The inheritance of susceptibility to infection with Schistosoma mansoni in Australorbis glabratus

The Inheritance Schistosoma

of Susceptibility to Infection with mansoni in Australorbis glabratusl Walter

L. Newton2

U. 8. Department of Health, Education, and Welfare, Public National Institutes of Health, National Microbiological Bethesda, Maryland

(Receivedfor publication, 13

June

Health Service, Institutes,

1952)

The existence of strains of parasite and intermediate host species, differing in infectivity and susceptibility, respectively, has been demonstrated for several parasitic diseases. The studies of Vogel (1942), Stunkard (1946), Cram et al. (1947), Comper (1947), Files and Cram (1949), and Files (1951) have demonstrated that a snail serving as an intermediate host for a schistosome in one geographical area may be refractory to infection with the same speciesof parasite from a different area. Of particular interest in this connection was the report of Files (1951) t’hat whereas Australorbis glabratus from Puerto Rico is susceptible to a local strain of Xchistosomumunsoni, a strain of this speciesof snail from SBo Salvador, Bahia, Brazil was shown to be completely refractory to the Puerto Rican strain of parasite. Information relative to the susceptibility of the Brazilian snail Do its local strain of parasite was not obtained. The two strains of A. glubrutus were morphologically indistinguishable, according to Dr. E. G. Berry, malacologist of the Laboratory. It was concluded from these findings that there were physiological differences between the two strains. It was of interest t’o ascertain the nature of some of the factors which influence t’he susceptibility of snails to schistosomeinfect’ion and which might account, at least in part, for the difference observed between these strains. The possibility presented itself that some of the factors are under genetic control. The relationship between t’he genetic constit,ution of an 1 The material presented in this paper is part of a thesis submitted to the Graduate Council of the George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2 The author is indebted to Mr. Lk~rvin Schneiderman, ljiomet.rics Section, National Cancer Institute, for his advice on t hc stat.isticnl aspects of this study. 3 1,aboratory of Tropical Diseases. 242

INHERITASCE

OF SI-SCEPTIBILITY

TO S. JfUAXVSONZ

243

animal and its susceptibility to infection with a particular disease organism has been the subject of considerable study, most of the investigations being concerned with vertebrate hosts. This field is well covered in reviews by Crew (1928), Kozelka (1929), Hill (1934), and Gowen (1948). With regard to invertebram vectors of disease, an association between genetic constitution and susceptibility has been deomonstrated for mosquitoes and malaria (Huff 1927, 1929, 1931; Trager, 1942; the Report of the Rockefeller Foundation, 1948; Micks, 1949); for leafhoppers and plant viruses (Storey, 1932; Black, 1943); and for mosquitoes and Dirofilaria immitis (Roubaud, 1937; Kartman, 1953). Genetic studies on the susceptibility of snail vectors have not been encountered in a review of the literature on this subject. It was felt that the two above-mentioned strains of A. gkhratus studied by Files (1951) might represent two genetically different populations with regard to susceptibility. In order to provide information in this regard, crosses mere made between members of the susceptible Puerto Rican and nonsusceptible Brazilian strains. Subsequent generations of progeny were then t’ested for susceptibility to the Puerto Rican strain of S. mansoni. MATERIALS

AND METHODS

Preliminary observations confirmed Brumpt’s (1941) conclusion that the hermaphroditic A.glabratus can self-as well as cross-fertilize. Consequently, the problem arose as to the ancestry of offspring obtained after a pairing. Fortunately, the writ,er was able to obtain a red mutant variety which appeared in a stack colony of the Brazilian strain maintained by Dr. Berry. This variety was found to breed true and to be recessive to the wild-type brown pigmented condition. Preliminary susceptibility tests also demonstrated that it was as refractory to infection with the Puerto Rican strain of S. nzansoni as was the pigmented variety. When pairings were made between a pigmented Puerto Rican and a red Brazilian snail, pigmented offspring from the latter would be the result of crossfertilization and would represent a “hybrid” of a susceptible-nonsusceptible cross. Offspring from the pigmented Puerto Rican parent were unsuitable for purposes of this study since they would he pigmented whether the parent had selfed or had been fertilized by the red Brazilian snail. Sot until the second generation could the ancestry of progeny of t,he Puerto Rican parent be told with certainty. Several pairings were made between l- to 2.mm diameter snails of the two strains. After several weeks, when eggs appeared on t.he side of the beaker in which a pair was kept, the snails were separat,ed. The red snail was then observed for the production of eggs which hatched as pigmented snails. As soon as the latter, which will be referred to as “F,“, appeared they were separated from the parent. Some were isolated individually for self-fertilization and FP production,

244

WALTER

L.

NEWTON

and some kept for exposure as a group. All Fr snails were exposed to 10 miracidia of a Puerto Rican strain of 6’. mansoni when a size of 7 to 9 mm was attained. Those serving as parents of Fz progeny were rc-exposed to 20 miracidia after studies with their offspring were completed, if negative after t.he first exposure. As the Fz were obtained, they were transferred to battery or museum jars for rearing. Considerable care was taken in this matter to insure that a parent continued to self and was not fertilized by one of its offspring. The Fz were observed every 7 to 10 days and all of a size of 7 mm or more (usually 7 to 9 mm) were removed from the rearing jars and exposed to 10 miracidia. This was continued until information had been obtained on the susceptibility of at least 100 F1 from each Fr parent, or until no further offspring were produced. As soon as sufficient data on the susceptibility of an Fz “population” (the progeny from a single selfed F1) were obtained, 10 to 14 young unexposed F2’s from that population were isolated at random for selling and F, production. The latter were also exposed to 10 miracidia at a size of 7 to 9 mm. However, the Ft parents were not exposed until after tests on their Fa progeny had been completed, at which time they were several months of age. It was found that if a snail acquired a fairly heavy schistosome infection before egg-laying occurred, very few if any young would be produced. This was the result, perhaps, of invasion of the gonads by sporocysts, a rather common occurrence in heavily infected snails. With the exceptions indicated above, all snails were routinely exposed at a size of 7 to 9 mm in diameter, i. e., juveniles to young adults 6 to 8 weeks of age. Snails of this size range were chosen because they were relatively easy to handle, and because it was felt that there would be a minimum amount of age variation in this range. Also, shortly after a size of 9 mm was attained egg-laying sometimes occurred. Livers from female white Swiss mice (NIH general purpose strain) exposed to cercariae 7 to 12 weeks previously served as the source of schistosome eggs. The latter were freed from the tissues by the usual procedures of comminution and sedimentation in saline and water, and hatching was stimulated by a desk lamp. The snails were exposed by placing 10 actively swimming miracidia into a small Syracuse watch dish to which a snail was added, and the dish then filled with aerated tap water. The dishes were then covered with glass slides and left for 3 to 10 hours, or occasionally overnight. Miracidia were not used if more than 3 hours time had elapsed after the eggs were set under the lamp for hatching. The studies of Maldonado and Acosto (1948) have shown that the miracidia can lose their infectivity rather rapidly after this time. A challenge of 10 miracidia per snail was chosen on the basis of results of a few preliminary experiments. The percentage of Puerto Rican snails which became infected increased as the number of miracidia used for exposure was increased. In a typical experiment, 12 of 20 snails exposed to five miracidia each were positive, whereas all of 19 snails were positive after exposure to 10 miracidia each. These findings are in keeping with the results of Schreiber and Schubert (1949) and Stirewalt (1951). During the summer months the temperature remained above 90°F most of the time. During the winter months, it was slightly more variable, but was usually around 85°F. The various parents and their progeny, both before and after

INHERITAKCE

OF SuSCEPTIBILITY

TO S.MANSONI

245

exposure, were maint,ained in glass containers to which aerated tap water only was added. They were fed daily on raw washed lettuce. Thirty to thirty-two days after exposure, the surviving snails were checked for infection in the following manner: Each snail was placed into a 20 X 150 mm test tube containing 15 to 20 ml of aerated tap n-ater. The tubes were placed under a lamp and examined throughout the day for t,he emergence of cercariae. Those failing to shed were dissected within the following 3 days. The hepatic gland and ovotestis were teased apart, and, if negative, the surfaces of the anterior portion of the snail were examined for sporocyst,s. All examinations were made with a binocular dissecting microscope. Twelve to twenty-four Puerto Rican snails 7 to (3 mm in size were usually exposed to infect,ion along with the progeny of t)he crosses. These served as cont,rols for the experimental procedure and for the viability of t’he miracidia. Similar, but less frequent, checks were made on the susceptibility of the Brazilian strain. RESULTS

AND DISCUSSION

Parent Strains Before the findings obtained with the various crossesare presented, the susceptibility behavior of the two parent strains will be discussed. Of 514 Puerto Rican snails exposed to IO miracidia each, in 32 tests, 463 (90 %) survived, and 95 y0 of the latter were positive for cercariae. The high susceptibility of t,his strain is apparent. While t,here was some variation in t#heapparent degree of infection in individual snails, in no instance was an obviously ret,arded infection found. Snails either were entirely negative or contained cercariae. In an effort to detect any marked influence of age upon susceptibility, eight parallel exposures of 7- to g-mm snails and 12- to 15-mm snails were made. Of 84 of the smaller snails, 75 (89 %/o)survived and of these 93 y0 were positive for cercariae. Of 92 of the larger snails, 88 (96 %) survived but only 66% of t’hese were positive. The infection rate of the larger snails was significant’ly lower (P < -01). However,

when

19 of t,he negative

large

snails Jvere re-exposed

to 20

miracidia, 17 of them became positive. It was also observed in some instances that miracidia appeared to effect entry into the larger snails lesseasily, and there was more mucus, which seemedto have a diverting effect. It is difficult, therefore, to separate the influence of an age effect from a size effect in lowering the infection rate among the larger snails. These tests demonstraM that when snails 7 to 9 mm in size were exposed t,o IO mirncidia no more than 3 hours of age, the Puerto Rican st,rain of ;t . glahralu

had a11 infcct.il)ilitjy

rate of approximately

95 %,

None of over 450 snails of Ihe nonsnsc~ept~i~)le Brazilian strain, both

246

WALTER

L.

NEWTON

red and pigmented varieties, was positive when exposed at a size of 7 mm or larger at various times t,hroughout t’hese studies. No sporocyst material was found upon dissection even though miracidia were observed to penetrate. This was true even when exposures to as many as 50 miracidia per snail were made. However, as shown in Table I, when snails of this strain were exposed at very early ages, some as young as 1 to 2 days, some positives could be obtained. In one experimental series, each snail was exposed to 10 miracidia, and in another to 5 miracidia. The great majority of the “positives” shed cercariae. A few positives which did not shed contained sporocysts in the hepatic gland. Some of the snails which shed were no larger than I Australorbis glabratusafter Exposure at Various TABLE

Infection

Results with Brazilian Ages to Puerto Rican -. ~~~-

“,~~~; S;;p

Nynber,of nuracldn

Group

Age at exposure (days)

Schistosoma Number snails exposed

mansoni

Number surviving

Number positive

Percent survival

Percent survivors positive

1

10

A B

1 to 9 11 to 24

155 112

68 74

24 29

44 66

35 39

2

5

A B

2 to 16 39 to 67

80 67

53 62

9 0

66 93

17 0

-

1 to 2 mm. Because the proportion of snails surviving an exposure at a particular age was sometimes small, each series is divided into a “younger” and an “older” half with approximately the same number of survivors in each half. These are referred to as groups A and B, respectively. It can be seent’hat a fair proportion of the very young Brazilian snails exposed to 10 miracidia could be infected. There did not appear to be any difference in infection rate between groups A and B. However, the significant difference (P < .Ol) in survival rate between these two groups suggeststhat the higher mortality in group A might well have been an indication that these younger snails could not survive the infection as well as the older onesand thus several potential positives may have been lost. This idea is supported by the fact that,, in Group A only 17 (24 ‘%) of 72 snails exposed at the ages of 1 and 3 days survived; whereas of 83

snails exposed at the ages of 3 and 9 days, 51 (61 yoj survived the 30-day developmental period. In the second experimental series, \vider age iutervals \verr used iii order to fiir(l ali cudpoint, of suwcptil~ility. Also, only 5 miracidia per snail used in a11 attempt to iiwcasc the sur\ivnl rate iu Ihc youugcr snails. As is apparent from Table I, louver iufection rates and greater survival resulted. Ko posit.ives were obtained among snails over 5 weeks of age (4 to 5 mm in diameter) after a challenge of 5 miracidia. These results are in keeping with t,he observation that snails of this strain are uniformly nonsusceptible when exposed at a size of 7 to 9 mm. In this same connection, Kendall (1950) observed that 23 of 63 Lymnaea staynalis became infected with Fasciola hepatica when exposed immediately after hat’ching. Of a second group of 33 snails from the same parent exposed 8 days after hatching, none became infected. The question arises as to whether other species and genera of snails which have been found experimentally to be refractory to infection with S. mansoni when exposed as juveniles and young adults might not become infected if exposed at a very early age and t’hus be pot’ential vectors. The Brazilian snails which became infected at, a very early age retained the infection long after a size and age was attained at, which they normally are refractory. Three snails exposed at the age of 1 day were still shedding cercariae 8 months later, at which time they were 15 to 17 mm in diameter. Cercariae obtained from the infected Brazilian snails were used for infecting mice. Eggs were obtained from the livers of the latter, and the miracidia were used for exposing 7- to g-mm snails of both the Brazilian and Puerto Rican strains. The same strain difference occurred, i.e., all the Puerto Rican snails became infected whereas none of the Brazilian did. Thus, there was no obvious change in the infectivity of the parasite after development in the young Brazilian snails. In other studies with snails of the Brazilian strain (Newton, 1952), it was found that miracidia of the Puerto Rican strain are destroyed within 24 to 48 hours after penetration of juveniles and young adults with a marked cellular reaction to t’he parasit)e. In very young Brazilian snails this response is presumably either absent or inadequate. In summary, the two strains serving as parents for the crossing studies had infection rates of 0 and 95 % when exposed and examined under a relatively standardized set of conditions. The infection rates of offspring

248

WALTER

L.

NEWTON

of crosses between these &rains were det,ermined under the same con&tions of exposure, as nearly as they could be duplicated.

Progeny of Crosses Information was obtained on progeny from five crosses.While several pairings were made, some did not result in crosses,i.e., only red offspring were obtained from the red Brazilian parent. After other pairings only one or two Fl’s were obtained and these did not produce offspring. The five crossesfrom which a satisfactory amount of data was obtained will be referred to by the Roman numerals, I, II, III, XXV, and XXIX. F1 Progeny. The infection results obtained with the various Fl’s are shown in Table II. Susceptible Fl’s were obtained in three of the five

Infection

Brazilian

TABLE II Results with Fl’s Obtained from Crosses between Brazilian Rican Australorbis glabratus parent

and cross No. ____~-

I II III xxv XXIX

Number of FI surviving incubation period

23 G G 25 64

Number

and Puerto

positive

4 1 0 0 10

crosses,but too few Fl’s were obtained in most instances for conclusions regarding genotypes or differences between crosses.A comparison even of the extremes, cross numbers XXV and XXIX, did not show a significant difference. It is apparent, nevertheless, that susceptibility to schistosome infection is a heritable character, inasmuch as susceptible offspring were obtained from nonsusceptible Brazilian snails after fertilization by susceptible Puerto Rican snails. Fz Progeny. Not all isolated Fl’s produced Fz populations in quantity adequate for study. A total of 40.F~ populations from the five crosseswas studied. It developed that all these populations were derived from nonsusceptible F1 parents. This was due partly to the fact that the susceptible F1’s died, probably from the infection, before producing offspring, and partly to the relatively low incidence of susceptibles among the F1. The detailed findings with the F, populations are presented in Table III. Before the data for individual crossesare discussed,certain general

Fn Progeny Homogeneity data--P Brazilian parent and cross No.

FL” parent

Total Number sarnnumber pies of FP snails survipopulation ving 30 days

173 212

45 47 0)

26.0 22.2

.2&.10 .5&.30

.50-.30

8 6 7 7

130 140 130 110

14 14 (1) 19 (1) 15

10.8 10.0 14.6 13.6

.5c-.30 .70-.50 .2&.10 .8&.70

.70-.50

A

7 3 5 8 4 5 4

136 12s 1YZ 197 98 85 105

27 (1) 4 11 32 7 (1) 11 (1) 5

19.8 3.1 9.0 16.2 7.1 12.9 4.8

.50-.30 .9(r.80 .2&.10 .5(r.30 iG.50 :7ck50 .80-.70

<.Ol

B c D I K 1, A I3 c 1) F G H I K L M N 0 P It s T u v

8 5 10 6 6 6 5 7 7 4 8 6 3 5 2 7 6 7 8

147 166 157 162 130 157 67 152 137 88 164 144 79 84 82 144 1.54 11s 126

25 10 91 30 8 9 13

17.0 6.0 58.0 18.3 6.2 5.7 19.4 14.5 7.3 26.1 32.9 27.8 7.6 20.2 6.1 22.2 14.3 47.5 39.7

.20-.lO .80-.70 .0&.02 .02-.Ol .50-.30 .5&.30 .3O-.20 .05-.02 .9CL.80 .8&.70 .80-.70 .30-.20 .9&.80 :70-.50 7G.50

<.Ol

c D

8 7

III

A B D F

xxv

II

XXIX

Among populations of a cross

19 29 6 30

107 86 142 129 107 123 76 120

-~

Among samdes of a population

.6&.30 .30-.20 O5S. 02 70-.50 :5O-.3” .30-.20 .X&.70 .313.20

5 6 6 4 6 7 3 5

c

a All FL parents nraative after two Pxposures ’ ( ) indicntcs nnmhrr of snails witb retarded

14 (I)” 22 (2) 43

Percent positive

13.1 2.5 .6 30.3 17.0 17.8 23.6 7.9 25.0

u E F G H I J

I

Nu~+r posltwe

of infection values

22 (2)

22

(1) 10 23 54 (2) 40 (3) 6 (1) 17 5 32 22 (2) 56 (I) 50 (3) to infection. infectiun.*.

249

[email protected] .5&.30 [email protected] .50-.30

<.Ol

250

WALTER

L.

NEB’TON

observations are in order. All Fz populations were composed of both pigmented and red individuals, in the approximate ratio of 3 : 1, respectively. There was a significantly higher proportion of susceptibles (.05 > P > .02) among the red snails (21.0 % of 1,259) than among the pigmented snails (18.2 y0 of 3.692) of the F, populations. Whether lack of pigmentation increased susceptibility, or whether there was linkage between the gene for pigmentation and a susceptibility gene was not apparent. Inasmuch as the 3:l ratio was consistent throughout all Fz populations, the color of the individual snails will be ignored in the present discussion. The number of snails exposed for each population is not presented because it closely approximated the number that survived 30 days. Usually, well over 90 % of snails exposed to infection survived this period. There was no indication of a higher mortality rate among snail populations with higher infection rates, prior to the end of the 30-day developmental period of the parasite. Several snails contained infections which had not attained the cercarial stage by the end of the 30-day period. In some instances, infections may have reached the hepatic gland, but mere no further advanced than the “germ-ball” stage. In a few infections, findings were limited to a small mother sporocyst on the anterior portion of the snail. Because of the difficulty in establishing objectively stable criteria suitable for phenotypic classification of these variants from the normal infection, it was decided to set the phenotypic segregation at an early stage of infection. All snails containing at least one mother sporocyst with daughters were considered to be positive, even though such an infection was obviously retarded. Only two phenotypes, i.e., positive and negative, were recognized. Such a segregat’ion, of course, precludes recognition of grades of susceptibility. However, the number of ret’arded infections was usually rather small so it is felt that no great error is occasioned by such an arrangement. The numbers of these retarded infections are presented in Table III and are included in the “positive” category. Considered as “retarded” infections were t,hose in which t.he parasite had not attained at least the germ ball or early cercarial stage in the hepatic gland. The infection result presented for each Fz population is a composite of dat’a obtained from the exposure of several small samples drawn from the population, usually over a period of several weeks. Thus, it was essential to ascertain the uniformity of the infection data obtained with these samples. A test for homogeneity, as presented in Snedecor (1946),

was applied to the data obtained with the several samples of each population, and the resulting P values are presented in Table III. These values show that the variation in t,he infection results obtained with the samples of an Fz population was essentially consistent with what might be expected to occur as the result of cahance alone. The same test was applied to the infection data obtained for the several Fz populations of a cross. This was done to ascertain the probability that all Fz populations wit’hin a cross belonged to a single uniform population, and that their respective parents were, therefore, genetically alike. The P values derived from these calculations are also shown in Table III. Wit’h regard to the findings with specific crosses, the infection rates of the eight Fz populat’ions in Cross lir’o. I varied from 7.9 to 30.3 %. Analysis showed that the probability of getting differences as great as those obtained due to chance alone, had the eight Fz populations been samples of a single uniform population, was less than 1 in 100. Thus, the assumption can be made that the R parents were of mixed genotypes. However, the infection rates of some of the F, populations (e.g., I-D, I-H, and I-J) were quite similar, and this suggests that the F1 parents of these particular populations may have been of the same genotype. Little can be said of Cross No. II, except that the two F1 parents may have been genotypically similar. In Cross No. III, also, the four F1 parents may have been of similar genotype. In Cross No. XXV, infection rates among Fz populations ranged from 3.1 to 19.8 %. As with Cross No. I, variation in Fi genotypes appeared likely. The average infection rate of t’he combined Fz populations from No. XXV was lower than the average rat’e of the combined Fz populations from No. I. In the case of the latter, 185 of a total of 890 Fz snails (20.8 %o) became positive. After Cross No. XXV, only 97 of a total of 871 Fz snails (11.1%) were positive. All the F1 parents in both crosses were phenotypically alike, however, in that they were negative. It would appear, therefore, that the negative F1 parents in Cross No. I had in their gene pool more factors favoring susceptibility t,han did the negative F1 parents in Cross No. XXV. In keeping with this idea, it is t,o be recalled (Table II) t’hat four of the 23 F1’s obtained in Cross No. I were positive, whereasno positives lvere obtained in 25 Fl’s of Cross No. XXV. Cross No. XXIX provided t’he best opportunity for analysis because of the large number of F, populations obt#ained. Susceptibility tests were made on a total of 2,454 Fz progeny from 19 selfed nonsusceptible Fl’s. As in some of the other crosses, there was considerable variation in in-

252

WALTER

L.

NEWTON

fection rate from one Fz population to the next, ranging from 5.7 to 58.0 %. Several infection rates were repeated; 6 of the 19 populations showed infection rates in the narrow range of 5.7 to 7.6%. This finding suggests that the six f parents of these populations were genotypically alike. Attempts to group all 40 of the Fz populations into a few distinct classes based upon infection rates, and thereby to estimate the number of different genotypes represented by the various Fi parents did not give satisfactory results. Many of the populations appeared to fall into classes which differed in infection rate from one another by multiples of 6%. However, the rather continuous type of distribution, apparently reflecting the existence of several genetic factors together with possible interaction, linkage, and crossing-over, precluded such an arrangement. It was apparent, nevertheless, that the nonsusceptible condition can be represented by several different genotypes. FO Progeny. A large number of Fz snails from all five crosses were isolated randomly for selling and FS production. Many of these, however, did not produce offspring or had produced only a few, by the time this study was terminated. Information was obtained on forty-four FB populations. It developed that all these populations were derived from Fz parents which eventually proved to be nonsusceptible. Generally, the fact that a particular Fz parent turned out to be negative was consistent with the infection rate of the Fz population from which it was randomly chosen. In a few instances, however, one might have anticipated obtaining a susceptible Fn parent. Whether or not the fact, as indicated earlier, that the Fz parents were not exposed to infection until they were considerably older and larger than usual influenced this finding could not be ascertained. In general, little additional information suitable for determining genotypes was obtained from studies on F, susceptibility. Greater extremes in infection rate were encountered. Several F, populations had infection rates of 0 y0 (identical with the rate of the parent Brazilian strain), and one showed a rate of 82 y0 (approximating the rate of the parent Puerto Rican strain). F, populations derived from snails of Fz populations with the higher infection rates showed, on the average, a higher incidence of susceptibles than F3’s from snails of Fz populations with the lower rates. In Table IV are presented the findings with the 14 F3 populations of Cross No. XXIX for which data on at least 40 snails were obtained.

Ih-HERlT.kNCE

OF

SUSCEPTIBILITY

TO S. MA.VSfJ.VI

253

These results may be considered as typical of the findings with the Fa of other crosses, with differences, of course,, in absolute values. Statistical values are not presented; the data behaved in much the same manner as did those of the F, populations. It can be seen that the FS populat,ions derived from line XXIX-M, which had an F, infection rate of 32.9 %, TABLE Znfect.ion

Results

with Fa Progeny f&n Australorbis glnbrntus

IV Selfed Fz’s Cross No.

of Puerto XXIX

Rican-Brazilian

B

6.0

1 6 7 9

41 58 105 95

0 0 2 3

3 0 14 40

3 0 16 43

7.3 0.0 15.2 45.3

I<

7.3

3 4 6 8 11

79 119 10s 109 108

0 0 0 0 0

1 41 27 12 0

1 41 27 12 0

1.3 34.4 25.0 11.0 0.0

M

32.9

1 2 7 9 10

94 95 74 87 100

52.1 15.8 27.0 19.5 82.0

* All parents negative after exposure to 20 miracidia

showed, on the average, higher infection rates than those of lines XXIX-B and -K, the infection rates of which were 6.0 and 7.3 %, respectively. Population XXIX-M 10 had an infection rate approaching that of the Puerto Rican strain. One might have anticipated that the parent of this population would have been positive. There was a relatively high proportion of retarded infections in FO populations M-l and M-10. The infections iii several of these snails were limited to early mother sporocysts in t,he head-foot region. h comparison could be made between retarded development, of t,hc parasit*e and t’he

254

WALTER

L.

NEWTOK

“poor 4-toe” condition described in guinea pigs by Wright (1934). The latter, in a study of the inheritance of polydactylism in guinea pigs, has suggested that a physiologic threshold separates the alternative expressions of a character controlled by multiple genetic fact,ors. Heston (1951) has applied this same concept to the inherit’ance of susceptibility to tumor development in mice. Perhaps the same interpretation may apply to the inheritance of susceptibility to schistosome infection in snails. The snails wit’h retarded infections may have fiad combinations of factors which placed them astride a physiologic threshold. The establishment of snail lines known to be genetically homogeneous will undoubt,edly be required before more exact information relative to the genetic behavior can be obtained. HoweVer, these studies on the progeny of susceptible-nonsusceptible crosses have demonstrated that in A. glabratus susceptibility to infect’ion with a strain of S. mansoni is a heritable character. The continuous distribution of infection rates among Fz and FS populations also indicates that multjiple genetic factors are probably involved, rather than a single factor type of inheritance, such as was reported by Huff (1931) for Culex pipiens and Plasmodium cathemerium and by Storey (1932) for a leafhopper and a plant virus. The Puerto Rican and Brazilian strains of A. glabratus used in these studies may thus be considered as two populations with markedly cliff erent genetic constitutions affecting susceptibility. The manner in which this difference is expressed is undoubtedly complex and purely a matter for speculation at the present time. However, in view of the marked difference betmeen t,hese strains in tissue response to the miracidia (Newton, 1952), the genotype of the snail may have a comparatively direct association with its tissue reactivity to a particular strain of parasite. Other factors, which may be grouped in a broad, “non-genetic” category for t’he present discussion, have been found in t.his study and in those of others, mentioned earlier, to be influential in determining whether or not a positive snail will be obtained after an exposure. These are both extrinsic and intrinsic, insofar as t.he snail is concerned. Among the extrinsic factors can be included the strain, age, and number of miracidia to which the snail is exposed. Among intrinsic factors, the age of the snail was shown to influence its susceptibility. Also, the condition of pigmentation appears to exert some influence, although its relationship to susceptibility is not clear. These findings t*hus emphasize the importance of attempts to control “non-genetic” sources of variation in studies

INHISRIT.\XCE

dcsigiied to elucidate constitution.

OF

Sil-Si('EP1'IHII,I'I7-

an nsswiution

"55

TO S. .lI.lSSO.\'I

bet\vecn xuscc~ptibilit~y and genetic

Studies I\-ere conducted t,o uscertaill \\-het~lier genetic factors were involved in the susceptibility differewe between a Brazilian and a Puerto Rican &rain of Australorbis gbabratzrs t,o a Puerto Rican strain of Schistosoma mansoni. Since it was found that non-genetic factors such as the number of miracidia used for exposure and the size and age of Ihe snails exerted some influence upon susceptibility atld infection rate, a standard set of exposure conditions was used. Vllder t,hese conditions 95 y0 of the Puert’o Rican snails were posit.ive for cercariae after 30 to 32 days, whereas none of the Brazilian snails became infect,cd. Crosses were made between members of the t,wo snail strains, and the progeny therefrom were exposed to infect,ion under the conditions mentioned above. Because A. glabratus can self- as well as cross-fertilize, a red mutant variety of t,he Brazilian strain which was recessive to the wild-type pigmented condit,ion was used as a member of a pair to ascertain when crossing had occurred. F, progeny from some Brazilian parents which had crossed with Puerto Rican snails included susceptibles; Fl’s from other crosses did not. Fa populations derived from selfed nonsusceptible F1’s had infection rates which she\\-ed a cont,inuous type of distribution from 3 to 58 %. The red members of the F2 populations showed a slightly, but’ significantly, higher infection rate than did the pigmented members. Fa populations derived from nonsusceptible Fz parent’s had infection rates varying from 0 to 82 %. Particular infect’ion rates were replicated in several of t,he populations. The findings demon&rated t.hat suscept,ibility to S. munsoni infection in A. glabratus is a heritable caharacter, and that several genetic factors are probably involved. The Puerto Rican and Brazilian strains of A. glabratus used in these studies apparently represent different genetic populations with regard to this character, at least insofar as the Puerto Rican strain of parasite is concerned.

BLACK, I,. AI. 1943. Genet,ic variation in the clover leafhopper’s potato yellow-dwarf virus. Genetics 28, 200-209. BRUJIPT, E. 1941. Observations biologiques diverges concernant

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(Aus-

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7, 706-734.

HUFF, C. G. 1929. The effects of selection upon susceptibility to bird malaria in Culex pipiens Linn. Ann. I’rop. Med. Parasitol. 23, 427439. HUFF, C. G. 1931. The inheritance of natural immunit,y to Plasmodium cathe merium in two species of Culex. J. Prev. Med. 6, 249-259. HESTON, W. E. 1951. Genetics of ncoplasia in mice. Growth 16, Suppl., 2346. KARTMAN, L. 1953. Factors influencing infection of the mosquito with Dirofilaria immitis (Leidy, 1856). Exptl. Parasitol. 2, 27-78. KENDALL. S. B. 1950. Snail hosts of Fasciola hepatica in Britain. J. Helminthol. 24, 63-74. KOZELKA, A. W. 1929. The inheritance of natural immunity among animals. J. Heredity 20, 519-530. MALDONADO, J. F., AND ACOSTA-MATIENZO, J. 1948. Biological studies on the miracidium of Schistosomu mansoni. Am. J. l’rop. Med. 28, 645-657. MICKS, D. W. 1949. Investigations on the mosquito transmission of Plasmodium elongatum Huff, 1930. J. Natl. Mal. Sot., 8, 206-218. NEWTON, W. L. 1952. The comparative tissue reaction of two strains of Australorbis glabratus to infection with Schistosoma mansoni. J. Parasitol. 38, 362-366. THE ROCKEFELLER FOUNDATIOS, 1948. Ann. Rept. Internat. Health Div. 6-7. ROUBAUD, E. 1937. Nouvcllcs rccherches sur l’infection du moustiquc de la fievrc jaunt par Dirojilaria immitis Lcidy. Lcs races biologiqucs de Aedes aegypti et l’infcction filaricnnc. Bull. sot. path. exotipue 30, 511-519. SCHREIBER, F. G., AND SCHUBERT, M. 1949. Results of exposure of the snail Australorbis glabratus to varying numbers of miracidia of Schistosoma mansoni. J. Parasitol. SNEDECOR, G.

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