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T H E PRODUCTION OF A MELANOTIC NEOPLASTIC DISEASE I N FISHES BY SELECTIVE M.4TINGS

IV. GENETICS OF GEOGRAPHICAL SPECIES HYBRIDS l

MYRON GORDON ASD G. M. SMITH

(From the Zoological Laboratory, Cornell University, and the Department of Anatomy, Yale University School of Medicine)

When a black-spotted female platyfish (Platypoecilus maculatus) is mated to an unspotted (genetically recessive) male platyfish, one of three hereditary expressions may be expected. If the female is genetically pure (homozygous dominant) for the spotted factor, all of the young may be expected to be spotted. The genetic formula representing this situation is (W)Sp(Z)Sp, for the mother, where W and Z represent the sex chromosomes upon which the two hereditary factors (Sp, for spots) are carried. The recessive father’s constitution is (Z)sp(Z)sp.

When another type of spotted female is mated to a similar unspotted male platyfish, only her sons are spotted, like their mother; the daughters are unspotted, like their father. This is the well known type of criss-cross inheritance and indicates the presence of sex-linked factors. The formula that represents the spotted mother of this type is (W)sp(Z)Sp where the dominant factor S p is carried on the Z chromosome and the recessive s p is on the W. The father’s formula is (Z)sp(Z)sp, the same as before.

When still another type of female spotted platyfish is mated to an unspotted platy male, (Z)sp(Z)sp, the following results are obtained: all the daughters are spotted like their mother while all the sons are spotless like their father. In this case, analysis shows that the spotted mother’s constitution is (W)Sp(Z)sp where the dominant factor for spots S p is carried on the W chromosome. The \V chromosome presumably carries hereditary factors po- tent for the development of the female sex. The origin of these three types of spotted females is described by Fraser and Gordon (1929).

Spotted female platyfishes (Platypoecilus maculatus) have been mated to males of a different species and genus, the Mexican swordtail (Xiphophorus hellerii) ; the spotted character of the mother appears in her hybrid offspring. If the mother’s constitution is (W)Sp(Z)Sp, the spotted character appears in all the offspring; if the mother is (W)sp(Z)Sp, it appears only in the sons; if the mother is (W)Sp(Z)sp, the spotted character appears only in the daughters. Only rarely do exceptions appear. The subject is reviewed by Gordon (1931, 1937).

In the black spotted hybrids resulting from these crosses the pigment cells, which form the spots, grow at an abnormal rate. At birth there is some im- mediate indication of accelerated growth of the large black pigment cells.

1 This work was aided by a grant from the International Cancer Research Foundation and the Atypical Growth Study Unit of Yale University School of Medicine.

At present, Fellow of the John Simon Guggenheim Memorial Foundation (1938-1939).

543

544 MYRON GORDON A N D G. M. SMITH

These cells or macromelanophores continue to grow rapidly and eventually envelop the hybrids in a black integumentary sheath. In many cases the growth of these pigment cells does not stop at the cutaneous areas, which is their normal environment, but the black cells invade the muscular regions as they travel along the connective-tissue fascia of the muscles. A preliminary histologic account of pronounced melanotic overgrowths in adult fish hybrids was given by Reed and Gordon (1931). It is quite evident that a definite

FIG. 1 . CROSS BETWEES T H E SPOTTI:D RIO P.AF.AJ.OAPAN PLATYFISH Axn THE Rlo GRASDE PLATYFISH A. P. niaculaltrs female (macromelanophores and micromelanophores). B. P. couchianus male

(micromelanophores only). C. First ’generation hybrid, normal female (micromelanophores only). D. First generation hybrid, melanotic male (macromelanophores and micromelanophores) . For interpretive diagram, see Fig. 2.

neoplastic condition develops in hybrid fishes when one of the parents is a black spotted platyfish and the other is a Mexican swordtail. Gordon and Smith (1938) have described similar overgrowths in very young fishes produced by genetic experiments.

While melanosis in hybrids between the common platyfish (Platypoecilus maculatus) and the Mexican sword tail (Xiphophorus hellerii) have been described by several workers (for bibliography see Gordon, 1931b and 1937), no other species of the tribe to which these two genera belong (Xiphophorini) were hybridized prior to 1930. This was because the remaining species (P. couchianus, P. xiphidiztm, P . variatus and X. montezumae) were unavailable alive.

As a result of the University of Michigan’s 1930 Mexican expedition for the study of fishes of the Atlantic drainage (Hubbs and Gordon, unpublished) P . couchianus was obtained at Santa Catarina, Nuevo Leon, in a small stream flowing into the Rio San Juan, a large tributary of the Rio Grande system (Gordon, 1935). In the second Mexican expedition, in 19.32, representatives of all the remaining platyfishes were taken alive and were shipped to the zoological laboratory at Cornell University (Gordon, 1933). The geographical distribution of the four species of Platypoecilus is presented in Fig. 16.

MELANOTIC NEOPLASTIC DISEASE I N FISHES 545

DATA FROM GENETIC EXPERIMENTS

Hybrids betwcen the Rio Grande Platyfish, P . cnuchianus, and the Rio Papaloapan Platy fish, P . maculatus

First Gcncration: The most northern of Mexico's platyfishes ( P . couchianus) has been hybridized successfully with the most southern species ( P . macukatus). In nature these species are separated by at least two great drain-

PI a%j poe ci \us macu I atus P \ Rtq poec; lus couchi anus from RioPapaloap.an from Rio Grade

n 'I

X

Normal female Melonotis mola

FlO. 2. CROSS BETWEEX T I I E SPOTTED KlO PAPALOAPAN PI.ATYFISII AND THE RIO CRANDE PLATYFISH

In this cross hetween the spotted (Sp) P. niacitlattts and the non-spotted ( s p ) P. cottchiotzus, the spotting factor (Sp) is transferred from the female parent to all her sons. The sons develop melanotic overgrowths-the daughters are normal.

Upon inbreeding the first generation (F,) hyhrids half of the next generation develop melanotic neoplasms, the other half are normal. Melanosis follows the inheritance of the Sp factor for macromelanophores introduced by the spotted P. nincirlolrts. In the second generation melanosis appears in half the male and half the female offspring.

age systems, the Rio Sota la Marina and the Rio Panuco. The distance be. tween the habitats of the two species is about one thousand miles. From four different crosses a total of 2 2 7 interspecific hybrids have been obtained.

The peculiar association of melanosis with sex in the hybrids listed in Table I (page 560) may be understood readily on the basis of the previous breeding behavior of the genetic lines to which the mothers of the hybrids

5 46 MYRON GORDON AND C. M. SMITH

belong. following matings melanosis appears preponderantly in the daughters.

Z chromosome of the P . maculatzis mother.

In the first cross melanosis appears only in the sons, while in three

In the first mating the S p factor for macromelanophores is carried on the Criss-cross inheritance of this

Fic. 3. CROSS BETWEEN TIIE SPOTTED RIO PAPALOAPAS P L . x r Y Y w I ASD THE RIO GRAXDE PL. \TYFISII

A . P. macirlal u s female (macromelanophores only). B . P. coirchianits male (micromelanophorrs only). C. First generation hybrid melanotic female (macromelanophores and micromelanophores). D. First generation hybrid normal male (micromelanophores only ). B . First genrrzi- lion hybrid melanotic male, exception (macromelanophores and micromelanophores) . F. Second generation hybrid melanotic female young (macromelanophores only). C. Second generation hybrid normal male (micromelanophores only). For interpretive diagram see Fig. 4.

factor might be expected and the sons have inherited the capacity to develop the large black pigment cells. These melanophores initiate the development of melanosis in the males (Figs. 1 and 2 ) .

In the next three matings ( 2 , 3 and 4 ) , the P . maculatus mothers belonged to a genetic line in which the S p factor is carried on the Mr chromosome, Con- sequently it is in accordance with theory that macromelanophores appear in the daughters (Figs. 3 and 4 ) . Once the macromelanophores start their de-

MELANOTIC NEOPLASTIC DISEASE IN FISHES 547

velopment in the hybrids, they eventually produce melanotic overgrowths. The appearance of three melanotic males from these ( W ) S p ( Z ) s p mothers is not entirely unexpected. In previous experiments with these P . maculatus strains Fraser and Gordon, 1929, showed that exceptional black-spotted sons

Plat9 oecilus maculatus Pldypoacilus C O L L C ~ ~ L I O

from k i opapol o q a n fromRioGrMde

FIG. 4. CROSS BETWEEN THE SPOTTED KIO PAI’ALOAPAH PLATYFISH ASD THE RIO GRANOE PLATYFISFI

This cross differs from the one illustrated in Fig. 2 in that the spotted P. nrarttlatus parent carries the Sp factor on the W chromosome instead of the Z. The male parent is the same as before, P. couchianzts. Instead of criss-cross inheritance, mother to daughter inheritance is displayed here. The daughters have melanotic overgrowths while the sons are normal.

Upon inbreeding the first generation (F,) hybrids, half of the next generation develop melanotic neoplasms, the other half are normal. Jn this case only the femalcs are melanotic, the males are normal.

Since the Sp factor is carried on the W chromosome, and the W chromosome denotes female- ness, melanosis is ~eneral ly associated with the females. In rare instances, where crossing over of the W and Z takes place, males develop melanosis. Three instances are reported in the text and one is shown in Fig. 3 ( E ) .

For explanation of the factors S1 and st see legend for Fig. 6.

occasionally appear from a ( W ) S p ( Z ) s p mother. This has been explained by crossing over of the sex chromosomes. In this instance the exceptions occur in about 4 per cent of the total population.

The production of melanotic hybrids in the species cross of P . maculatus and P . couchianus practically parallels the results of mating Platypoecilus

5 48 MYRON GORDON AND C. M. SMITH

maculatus with Xiphophorus hcllcrii, the Mexican swordtail, although the number of interspecific hybrids with definite melanotic overgrowths is not as great as in intergeneric hybrids. The appearance of melanotic hybrids in this species cross indicates a fundamental physiological difference between the species.

The genetic interpretation of the appearance of melanosis in the species cross is similar to the one offered for the generic cross previously given by Gordon (1931 and 1937). The southern species, P. maculatus, transmits the hereditary factor for large black pigment cells or macromelanophores (Sp), while the northern species transmits physiological or modifying factors, or both, which determine the rate and type of growth of these pigment cells. The hereditary contributions of both parents interact in the hybrid and bring about the unrestrained growth of the macromelanophores.

Second Generation: The results as indicated in mating number 5 show that only about one half of the second generation are melanotic; the results show further that melanosis appears in the sons as well as in the daughters; a study of the advance of melanosis among the melanotic offspring indicates that the intensity of this neoplastic disease varies greatly.

Melanosis, intense or mild, should appear in one half of the offspring because the father carries the S p factor in the heterozygous state as (Z)Sp(Z)sp and the mother is recessive, ( \ V ) s p ( Z ) s p . The discrepancy between the observed and expected results is attributed to prenatal death of some intensely melanotic fishes. Among some of the second generation young the advance of melanosis is remarkably early. Definite overgrowths have been seen in day-old hybrids. I t is thought, therefore, that in some cases melanosis may appear before birth and bring about the death of the embryo.

An examination of the four phenotypes in the second generation hybrids reported in mating number 6 will reveal an important relationship that exists between the hereditary factors for micromelanophores (stipples) and macromelanophores (spots).

The female parents (Ped. 175) of the F, hybrids (Ped. 226) were spotted (Sp) but not stippled ( s t ) . Their genetic formula may be written as Spsp stst . The male parents (Ped. 200 and 218) of the F, hybrids (Ped. 226) were not spotted (sp) but they were homozygous for stipples ( S t ) . Their genetic formula may be written as spsp S t S t . The hybrids (Ped. 226) were of two types, melanotic males Spsp Stst and normal females, spsp Stst. Both had stipples and were heterozygous for the stipple factor (Stst) .

When the hybrids were inbred (226 X 2 2 6 ) , as indicated in mating number 6, four classes appeared in the second generation (Ped. 239). Using spots and stipples for classification there were 10 melanotic females, spotted and stippled (Sp S t ) , 5 melanotic females spotted only ( S p s t ) , 16 normal males without spots but with stipples (sp St ) , and finally 3 normal males without either spots or stipples (sp s t ) . It is well to remember that the dominant stipple factor originated in P. coztchianus, while the dominant spot factor originated in P. maculatus.

When micromelanophores (autosomal factor S t ) and macromelanophores (sex-linked factor Sp) are present together (as St Sp), the degree of melanosis is more intense than when macromelanophores ( S p s t ) are present alone. The


presence of micromelanophores alone (St sp ) in the hybrids produces no melanotic conditions.

In this connection Gordon (1928) indicated that when a spotted ( S p ) platyfish, P. maculatus, is mated with a stippled ( S t ) platyfish (also P . maculatus) the spotted and stippled ( S p S t ) platyfish intervarietal hybrid has a greater number of macromelanophores than its spotted parent. In no individual, however, is melanosis evident. The S p factor, then, is affected by the St factor and its phenotypic expression is extended. In intervarietal matings in Xiphophorus hellerii the same phenomenon is displayed. This has recently been demonstrated by Gordon ( 1938).

From the point of view of comparative genetics it is interesting to note that the stippling factor of P. couchianus reacts with the spotting factor of P. maculatus in interspecific hybrids in the same manner as it does in intervarietal hybrids. Gordon (19.31, 1937) has shown that the St factor of Xiphophorus hellerii reacts in the same manner in intergeneric hybrids. While the stippling factor of P. couchianus enhances the effect of the spotting factor of P. maculatus in the hybrids of these two species, it must be remem- bered that the stippling factor itself is not responsible for the neoplastic ac- tivity on the part of macronielanophores. Melanosis is due to many P. couchianus factors working with the S p factor of P . macadatus.

Among the stippled and spotted F, hybrids were several young fish in which melanosis had progressed so rapidly that the hybrids showed definite neoplastic overgrowths on the day of birth. The hybrid fishes with tumors at one day old indicate that the melanotic neoplasms appear about thirty days after the fertilization of the ovum. Further studies, particularly with greater numbers of gravid hybrid females, may be expected to yield some F, hybrids which develop melanotic lesions during embryonic life. Studies of the tumors in embryonic animals will be available in tracing the early origin of the cells responsible for the neoplastic disease.

Hybrids between the Rio Sota la Marina Platyfish ( P . xiphidium) and the Rio Papaloapan Platyfish ( P . ntaculatws)

First Generation: The Rio Sota la Marina platyfish derives its name from the river system in which it is found. The mouth of the Rio Sota la Marina is about three hundred miles south of that of the Rio Grande. Between these two rivers is the Rio San Fernando (also called the Rio Conchos) but as yet no platyfish have been taken from this stream (Gordon, 1933).

P . xiphidium Hubbs and Gordon, as a distinct species, is of recent dis- covery. While Meek (1904) collected the species, he grouped it with P. variatus. Hubbs and Gordon have given the reasons for the separation of this species, their argument being based upon the examination of over two hundred specimens collected in 1930. For a preliminary species diagnosis see Gordon ( 1935).

Three P. maculatus females of the constitution ( W ) S p ( Z ) s p were mated with male P. xiphidizrm recessive for the spotted factor (crosses number 7, 8 and 9). The results obtained are almost identical with the results obtained when P. maculatus females of the same genetic type were mated with P.

5 50 MYRON GORDON AND G. M. SMITH

couchianus males (Figs. 5 and 6) . Here again melanosis predominates in the daughters, while the sons, with only a few exceptions, are normal. The 5 exceptional melanotic males have all been derived from one of the spotted fe-

FIG. 5 . CROSS BETWEEN THE SPOTTED KIO PAP, \ \r .o . iPns PLATYPISII AND T H E Rxo SOTA LA MARINA PLATYFISH

A . P. ntacrrlofus female (macromelanophores) . B . P. .ruipliidirtnt male (micrornelanophores) . C. First generation melanotic female hybrid (macromelanophores and rnicromelanophores). D. First generation normal malc hybrid (micromclanophores only) . E . First generation melanotic male hybrid, exccption (macromelanophores and micromelanophores). F. Second generation melanotic hybrid female (macromclanophores and micrornelanophores) . C. Second generation normal hybrid male (micrornelanophores), For interpretive diagram see Fig. 6.

males, mating number 9. This requires further investigation from the point of view of genetic interpretation of the actual constitution of that female. The significant fact gained from these experiments is the close association of the development of melanosis with the heritable factor for the development of macromelanophores.

Second Gencration: The first generation hybrids were mated together,


mating number 10: the mothers (Ped. 262) were melanotic but this did not interfere with their reproductive capacities. The fathers (Ped. 262) were non-melanotic. Since the original mothers were dominant but heterozygous

Plotljpoecilus rnwuIatus Platypoecilus xiphidium from R i o b p a l o a p a n from RioSota la Marina

I I I

Melanotic feno le Normal male

F2 sp-now s; male spolted

Id st-non-stippled

FIG. 6. CROSS BETWEES THE SPOTTED R l O P.\PM.OAPAN PI.ATYFlSII ASD TIIF. R l O SOTA LA MARINA PLATYPlSlI

In this mating the P. mncrtlntirs female parent carries the dominant S p factor for macromelanophores on the W chromosome. but it is recessive for the autosonial factor for micromelanophores, SL. The P. xiphidium male is recessive for macromelanophores s p but is dominant for the auto- soma1 factor St for micromelanophorc~s.

The P. maculafus parent transmits the Sp factor to her daughters only. The P. xiphidium male transmits the S1 factor both to his daughters and sons. The first generation females carry both micromelanophorrs and macromelanophores while the males carry micromelanophores only.

Upon inbreeding the first Keneration ( F , ) hybrids, half of thr second generation are melanotic. These are all females. Half of these females have macromelanophores only. All the males of the second generation are normal-some have micromelanophores-others have no melanophores a t all.

Only those hybrids which inherit macromrlanophores S p develop melanotic tumors.

for the spotted factor and the fathers were homozygous dominant for the stippling factor, the formulae of the parents may be written as:

PI PI Mother ( W) sp (Z ) s p sts t X ( Z ) s p ( Z ) s p StSt Father

Fl Fl Daughters ( W ) s p ( Z ) s p Stst X ( Z ) s p ( Z ) s p Stst Sons

552 MYRON GORDON AND G. M. SMITH

The types of fishes in the second generation that are expected are listed below according to their phenotypic constitutions.

F, daughters: melanotic Spotted and stippled, (W)Sp St, 9 Spotted only, (W)Sp s t , 3

F2 sons: normal Stippled only, ( Z ) s p St , 9 Gold only, ( Z ) s p st , 2

FIG. 7 . CROSS BETWEEN THE SPOTTED Rto PAPALOAPAN PLATYFISH AND THE RIO SOTA LA MARINA PLATYFISH

B . P. riphidium male (macromelanophores and micromelanophores, twin-spot) . C. First generation normal female (micromelanophores). I). First generation normal male (micromelanophores). E . First generation normal female (macromelanophores and micromelanophores, crescent, twin-spot) . F. First generation normal male (macromelanophores and micromelanophores, crescent, twin-spot). For interpretive diagram see Fig. 8.

A. P. maculatus female (micromelanophores, crescent).

Again it is possibIe to get melanotic second generation hybrids with and without the micromelanophores, as ( W ) S p St or (W)Sp st . The fact that melanotic conditions result even when no micromelanophores are present is significant; but melanosis in hybrids without micromelanophores is not as intense nor does it appear as early as in those in which both types of melanophores occur.

The genetic interpretation of the results of matings between the Rio Sota la Marina and Rio Papaloapan platyfish may be stated in terms similar to those previously given for the crosses of Rio Grande platyfish or the Mexican swordtail with the Rio Papaloapan species. The Rio Papaloapan platyfish contrib- utes to the hybrid the factor S p for macromelanophores which, when com-


bined with the modifying factors transmitted by species other than its own, results in the abnormal development of the large pigment cells in the hybrid.

Spotted Factor of P . xiphidium Not Active in Production of Melanosis in Hybrids: When a spotted P . xiphidium (Z)Sp(Z)sp is mated to a non-spotted P. maculatus (see mating 11 and 1 2 ) the heritable factor Sp for the P . xiphidium macronielanophores segregates in the ratio of one to one, but no

Ptatypoectlus maculatus Platypoecilus wiphidiuro frornRio Papalocapan frornRRio Soto la Marina

. * F: X

I Xpplcd, Spited I SXppLd

I

Non malanotic female Non nelanoti'e male

Stpplcd %ppl.d

FIG. 8. CROSS BETWEES TIIE SPOTTED RIO PAPALOAPAN PLATYFISII AND THE RIo SOTA LA MARINA

I n this mating the P. xiphidium parent carries the Sp factor for macromelanophores; it also carries the St factor for micromelanophores. In addition to these two factors it has the twin-spot pattern (T) which is made up of closely associated micromelanophores.

The female parent, P. rnaculatus, has no macromelanophores, but it carries the factor for micromelanophores, St, and the factor for single crescent, C , which is made up of closely associated micromelanophores.

When the Sp factor comes from P. xiphidium, the hybrids are not melanotic a t all, merely lightly spotted. Half of them do not have macromelanophores, for the father carried the Sp factor in a heterozygous state. Some of the hybrids have both the twin-spot character and the single crescent; others have one or the other (these are not shown in the diagram) ; others have none of the patterns in the caudal peduncle.

The significant feature of this cross is that when macromelanophores come from P. riphidium. melanosis does not appear in the hybrids.

PLATYFISH

melanotic conditions are evident. Indeed, the actual number of macromelanophores as represented in the spotted father is actually reduced in his offspring (Figs. 7 and 8).

These interesting results apparently reveal that the spotted factor of P . maculatus (and its genetic associates) reacts differently than does the spotted factor of P . xiphidium. The spotted P . maculatus factor induces the macro-

5 54 MYRON GORDON AND G. M. SMITH

melanophores to neoplastic development in the specific hybrids, but the spotted P . xiphidium factor does not produce the same results; in fact, there is a per- ceptible decrease in the production of these large black pigment cells.

This preliminary experiment, naturally, calls for further tests of the same kind, as well as for tests where a spotted P . xiphidium female is mated to a P . maculatus male.

Back-cross of P . maculatus-P. xiphidium iliclanotic Hybrid to P . maculatus and I t s Significancr: Since in pure races of the spotted Rio Papaloapan platyfish the macromelanophores are held to a normal rate of development, it

FIG. 9. CROSS BETWEEN TIIE SPOTTEO RlO PAPAI.OAP.9N PLATYFlSH AND THE R I O P.ANUC'0 PLATYFISII

A. P. moculatrc.r female (macromelanophores and micromelanophores) . R. P. variatzrs male (macromelanophorrs and micromelanophorcs) . C. First generation melanotic female (macromelanophores of P. ntocttfaltts and P. vuriulus; micromelanophores of P. moculntus and Y. vorialrts) . D. First gencration normal male (macromclanophores of P. variatus; micromelanophores). For interpretive diagram see Fig. 10.

must be assumed that this species has certain constitutional large black pigment cell restraining factors. This assumption has been checked :

A melanotic hybrid male ( P . xiphidium y P . maculatus), pedigree 264-1 1, was back-crossed to a golden P . maculatus female, pedigree 160-4. Since the hybrid male was heterozygous both for the spotted as well as for the stippled factors, four phenotypes were obtained as follows:

Spotted and stippled ( S p S t ) . . . . . . . . . . . . . . . . . . 17 young Spotted only ( S p s t ) . . . . . . . . . . . . . . . . . . . . . . . . . . 18 young Stippled only ( s p S t ) . . . . . . . . . . . . . . . . . . . . . . . . . . 14 young Neither spotted nor stippled fsp s t ) . . . . . . . . . . . . . . 17 young

The results approach the 1 : 1 : 1 : 1 ratio expected. The significant point, however, is that in both the spotted-stippled group ( S p S t ) and in the spotted- non-stippled group ( S p s t ) there is no evidence of melanosis. Normality has been restored to the hybrids by the addition of the heritable pigment cell restraining forces of P . muculatus. On the basis of theory, one half of the

MELANOTIC NEOPLASTIC DISEASE IN FISHES 5 5 5

spotted back-cross hybrids should have shown some signs of melanosis, but none of them did. This cannot be explained cytologically on the basis that all P . maculatus chromosomes, and the hereditary factors they carry, of the hybrid united with only P . maculatus chromosomes of the female, because the stippling factor was originally brought into the hybrid from the P . xiphidium parent; and since approximately half of the back-cross offspring are stippled, at least one P . xiphidium chromosome must be present. Undoubtedly more P . xiphidium chromosomes entered the back-cross off spring, but they cannot readily be identified.

PlatypoeciIus macu\aTus Platcjpoecilus variatus f r o m RioPapaloapao f r o m RioPanuco

n ' I X

a w l

I

Normal male F, M c l a n o t k female

I IV IT

FIG. 10. CROSS BETWEES THE SPOTTED Rro PAPALOAPAN PLATYFISH AXD THE Rio PANUCO PLATYFISH In this mating the female, P. ntnctrla/rts, carries the S p factor for macromelanophores on the W

chromosome. On mating to P. vuriattts, melanotic iemale hybrids appear hut the male hybrids are normal. The P. vnrinttis had macromelanophores characteristic of its own species. These re- appeared in the hybrid males but these males were not melanotic, indicating that the S p factor for P. uuriulus is physiologically different from the S p factor of P. ntactrlofus. In the diagram only the Sp factor of P. maculalzrs is traced genetically.

Hybrids bctwern the Rio Panuco Platyfish ( P . variatus) and the Rio Papaloapan Platyfish ( P . ntaculatus)

The Panuco river system drains the largest part of the east-central area of Mexico. Within this general area, particularly near the Atlantic coast, a number of present-day streams are separated from this river, but from their platyfish contents it is evident that these streams form a part of the Panuco system and at some former time must have been an actual part of it. There are a number of subspecies of P . vuriatzis, but the ones used in these experiments are P . variatus variatus. These were taken from the Rio Axtla in 1932, fifty miles south of the village of Valles, San Luis Potosi (Gordon, 1933).

As in previous experiments only a few species crosses, out of many at- tempted, were effective between P . variatus and P . maculatus. The results obtained, however, are clear cut and in harmony with the results of previous species matings.

5 56 MYRON GORDON A N D G. M. SMITH

A female P. maculatus of the constitution ( W ) S p ( Z ) s p , when mated to a P. variatus male recessive for spots (mating 14): produced 37 melanotic hybrids, 20 of which matured, and all of these were females; 39 normal hybrids were produced by the same female and male, and of these 15 matured and were all males (Fig. 9 and 10). Melanosis, again, followed the hereditary trail of the spotted factor. Since the spotted factor was associated with the W chromosome, it was within the theory to expect only melanotic daughters and only normal sons. -4 few I;, individuals were inbred, and about half of the young obtained were melanotic while the other half were normal. These young were not carried to maturity for lack of space.

In another experiment some peculiar and as yet unexplainable results were obtained. The situation is of interest in the field of theories of the genetic

FIG. 11. CROSS B E ~ W E E S TIIE Rlo PAIVUCO PLATYFISH AWD THE SPOTTED Rio PAPALOAPAW PLATYPISII B . P. maculatus male (macromelanophores and

micromelanophores) . C. First generation normal female (micromelanophores only). D. First generation melanotic male (macromelanophores and micromelanophores). For interpretive diagram see Fig. 12.

A . P. uariolus female (micromelanophores).

control of sex. As far as the hereditary picture of melanosis is concerned no complications are seen.

A P . variatus unspotted female was mated with a P. maculatus male with a spotted belly pattern (Figs. 11 and 12). The spotted belly pattern ( S b ) is composed of macromelanophores, like the general spotted pattern, but the pigment cells are chiefly confined to the ventral areas, reaching about the middle of the body. The genetics of this pattern has not, as yet, been worked out. I t is one of the new patterns found in the 1932 expedition to the Rio Tonto, part of the Rio Papaloapan system. As mating 15 shows, all the melanotic hybrids resulting from the cross were males, while the normal fishes were females. Similar results, as far as the patroclinous behavior of the spotted belly pattern is concerned, have been observed in intervarietal crosses.

This species cross completes the series within the genus Platypoecilus. In each case, where P. maculatus with macromelanophores was crossed with a different species, the hybrids which inherited macromelanophores also in-


herited melanosis. Melanosis is the result of the hereditary interaction between the factor for macromelanophores of P . maculatus with the intensifying factors of some other species. Another way of interpreting the problem is to say that the species P. couchianus, P. xiphidium and P. variatus transmit to their hybrid offspring factors which remove a specific inhibitor that controls the normal growth and multiplication of the large black pigment cells of P . maculatus.

PRELIMINARY NOTES ON THE CONDITIONS OF MELANOSIS IN THE INTERSPECIFIC HYBRIDS

P, maculatus-P. couchitmus Hybrids: The interspecific hybrids between the spotted P. maculatus and P . couchianus develop large melanotic over-

PlaqTpoecilus variatus Platypoecilus rnaculatiis f r o m RioPanuco f rom RioPapoloapan

i?

Normal fenole F; Melanotic male I

FIG. 12. CROSS BETWEEN THE R I O PANUCO PLATYFISH AXD THE SPOTTED R I O PAPALOMAN PLATWISH In this mating the male P. moculdus carries the factor for spotted belly, the inheritance of

which is still incompletely known. This factor Sb seems different from the Sfi factor for spotting, although Sb and Sp involve macromelanophores. In this experiment only the male offspring showed signs of melanosis; the female hybrids were normal. The state of melanosis is relatively superfiaal. No overgrowths were detected in these species crosses.

growths that have the same general external and histologic features characteristic of overgrowths in the intergeneric hybrids of the spotted platyfish and the swordtail. When the interspecific hybrids are melanotic and are inbred, the second generation progeny develop melanotic overgrowths early in life. This genetic response parallels the reaction in the platyfish-swordtail matings as indicated in former papers of this series (193 1, 1937).

Fig. 13 represents a nine-day-old, 10 mm., second generation melanotic hybrid between a spotted P . maciilatws and a P . couchianus. When alive, this specimen, of culture number 230, displayed symptoms of the last stages of melanosis. A large melanotic overgrowth developed over the entire posterior half of the body. The increase in thickness of the integument was rather uni- form with no prominent individual nodules. This hybrid was deeply spotted

FIGS. 13-15. NINE-DAY-om 10 MM. SE(.OWD GENERATION MELANOTIC HYBRID RETWEEN P. BIACLILATUS AXL) P. ( ' O ~ C I I I A N U S , AND SERIAL SECTIOSS OF SAME

The entire posterior half of the body is involved in a state of melanotic overgrowth (Fig. 1 3 ) . The most anterior part of Fig. 14 shows the body cavity, with a small section of intestine

along the upper edge. The body wall at this level is rclatively thin and most of it is infiltrated by large black pigment cells. In the anterior part the scales lie flat upon the muscle mass; posteriorly they are forced out a t a 45' angle by the hyperplastic spindle cells that appear as the gray, outer layer. The black pigmentation within the musclc mass represents the invasive macromelanophores. In the extreme posterior part of the tail, the tumor consists chiefly of macromelanophores, spindle- shaped pigment cells. a reticular network, and some fat cells.

Fig. 15, under higher magnification, shows the details of pigment cell invasion of the muscles and their replacement by pigmented tissue. The corium is represented by spindle-shaped pigment

LLegend cont. at foot of neit pagt.1

( X 35)

558


with macromelanophores on the day of its birth. Between groups of macromelanophores there was a great profusion of micromelanophores and the scale type of melanophores. Later the groups of macromelanophores approached each other and formed a continuous mass of coal black tissue, Shortly after- ward the black coloring changed to a slightly lighter shade. This is to be correlated with the development of the true overgrowth. The caudal fin was intensely pigmented and thickened. Reed, Gordon and Lansing (1933) have made a preliminary statement on the post-embryonic growth in these melanotic hybrids.

The nine-day melanotic hybrid was cut in longitudinal sections; a continuous series of slides, representing the entire body, was available for study (series 814). Fig. 14 represents part of one section; this includes the posterior part of the body cavity and extends to the tip of the tail.

A histologic survey of the serial sections reveals a widespread condition of melanosis throughout the muscular and corial tissues of the body. In the posterior part of the caudal peduncle the muscles are completely destroyed and replaced by melanotic tissues. The corium of the entire caudal peduncle shows an intense hypertrophy of smaller pigment cells. The distal tips of the scales of the caudal peduncle are lifted from their normal flat position on the body by the newly formed cells of the overgrowth.

Within the muscle masses, the region of most intense pigmentation, the lesion is formed by an extensive macromelanophore hyperplasia. In the corium, which is less pigmented, there is a hyperplasia of smaller spindle- shaped pigment cells associated with a network of reticular fibers and fat cells. The capillaries are well developed and are engorged with red blood cells.

The macromelanophore invasion of the muscles is widespread, penetrating to the vertebral column (Fig. 15). The pigmentation of the meninges of the spinal cord is more intense than in the normal hybrid fish. The cell bodies of the macromelanophores, in the lesion, are greatly engorged with dense masses of melanin pigment granules. The dendritic processes of these cells are long and irregular and branch out in every plane, filling the intermuscular spaces to form a network of pigmented tissue within which muscle fibers are found, singly or in groups.

No abnormal pigmentation of the visceral organs was observed. There was no evidence of metastasis. The thin muscular mass surrounding the body cavity was almost entirely infiltrated with macromelanophores but no large pigment cells were found within the body cavity itself.

The histologic details seen in these melanotic interspecific hybrids agree, in principle, with those observed in the overgrowth of intergeneric hybrids. There is a widespread macromelanophore hyperplasia, first of the corium and then in the muscles below; then there follows a hyperplasia of pigmented spindle cells which are relatively small in terms of macromelanophores. These smaller pigment cells are characteristic of previously described melanotic neoplasms of intergeneric hybrids. In the interspecific hybrids, however, while they have been seen, they are not as well represented. Perhaps these spindle

cells and a reticular network. The scales are elevated. The epidermis is Seen on the left side as a thin strand of tissue. The middle of the photomicrograph shows the spinal cord and part of the vertebral column, which are reached by invading pigmented tissues.

Muscles suffer loss of striation and atrophy.

It is not hyperplastic.

5 60 MYRON GORDON A N D G. M. SMITH

cells appear in greater profusion in older interspecific hybrid specimens than the one available for the present report. The smaller pigment cells of the melanotic overgrowth in killifish hybrids resemble other teleost melanosarcoma cells and they also bear a likeness to cells of mammalian melanosarcoma.

P . maculatus-P. xiphidium Hybrids: It is noteworthy that relatively few of the P. maculatus-P. xiphidium melanotic hybrids reach the severe form of overgrowth development that is defined as the third stage of melanosis by Reed and Gordon ( 193 1). Although most of these hybrids are intensely pig-

FIG. 16. ~ E O G R A P I i l C DISTRIBUTION OF KXOWN SPECIES OF PLATYPOECILCS

Each species of platyfish is confined to a river system. I n some cases the headwaters of two adjoining river systems appear to be close together but mountain barriers effectively separate the aquatic habitats. The dots indicate collection stations.

In the laboratory, the species hybridize readily. When the spotted Rio Papaloapan platy fish is mated with any other species of platyfish or with the swordtail, their hybrid offspring dcvrlop melanosis. The intensity of melanosis within the interspecific hybrids varies directly with the distance hctwecn the habitats of the species. See Fig. 1 7 .

mented and exhibit niacromelanophore hyperplasia of the corium, the destruc- tion of muscular tissue has not been observed as yet.

When the macromelanophores are transmitted to the interspecific hybrid by the P. xiphidium parent, no melanotic conditions appear.

P . maculatus-P. variatus Hybrids: No melanotic overgrowths have yet been detected in these melanotic hybrids. These interspecific hybrid fishes show an intense macromelanophore hyperplasia of the corium, but the condition of melanosis is definitely less marked than that of the P. maculatus- P. xiphidium hybrids.

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HELANOTIC NEOPLASTIC DISEASE IN FISHES 563

DISCUSSION The observations presented in the text and tables may be discussed from

several points of view. The data are significant in adding further evidence for the heritability of melanotic neoplasms in fishes and indicating the special importance of hybridization as a prime factor in tumor development. They are significant, also, ‘in studies of evolution and speciation, for they indicate the extent of physiological as well as morphological and geographical separation of the species (Fig. 16). Were it not for the geographical barriers which confine each of the four species to individual river systems, a merging of their various hereditary differences would take place. This phase of the problem will be discussed elsewhere.

The observations recorded may also be discussed from the point of view of the hereditary basis of sex in this group of fish. Bellamy (1922) and Gor- dqn (1927) have shown that P. muculutus has the WZ (female) ZZ (male) type of sex inheritance. Kosswig (1935) and Bellamy (1936) claim that P . xiphidium and P . variatus have the XY (male) XX (female) type of inheritance. A discussion of this phase of the problem is reserved for a further paper, but a preliminary critical review has recently been presented (Gordon, 1937). Goldschmidt (1937) and Castle (1938) have just published reviews of the problem of the genetics of sex in fishes.

The following discussion will be confined to the hereditary aspects of spontaneous tumor development in hybrid fishes. On the whole, the data tell a consistent story. Starting with P. muculatus of the spotted variety-a variety whose spotted condition is due to the presence in its skin of many large black pigment cells-the influence of heredity is clear. The large black spots, here called macromelanophores, are definitely inherited. The hereditary factor involved is a sex-linked dominant, Sp. When a spotted platyfish of the species P . maculutus is mated with an unspotted variety of the same species, the offspring are spotted. The intensity of the spotted condition varies slightly but no melanotic conditions are detected among the young. From a spotted mother and an unspotted father a number of definite ratios may result. These have been mentioned in the introduction. I t is clear, however, that no matter what the ratio of the spotted to the unspotted offspring is, and no matter what the sex, the spotted fish are merely spotted and not pathologically melanotic.

When a spotted P . macdutus ( S p ) is mated with any other species, P . vuriatus, P . xiphidium, P . couchiunus, or Xiphophorus hellerii, the interspecific or intergeneric hybrids that inherit the macromelanophore factor from their P. maculutus parent have melanosis (Fig. 17). Melanosis once present in an individual may be transmitted to individuals in succeeding generations under suitable mating. This may be brought about by mating melanotic hybrids to one another or to a pure species other than P. muculutus. Back-crossing and inbreeding provide suitable hereditary conditions for the production of melanosis early in the life history of the individual. Special attention to this phase of the work on melanosis and heredity has been given recently by Gordon (1937).

While the data on melanosis in interspecific crosses given here are still

5 6 4 MYRON GORDON AND G. M. SMITH

inadequate for final statements, they seem to p i n t to a definite correlation between the intensity of melanosis of the first generation hybrid and the relative geographical distance that separates their parents in their natural habitat (Fig. 16). To explain: P . mucdatus of the Rio Papaloapan drainage area is about a thousand miles away from P . couchianus of the Rio Grande system. Matings between the spotted Rio Papaloapan platyfish and Rio Grande platyfish produce hybrids with a relatively severe form of melanosis. But the degree of melanosis is not as great in hybrids between the same spotted P . maculatus and P . xiphidium, whose habitat is some 750 miles away. The intensity of melanosis is least in platyfish hybrids between P . maculatus and P. variatus, whose habitats are in adjacent river systems (Fig. 17).

These observations seem to indicate that P . couchMnus and P . maculatus are not only far apart geographically but physiologically as well. Yet though Xiphophorus hellerii lives in the identical habitat with P. macdatus in Mexico ( Friedman and Gordon, 1934), their intergeneric hybrids suffer from melanosis (Gordon, 1931). Here the species are not geographically separate but their physiological constitutions are quite diverse. Can the explanation for these two apparently diverse results depend upon the possibility that Xiphophorus and Platypoecilus had been geographically separated at some time in the past, and only in recent times have returned to a common habitat?

The hereditary bases for spontaneous melanotic neoplasms in hybrid fishes lie in the genetic constitutions of both parental stocks. The genetic contribu- tion of one of the parents is known, for in each case of melanosis among hybrid fishes one of the ancestors was a macromelanophore-carrying P. macdatus. The other parent contributed the hereditary factors for modifying the normal growth habit of macromelanophores. From experiments conducted previously it has been shown that X . heUerii may have more than one of these modifying hereditary factors. Kosswig (1931) and Gordon (1937) have arbitrarily assumed at least two such factors. The data collected here, upon purely specific crosses, may be interpreted on the same basis.

I t might appear that, since the time of appearance of spontaneous melanotic neoplasms may be pushed forward, even to the point of late embryonic life, the modifying factors speed the rate of pigmented cell development or those melanoblasts that furnish macromelanophores with black pigment.

Apparently the modifying factors of P. couchianus, P . Xiphidiurn and P. vahtus vary in their ability to influence the development of macromelanophores controlled by the hereditary factor S p of P . macukrtus, since the three kinds of interspecific hybrids vary in the intensity of melanosis.

SUMMARY

(1) A melanotic neoplastic disease appears spontaneously in hybrid fishes produced by mating Rio Papaloapan platyfish, Pkatypoecilus macdatus (macromelanophorecarrying variety) with: ( a ) Rio Grande platyfish, P. couchicmtrs; (b) Rio Sota la Marina platyfish, P . xiphidium; ( c ) Rio Panuco platyfish, P. uahtus. This is summarized in Fig. 17.

(2 ) When the macromelanophore, sex-linked factor S p of P. mcrcdatus is carried on the Z chromosome of the female in an interspecific cross, the hy-


brid daughters are normal, while the hybrid sons are melanotic because they alone inherit the S p factor. When S p is carried on the W chromosome of the female P. maculatus, the hybrid daughters inherit the S p factor and develop melanosis; the sons are normal as a rule. This is illustrated in Figs. 2 and 4.

(3) In the melanotic hybrids of P. maculatus and P . couckianus, the melanotic neoplastic disease appears when the hybrid possesses macromelanophores only (Fig. 3 F ) . Micromelanophores are not necessary for the melanotic state.

(4) The degree of melanosis is most intense in the hybrids of P. maculutus and P. couchiunus, less in hybrids of P. maculatus and P . xiphidium, and least among hybrids of P. maculatus and P . variatus. This is correlated with the distance separating their natural habitats in Mexico (Figs. 16 and 17).

( 5 ) The neoplastic disease that appears within the species hybrids is similar to the melanotic conditions that appear among the generic hybrids of the spotted P. maculatus and X. kellerii. The cells of these teleost melanotic overgrowths resemble cells of mammalian melanosarcoma.

(6) In some second-generation hybrids the melanotic disease appears on the day of birth.

BELLAMY, A. W.: Anat. Rec. 24: 419-420, 1922. BELLAMY, A. W.: Proc. Net. Acad. Sci. 22: 531-535, 1936. CASTLE, W. E.: Sc. Monthly 46: 344-350, 1938.

-MAN, B., AND GORDON, MYRON: Am. Naturalist 68: 446-455, 1934. GOLDSCHMIDT, R.: Quart. Rev. Biol. 12: 426-439, 1937. GORDON, MYRON: Genetics 12: 253-283, 1927. GORDON, MYRON: J. Heredity 19: 551-556, 1928. GORDON, MYRON: Am. J. Cancer 15: 732-787, 1931, a. GORDON, MYRON: Am. J. Cancer 15: 1495-1523, 1931, 6. GORDON, MYRON: Aquatic Life 16: 518-522 and 17: 4-6, 1933. GORDON, MYRON: Fish Culturist 12: 143-146, 1933. GORDON, MYRON: @. Naturalist 67: 18, 1933. GORDON, MYRON: Home Aquarium Bull. 5: 7-10, 1935. CORDON, MYRON: Home Aquarium Bull. 5: 185-188, 1935. GORDON, MYRON: Genetics 22: 376-392, 1937.

REFERENCES

hASER, A. c . , AND GORDON, MYRON: Genetics 14: 160-169, 1929.

GORDON, MYRON: Am. J. Cancer 30: 362-375, 1937. GORDON, MYRON: Copeia, pp. 19-29, 1938. GORDON, MYRON, AND REED, H. D.: Proc. Sixth Internat. Congress Genetics 2: 245, 1932. GORDON, MYRON, AND SMITH, G. M.: Am. J. Cancer 34: 255-272, 1938. HUBBS, C. L., AND GORDON, MYRON: Fishes of Northeastern Mexico, unpublished. KOSSWIC, C.: Ztschr. f. ind. Abst. u. Vererb. 59: 61-76, 1931. KOSSWIC, C.: Der Zuchter 7: 4048, 1935. KOSSWIG, C.: Arch. f. Entwcklngsmechn. d. Organ. 133: 140-155, 1935. MEEK, S. E.: Field Columbian Museum, Zool. Ser. 5: i-lx, 1-252, 1904. REED, H. D., AND GORDON, MYRON: Am. J. Cancer 15: 1524-1546, 1931. REED, H. D., GORDON, MYRON, AND LANSING, W.: Anat. Rec. 57: 107, 1933.

by - cancer research · this cross differs from the one illustrated in fig. 2 in that the spotted...

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