLIGOCHETES 435

ganglion, but the general ventrodorsal differential character- istic of the whole body wall is evidently independent of such local differences and indicates an intrinsic ventrodorsal physiological differential. When the intestine is stained parts lying ventrally usually reduce before dorsal parts. This differential, however, is doubtless due to the more rapid oxygen decrease ventrally inside the body and does not indicate an intrinsic intestinal differential. Reduction is apparently slightly more rapid locally about the ventral seta-groups, but whether a similar local differential occurs in relation to the dorsal groups is uncertain. With methylene blue the ventrodorsal differential appears clearly but it is difficult to determine through the stained body wall when loss of color occurs in the segmental ganglia.

Indications of physiological isolatiolz irz posterior regions. So far as known, Tubifex does not regenerate a head after removal of more than a few anterior segments, but is capable of regenerating a posterior end at all except extreme anterior levels. I t is not to be expected, therefore, that fission with development of new individuals, whether before or after separation will occur in this animal, but the possibility of some degree of physiological isolation and even of separation of a posterior part of the body is not excluded. If such separation occurs the posterior piece undoubtedly dies because a head does not develop and feeding is impossible, but the anterior piece regenerates a new posterior end.

Attention is called to this possibility because of conditions and behavior observed in a stock collected in early April from mud about a spring after a very severe winter. Most individuals of this stock were 25 to 40 mm. in length, some even longer, and possessed a clitellum in some stage of development and in almost every individual the posterior end had been lost and was regenerating (fig. 10, R). Differential reduction of dyes in these animals showed a condition not observed in other material collected in summer from the same and other localities during two successive years. In addition to the usual anterior and posterior reduction differentials

436 0. M. CHILD AND OLIN RULON

and early reduction of the clitellum a rather sharply limited region from one-fourth to one-third the length from the posterior end reduced almost as rapidly as the head and posterior end (fig. 10). This early reduction occurred chiefly in the ventral body wall. Dorsally there was little difference from ad joining regions. From this localized region reduction progressed posteriorly and met the differential progressing anteriorly from the growing region, but there was little progress of reduction anteriorly until the whole posterior fourth or third had reduced. Then reduction progressed anteriorly in the usual manner (figs. 4 and 5). In other words, there is in these animals a localized region of increased rate of reduction at' a somewhat varia.ble distance from the posterior end with a reduction gradient extending posteriorly

C Rv \

Fig.10 DiBerentiaJ reduction indicated as in figures 1 to 6 in animals with clitellum, C, region of rapid reduction in posterior third, suggesting physiological isolation and regenerating posterior end, B.

from it. Moreover, slight mechanical stimulation posterior to this region was generally followed by contraction of only the posterior fourth or third, but contraction of the whole body usually followed stronger stimulation. Also respiratory movements of the posterior region often stopped at this level. And finally, a few animals 12 to 15 mm. found in this stock showed only the usual anterior and posterior differentials with no indication of a localized region of rapid reduction elsewhere. As will appear below, the head regions of developing zooids in species which undergo fission at definite body levels also appear as regions of rapid reduction with a gradient of decreasing rate posterior to them.

All the facts at hand suggest that this localized region of rapid reduction may indicate some degree of physiological isolation of a posterior part in these long animals, resulting


in development of a new gradient with high end anterior in the isolated part. Such local change in condition probably concerns primarily the nervous system and determines some degree of independence in motor activity and some slight degree of dominance by the high end of the gradient. Since head development apparently cannot occur at these levels, physiological isolation does not result in development of a morphologically distinguishable zooid, but it may conceivably attain a degree which permits separation of the two parts. The presence of a regenerating posterior end in almost every individual of the stock concerned suggests the earlier occurrence of such a separation and the size of the most posterior old segments indicates that a considerable number of segments must have been lost. The fact that these animals had survived an exceptionally severe winter suggests further that the physiological depression during the winter, with perhaps little intake of food may have been factors in determining physiological isolation and loss of a posterior end and later a second partial isolation of another region anterior to that lost.

Reduction in animals with decreased motor activity. Intact Tubifex individuals usually show intense motor activity in the sealed preparations and in general when removed from mud to clear water. Motor activity is apparently greatest in the head region, somewhat less in the posterior region and least in intermediate parts. In view of the possibility that motor activity might be an essential factor in determining the reduction differentials, attempts were made to decrease or eliminate movement by anesthetizing with chloretone or ethyl urethane, In some cases animals were first stained, then anesthetized, in others Janus green was added to the anesthetic solution and the animals were washed and sealed in the anesthetic solution. In animals stained 1 to 2 minutes with Janus green 1/50,000' in chloretone 1/2000 and washed and sealed in the chloretone solution movement was greatly decreased. Slight, slow movements of the head and of other parts might occur and occasional heart beats and contrac-


tions of blood vessels were observed. In general motor activity was more decreased in the head and posterior region than in other parts, that is, the differential susceptibility to chloretone was the same as that to the dyes and to KCN (Hyman, '16). In some animals prostomium and a longer or shorter posterior region were injured so that reduction was retarded in them, but in other parts the usual differentials appeared, as in figure 7. In others reduction occurred before visible injury and the differentials were similar, though perhaps somewhat less steep than those in motile animals as indicated in figures 1 to 6. In concentrations of chloretone above 1/2000 injury and disintegration usually began before reduction and reduction was limited to the least susceptible regions or did not occur at all. In chloretone 1/4000 considerable movement persisted and the usual differentials appeared. Reduction usually occurred somewhat more slowly in the anesthetized than in active animals.

Ethyl urethane was less satisfactory than chloretone. A 1% solution did not appreciably decrease movement, but by gradual addition of crystals of ethyl urethane to a few drops of water containing one or more animals movement could be considerably decreased and staining and reduction carried out without visible injury. In such cases reduction differentials were similar to those of active animals. Complete elimination of movement by ethyl urethane was followed in a few minutes by the same gradients of injury and disintegration as with other agents. Apparently Janus green and these anesthetics are to some degree additive in their effects. Anesthetized animals seem to be somewhat more susceptible to the dye than normal animals, but further experiment con- cerning this point is desirable.

In the course of work with Tubifex it was found that motor activity could be greatly decreased without the use of anesthetics or other chemical agents. In order to determine possible effects on differential reduction of isolation with no possibility of aggregation or of burrowing in mud single animals were placed in finger bowls in clear water and kept


in darkness, except for brief periods of observation in day- light. Under these conditions the animals were at first in- tensely and continuously active, but movement gradually decreased and after a week or more, though intact and normal in appearance and capable of movement they were very in- active, remaining almost motionless for perhaps several minutes when unstimulated and showing relatively slight and brief motor reactions to stimulation. When pipetted to slides they contracted and the head became temporarily active, but its movements were much slower than in control animals and soon decreased. After 7 and again after 10 days of such isolation animals were stained as usual in Janus green and sealed either singly or two or three together. After sealing some movement of the head and occasional slow bendings or contractions of other parts occurred, but the animals often remained almost or quite motionless for several minutes. There was certainly no more movement than in chloretone, perhaps even less. With the shorter periods of staining (1 to 2 minutes) they remained intact during reduction and showed the usual anterior and posterior differentials, but in some individuals reduction was slower than in active animals and relatively slower in the head region than posteriorly. After 10 days’ isolation some decrease in size had occurred and the clitellum had disappeared. The isolated animals were from the stock collected in early spring and usually showed more or less clearly, at least ventrally, the region of rapid reduction one-fourth to one-third of the body length from the posterior end. Comparison of figures 11 and 12, which indicate stages in the course of reduction after 10 days’ isolation, with figures 1 to 4 shows the relatively slower anterior reduction in the isolated animals.

Whether the decrease in motor activity in the isolated animals is due to fatigue or gradual exhaustion resulting from the earlier intense motor activity in the altered environment and lack of food, or to a gradual acquirement of tolerance to the new environment, it is evident that it does not alter essentially the reduction differentials. Redu‘ction may become


evident somewhat later and progress more slowly and the head region may reduce relatively somewhat more slowly than in control animals, but the differentials remain the same as regards their ‘high’ regions and directions. In short, it seems evident, both from the anesthetized and the isolated animals that the reduction differentials are not primarily ex- pressions of differential motor activity, but indicate a more fundamental physiological differential underlying motor dif- f erences.

12 l-LIz- Fige. 11 and 12 Differential reduction indicated aa in figures 1 to 6, in animale

Figure 11, early, With motor activity decreaeed by anesthetics or by isolation. figure 12, later etage.

DIFFEBENTIAL REDUCTION IN OTHER OLIGOCHETES

A few additional data on differential reduction of methylene blue in relation to fission in Aeolosoma hemprichii, Stylaria lacustris and a species of Nais, apparently Nais elinguis, and results obtained with several species of earthworms are briefly presented. F o r staining Aeolosoma, Stylaria and Nais methylene blue 1/10,000 to 1/50,000 was used. Animals were either sealed immediately in the dye solution or remained a few minutes in the solution exposed to air before sealing in the same solution. Heads and posterior growing regions apparently stained less deeply than other parts at first, but with longer staining deeper staining and injury occurs first in these parts. Two-zooid chains of Aeolosoma in which the posterior zooid has developed beyond the earliest stages and the anterior zooid has begun to develop a new posterior end show the reduction differentials indicated in figure 13, early,


and figure 14, later stage. From the head regions of the two zooids which reduce at about the same rate the rate decreases posteriorly and increases again in the developing posterior end of the anterior zooid just anterior to the fission zone F and may also increase in the posterior region of the second zooid, as indicated in figures 13 and 14. In the fully developed Aeolosoma individual no increase in the posterior region has been observed, the increase evidently being correlated wit.h the development of new segments, which does not con-

E a-

13

F

14 a 15

Wgs. 13 to 15 Differential reduction indicated aa in figures 1 to 6, in two-zooid chains of Aeolosoma, Nais and Stylaria. Figure 13, early, figure 14, later stage in animals not visibly injured by the dyes; figure 15, Merential reduction after deeper staining with injury and retardation of reduction in anterior ends and posterior growing regions. F indicates fission zone.

tinue indefinitely. In the two-zooid chains the whole posterior zooid reduces slightly more rapidly than the anterior zooid (figs. 13, 14). In very early stages of zooid development little change in rate of reduction appears, but as development progresses the gradient of the zooid becomes evident. In Nais and Stylaria the differentials are essentially similar to those in Aeolosoma. In all three species, as in Tubifex, a distinct ventrodorsal reduction differential appears. With long-continued staining anterior and posterior regions become more deeply stained than other parts and are


first injured by the dye and reduction following such staining shows reversal of the differentials, that is, reduction is retarded or does not occur in the injured parts (fig. 15) and with sufficient staining disintegration begins in these parts and shows the same differentials as reduction.

In view of the considerable volume of work on physiological gradients in earthworms a brief preliminary statement of results obtained with the reduction method on several, not yet identified species is of interest. Reduction of J a m s green shows in some species differentials similar to those of Tubifex, that is, a short steep decrease in rate of reduction from the prostomium posteriorly and a long, much less steep decrease in rate from the posterior region anteriorly. In one species, however, a gradient decreasing from the anterior to the posterior end was observed. In this species a posterior growing and segment-forming region was apparently not present, for posterior segments were as large as others. In this connection it may be noted that according to Sun and Pratt ('31) the earthworm, Helodrilus foetida does not develop new segments after hatching. In all species in which reduction has been observed thus far the ventral body wall reduces much more rapidly than the dorsal and the differentials are much more distinct ventrally than dorsally and are clearly visible to the naked eye.

DISCUSSION

Differential reduction of vital dyes in decreased oxygen has proved a useful method for indicating some of the differences in physiological condition in different regions of various living, intact organisms, including protozoa, hydroids, tur- bellaria, annelids and the chick embryo (Child, '34 a, b ; Child and Watanabe, '35 ; Watanabe, '35 ; Rulon, '35) and has also given data of interest in connection with reconstitution (Child and Watanabe, '35; Watanabe, '35). The results obtained with oligochetes, briefly mentioned in an earlier paper (Child, '34 a) and more fully presented above constitute further direct evidence for the existence of quantitative physio-

DIFFERENTIAL REDUCTION IN OLIQOCHETES 44.3

logical gradients or differentials in these forms. A point of particular interest as regards these reduction gradients is their close similarity to the gradients of susceptibility, not only in oligochetes, but in other forms, so far as data are available,, and the correspondence between susceptibility gradients and gradients determined by other methods has been pointed out in various papers (see Child, '28 and literature there cited). A comparison of the susceptibility gradients in oligochetes observed by Hyman ('16) with the reduction gradients shows that they are essentially identical as regards regional differences and directions in the body. Hyman's graphs, however, show the progress of disintegration over the body in relation to time, while the curves of differential reduction in the present paper are attempts to show the differentials as they appear at particular moments in the course of reduction. Moreover, the reduction gradients are identical with the gradients of susceptibility to vital dyes in the same species. And finally, the reduction gradients are similar to the gradients of oxygen consumption found by Hyman and Galigher ( '21) and Hyman ( '32) in Lumbriculus and two species of Nereis and to gradients of C.0, production and of oxidizable substance as determined by the Manoilov reaction in the earthworms, Pheretima hilgendorfi and Allolo- bophora foetida (Watanabe, '31). Apparently gradients of electric potential also correspond at least ventrally to these gradients (Morgan and Dimon, '04; Watanabe, '28). Agree- ment, however, is not complete as regards the annelids. Shearer ('24) found a gradient of oxygen consumption decreasing from anterior to posterior end in an earthworm, but gives no information as to presence or absence of a posterior growing and segment-forming region in the species concerned. In a later paper (Shearer, '30) he regards the existence of any gradient as doubtful, but presents no further data on the

'Compare, for example, Child and Deviney ('26) and Uhild ('34a, b) for protozoa; Child and Hyman ('19) and Child ('34a) for hydra; Child ('26) and Child and Watanabe ( '35) for Corymorpha; H y m n ( '27)' Hinrichs ( '27) and Rnlon ( '35) for the chick embryo.

444 C. M. CHILD A N D OLIN RULON

earthworm. Parker ('29) found in Nereis virens a lower GOz production at the two ends than in middle regions. Shearer's and Parker's results have been discussed and criticized elsewhere (Hyman, '32 ; Watanabe and Child, '33) and need not be further considered here. Maloeuf ('35) has found in very short isolated pieces of earthworm no con- sistent differences in oxygen consumption in the body wall, except that the anterior end apparently has a lower rate, but whether, or to what extent the oxygen consumption of these pieces differs from that of longer pieces and of the body wall as a whole and whether a posterior segment-forming region is or is not present in the material, he has not determined.

Except when formed anew in consequence of fission, frag- mentation or loss of posterior parts by injury or otherwise, the posterior growing region apparently becomes less active in formation of new segments as development progresses and in various species which do not undergo fission, as in the earthworm mentioned above (p. 442)' and in some which do divide (e.g., Aeolosoma) may disappear when a certain number of segments or a certain length is attained. In such cases the posterior arm of the longitudinal gradient may be expected to disappear or become less marked, or in case the posterior end performs respiratory movements as in various microdrilous forms, may persist in greater or less degree. Comparative study of different stages of the life cycle of different species is necessary to determine whether, or to what extent such changes occur, but in any case we need not expect to find a gradient with two arms in opposite direction (often called for convenience U-shaped gradient) present throughout life in all species of oligochetes or annelids.

Evidently, however, by far the greater part of the data at hand agree in indicating the presence of a gradient in annelids, involving respiration, reduction rate, susceptibility and electric potential, decreasing from the head posteriorly and increasing again toward the posterior growing region when such a region is present. As Hyman ( '16) has pointed out, the two arms of this gradient are certainly not identical


physiologically in the adult animal even though certain of their differential characteristics are identical. The anterior arm is an extension posteriorly of the primary gradient of early developmental stages and a number of later formed segments may be included in it as they become fully differen- tiated and subordinate to anterior dominance. The posterior arm arises secondarily in relation to growth and development of segments, the posterior growing region being its high end (Child, '17).

Even when the posterior arm of the gradient is present we need not expect always to find the same relations between reconstitution and the two arms of the gradient, as Castel- nuovo ('32) has assumed. Actually we find that they differ in different species and we may fhd that they differ at different stages in a single species. In some species heads regenerate from the anterior ends of pieces at all except perhaps extreme posterior levels. In others heads regenerate only from anterior body levels within a short distance from the original head and posterior ends may develop from both anterior and posterior cut ends of pieces from more posterior levels, apparently in the posterior arm of the gradient. The preexisting gradient in a piece, not only in annelids but in other forms, is very commonly an important factor in determining where a head or a posterior end shall develop, but it is by no means necessarily the only factor. The change in conditions of the cells adjoining a cut end may bring about obliteration of a preexisting gradient and determine a new one opposite in direction to it, as in Corymorpha (Child and Watanabe, '35 ; Watanabe, '35). In such a case a head may arise a t the low end of the preexisting gradient. If, on the other hand, the change in condition of the cells adjoining the cut end is slight in relation to conditions in the rest of the piece, a basal or posterior end may arise from the high end of the preexisting gradient. In the posterior arm of the annelid gradient, while growth and development of segments continue, dominance of any part over any other is undoubtedly less effective than in anterior regions with fully differen-

TRB JOURNAL OF EXPERIMENTAL ZOOUJGY, POL. 74, NO. 3


tiated nervous system, and obliteration of a preexisting gradient and determination of a new one can occur more readily there than anteriorly. I n short the differences in reconstitution in relation to the posterior arm of the gradient appear merely as special cases of the relation between preexisting gradients and the physiological condition and potencies of the cells which give rise to the new part, whether a head or a posterior end, or fail to regenerate.

SUMMARY

1. Reduction of Janus green and methylene blue by Tubifex tubifex shows differentials in rate of reduction in the body wall. Prostomium and posterior growing region immediately anterior to the anal segment reduce most rapidly in animals not injured by the dye. From the prostomium rate of reduction decreases posteriorly in a steep, short gradient and from the posterior growing region anteriorly in a long, much less steep gradient. The clitellum, when present, reduces almost as rapidly as prostomium and posterior growing region.

2. The anal segment usually reduces much less rapidly than the growing region immediately anterior to it.

3. A distinct ventrodorsal gradient appears at all levels posterior to the prostomium and the segmental ganglia of the ventral nerve cord apparently reduce more rapidly than the ventral body wall. 4. With continued staining differential susceptibility to the

dye, as indicated by injury and following disintegration and death, shows the same differentials as reduction in less stained uninjured animals. Reduction is retarded or does not occur in the injured parts, consequently the reduction differentials gradually become reversed in direction, the anterior end and posterior growing region reducing less rapidly than intermediate regions or not at all.

5. Two-zooid chains of Aeolosoma hemprichii, Nais elinguis and Stylaria lacustris, with posterior zooid somewhat ad- vanced in development show reduction gradients in each zooid


decreasing posteriorly from the anterior end and increasing again in the posterior growing region where new segments are developing. Intermediate regions usually reduce slightly more rapidly in the posterior, than in the anterior zooid.

6. Application of the reduction method to several, not yet identified earthworm species shows in one a decrease in reduction rate from anterior to posterior end and apparent absence of a posterior growing region. In others a gradient with two arms opposite in direction, like that of Tubifex, and a very steep ventrodorsal differential appear.

LITERATURE CITED

CASTELNUOVO, G. 1932 Sulle rigeneraeione e suscettibilith differenziale (KCN)

CHILD, C. M. 1917 Differential snsceptibifity and differential inhibition in the

Studies on the axial gradients in Corymorpha. 11. Biol. Gen.,

di Limnodrilus hofmeisteri Clap. Arch. Zool. Ital., vol. 17, p. 323.

development of polychete annelids. J. Morph., vol. 30, p. 1. 1926

1928 The physiologkal gradients. Protoplasma, vol. 5, p. 447. 1934s Differential reduction of methylene blue by living organ-

isms. Proc. SOC. Exp. Biol. and Med., vol. 32, p. 34. 1934 b The differential reduction of methylene blue by Paramecinm

and some other ciliates. Protoplasma, vol. 22, p. 377. CHILD, C. M. AND E. DEVINEY Contributions t o the physiology of Para-

mecium caudatum. J. Exp. ZOiil., vol. 43, p. 257. CHILD, C. M. AND L. H. HYMAN 1919 The axial gradients in Hydrozoa. I.

Biol. Bull., vol. 36, p. 183. CHILD, 0. M. AND Y. WATANABE 1935 Differential reduction of methylene blue

by Corymorpha palma. Physiol. Zool., vol. 8, p. 395. HImcHS, M. A. 1927 Modification of development of the basis of differential

susceptibility to radiation. IV. J. Exp. Zoiil., vol. 47, p. 309. HYNAN, L. H. 1916 An analysis of the process of regeneration in certain

microdrilous oligochetes. J. Exp. Zool., vol. 20, p. 99. 1927 The metabolic gradients of vertebrate embryos. 111. Biol.

Bull., vol. 52, p. 1. 1932 The axial respiratory gradient : experimental and critical.

Physiol. Zool., vol. 5, p. 566. Direct demonstration of the existence

of a metabolic gradient in annelids. J. Exp. Zoiil., vol. 34, p. 1. Peristalsis and physico-chemical differences along the

longitudinal axis of the earthworm. Proc. Am. SOC. Zoologists, Anat. Rec., vol. 64, p. 79.

MORQAN, T. H. AND A. C. DIMON 1904 An examination of the problem of physiological ‘polarity’ and electrical polarity in the earthworm. J. Exp. Zool., vol. 1, p. 331.

vol. 2, p. 609.

1926

HYMAN, L. H. AND A. E. GALIQREB 1921

MALOEUF, N. S. R. 1935


PARKER, G. H. 1929 The metabolic gradient and its applications. Brit. J. Exp. Biol., vol. 6, p. 412.

RULON, 0. Differential reduction of Janus green during development of the chick. Protoplasma, vol. 24, p. 346.

SHEARER, C. 1924 On the oxygen consumption rate of parts of the chick embryo and fragments of the earthworm. Proc. Roy. Aoad., Ser. B, vol. 96, p. 146. 1930 A re-investigation of metabolic gradients. J. Exp. Biol.,

vol. 7, p. 260. SUN, K. H. AND K. C. PUTT Do earthworms grow by adding new pleg-

mentsP Am. Nat., vol. 65, p. 31. WATANABE, Y. 1928 On the electrical polarity in the earthworm, Perichaeta

communissima goto et Hatai. Sci. Reports Tohoku Imp. Univ., Ser. 4, Biol., vol. 3, p. 139. 1931 On the physiological axial gradients of chaetopod annelids. 11.

Ibid., p. 437. 1935 Physiological dominance in Corymorpha palma in relation to

reconstitution and methylene blue reduction. Physiol. Zoiil., vol. 6, p. 417.

The longitudinal gradient in Stylochus ijimai: with a critical discussion. Ibid., vol. 6, p. 542.

1935

1931

WATANABE, P. AND C. M. CHIID 1933

differential reduction of vital dyes in certain oligochetes

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