INCREASING TREND IN FREQUENCIES OF LETHAL AND SEMILETHAL CHROMOSOMES IN A NATURAL POPULATION OF

JAPAN. J. GENETICS Vol. 48, No. 1: 41-51 (1973) INCREASING TREND IN FREQUENCIES OF LETHAL AND SEMILETHAL CHROMOSOMES IN A NATURAL POPULATION OF DROSOPHILA MELANOGASTER SUMIO MINAMORI, KAZUKO ITO, AKIKO NAKAMURA, YASUHARU ANDO AND HATSUMI SHIOMI Zoological Laboratory, Faculty of Science, Hiroshima University, Hiroshima 730 Received November 21, 1972 Genetic changes in natural populations have been observed in several insect species; e.g., lady beetle, Harmonia (Komai 1954) ; butterfly, Maniola (Ford 1968) ; or Drosophila. Dobzhansky, Anderson and Pavlovsky (1966) reported a directional change in the frequencies of certain inversions of the third chromosomes in populations of D. pseudoobscura found in the Pacific Coast of the United States. Certain inversions had grown and others had dwindled in the populations between 1940 and the 1963-1965 period. Although the cause of the genetic changes observed remains unknown, they pointed out the contamination of the fly environments with insecticides as a possible factor. Ives (1945, 1954, 1970), Band and Ives (1961, 1963, 1968) and Band (1972) have annually surveyed the genetic structure of a natural population of D. melanogaster by sampling flies from the same station located in South Amherst, Massachusetts since 1938. In the 1938-1946 period, a high frequency of lethal and semilethal second chromosomes and a low frequency of allelism between lethal genes were found in the population. However, the high frequency became lower during 1947-1952 and dropped a little more in the 1953-1969 period. These authors found a correlation of the frequency changes with certain weather factors; positively with summer rainfall and negatively with the average daily temperature range of the week prior to collection. The genetic structure of a natural population of D. melanogaster has also been surveyed annually since 1961 in our laboratory through a station in Hiroshima City. The results obtained in surveys made in the 1961-1968 period were reported (Minamori and Saito 1964; Minamori and Tatsukawa 1968; Minamori et al. 1970). The characteristics observed in this population during the period are as follows. 1) This population shows a seasonal change in actual size; increases from early summer to fall and dwindles in winter. 2) The frequency of lethal plus semilethal second chromosomes was lower and that of allelism between lethal genes was higher in earlier surveys; hence, the effective number of this population was assumed to be small. 3) The lethal plus semilethal frequency increased and allelism frequency decreased from early summer to fall in each

42 S. MINAMORI, K. ITO, A. NAKAMURA, Y. ANDO AND H. SHIOMI year. 4) An infective agent was discovered in this population in 1964, and it was recognized to reduce lethal frequencies, but not semilethal frequencies (Minamori 1967; Minamori and Tatsukawa 1968). 5) An extrachromosomal element denoted by delta was found in this population in 1965 (Minamori 1969). An important nature of delta may be its mutagenic action to its carrier's chromosomes; it induces frequent recessive lethal mutations in definite regions of the second chromosome, possibly at an embryonic stage of the carrier (Minamori and Ito 1971). The population has annually been surveyed as to the frequencies of lethal and semilethal second chromosomes, allelism, infected flies with the killing agent, and of delta-associating second chromosomes. The surveys made through 11 years (1961-1971) disclosed an increasing trend in frequencies of lethal and semilethal chromosomes, and a decreasing trend in allelism frequencies. In the present report, the data obtained during from 1969 to 1971 are presented and the trend is analyzed. MATERIALS AND METHODS Flies used in this study were collected at a station located in a pickle factory on Tera Street in Hiroshima City. Collections were made once or twice, summer (July, August) and fall (September, October), each year. The frequencies noted above were examined by the same procedure employed in an earlier study (Minamori et al. 1970). The procedure is briefly noted below. Isolation of second chromosomes and estimation of homozygous viabilities : A single wild male or a single daughter of individual wild females was crossed with Cy/Pm females or males (Cy, Curly wing gene; Pm, Plum eye gene). A single Cy/+ (+, wild second chromosome tested) F1 male or female of this mating was crossed again with Cy/Pm females or males, and when the Cy/+ sons emerged they were mated with their sister Cy/+ females. In the subsequent generation, the percentage of homozygous non- Curly flies which emerged was obtained. Lines producing all Curly flies were classified as lethal. Those producing less than 16.7% non-curly flies were classified as semilethal. Allelism of lethal genes : Lethals obtained were maintained as a balanced strain with a Cy chromosome, and were employed for half-diallel crosses. The percentage of allelic crosses in which no non-curly flies emerged was obtained (allelism frequency). Testing for infection : In the course of estimating the homozygous viability, the number of F1 offspring of the mating, Cy/Pm x +1+', was tallied, and the percentage of non-curly (Pm/+) flies was obtained. When the percentage was significantly higher than expected (50%), the wild male or the wild female from which these offsprings were descended was assumed to be infected since the infective agent kills more Curly zygotes than Plum zygotes (Minamori 1967). Determination of S and ID chromosomes : There are two second chromosomes in the population, sensitive and insensitive, related to the killing action of delta. The

INCREASE IN LETHAL FREQUENCY 43 sensitives are further classified into three categories, Se', Sr and Sc; Sb and Sr steadily retain each specific delta, delta b or r, while Sc retains delta b temporarily (Minamori 1971) The insensitive chromosomes are also classified into three types, IDb, ID and I. IDb retains delta b steadily, IDC retains delta b temporarily, and I retains no delta. In the present study, the chromosomes were classified into 3 categories: sensitive (S), insensitive retaining delta b (ID) and insensitive (I). For testing the sensitivity of chromosomes, Curly flies heterozygous for the individual second chromosomes tested were crossed with females of Cy/bwD stock (bwd, a dominant brown eye gene on a sensitive chromosome Sb, retains delta b steadily), and the percentage of non-curly flies (bwd/+ ) among the total number of flies of this mating was obtained. The frequency of non-curly flies was expected to be lower when the + chromosome was sensitive. Lines showing a significantly lower percentage compared with that of insensitive lines (average, 33.84%) were assumed to be sensitive. Insensitive chromosomes classified by the above test were examined as to whether they could retain delta b. The bwd/+ males of insensitive lines obtained in the above test were crossed with Cy/bwD females which retained delta b. The Cy/+ daughter of this mating would inherit delta from the Cy/bwD mother, and retain an appreciable amount of delta, when the + chromosome carried delta-retaining gene Da (located at 24.9 map-unit; Minamori and Sugimoto, in press). To test whether or not this Cy/+ daughter carried delta, the insensitive chromosome carried by her was replaced with an Sb-5 chromosome (isolated in 1965 from a wild fly) by mating with L/Sb-5 males (L, Lobe eye). The Cy/Sb-5 females obtained would retain an appreciable amount of delta when they inherited delta from their Cy/+ mothers. The retention of delta of the Cy/Sb-5 females were tested by mating the females with Cy/bwD males. The number of bwd/sb-5 flies recovered from this mating was none or few when their mother carried an appreciable amount of delta. Lines showing a significantly lower frequency of bwd/sb-5 flies in this mating were assumed to have an ability to retain delta, classified as ID chromosome. RESULTS AND DISCUSSION Data obtained in surveys made in the 1969-1971 period Data obtained in surveys made in the 1961-1968 period were reported (loc. cit.), and data obtained in the 1969-1971 period are noted in this section. In Table 1, frequencies of lethal and semilethal second chromosomes obtained in the three years are given. The frequency of lethal chromosomes in this period increased from 16.3% in 1969 to about 20% in 1971. The latter frequency was highest noted during the surveys made these past 11 years. The frequency of semilethal chromosomes also incresed significantly in every year. The frequency in 1971, 16.9%, was also the highest during the surveys made. Consequently, the frequency of lethal plus semilethal chromosomes (abbreviated as le+ sle frequency) increased significantly from 1969 to 1971. It must be

44 S. MINAMORI, K. ITO, A. NAKAMURA, Y. ANDO AND H. SHIOMI Table 1. Frequencies of lethal and semilethal second chromosomes Table 2. Allelism frequencies of lethal genes isolated in each collection Table 3. Frequencies of infected flies with a killing agent, and of S and ID chromosomes noted that the lethal frequency in 1971 was nearly equal to that obtained in the previous year, while the semilethal frequency increased significantly. The frequency of allelism between lethal genes sampled from each collection is shown in Table 2. The frequency was about 1 % during the three years. Most lethals appeared once, and there was no allelic gene which appeared more than 3 times in the test. As shown in Table 3, a certain proportion of flies of the population had also been infected with a killing agent in this period. The frequency of infected flies was lower in 1969; however, it increased significantly in the successive two years. The frequency of delta-associating chromosomes, S and ID, obtained in this period is presented at the right of Table 3. The frequency of S chromosome was low in 1969 and 1970, while it increased appreciably in 1971. The frequency of ID chromosomes was generally higher than that of S in each year; however, it dropped from about 32% in 1969 to 8% in 1971. Consequently, the frequency of S plus ID decreased significantly during this period,

INCREASE IN LETHAL FREQUENCY 46 Directional changes in the le+sle and the allelism frequencies during 1961-1971 period The frequencies of lethal, le+ sle, allelism, infected flies, and of S+ ID surveyed in the 1961-1971 period are summarized in Fig. 1. As shown in the figure, the lethal frequency increased gradually from the summer of 1961 (8%) to the summer of 1964 (15%) and then reached its first peak. Then, it began to decrease from the fall of 1965 Fig. 1. Frequency changes in the second chromosomes; - A- -A infected flies chromosomes. 1961-1971 period. AA lethal plus semilethal - lethal second chromosomes; allelism; with killing agent; - - delta-associating to the summer of 1967 (10%), again continually increased and reached about 20% in the summer of 1970 and 1971. The frequency increased about 2.5 times during the 11 years. The semilethal frequency showed little change from 1961 to 1968, ranging 5-7%, except for the fall of 1967 (12%). However, it increased appreciably in the last three years. The le+sle frequency generally showed an increasing trend in the 1961-1971 period. At the beginning of this survey (the summer of 1961) the frequency was about 13%, increased to 17% in the fall of the same year, and was maintained at a

46 S. MINAMORI, K. ITO, A. NAKAMURA, Y. ANDO AND H. SHIOMI nearly equal level until 1963, then it reached its first peak in 1965 (about 20%). However, it began to decrease in the fall of the same year, and then a second peak (23%) was observed in the fall of 1967. Although the frequency dropped once in the fall 1968, it again increased by 6-8% each year during the 1968-1971 period, and reached 37% in 1971. This frequency was about triple that of the summer of 1961. The allelism frequency also showed a systematic change during the period, though it fluctuated appreciably. It was higher in earlier surveys, and gradually decreased in later surveys. The prominently higher frequencies in the fall of 1962 (13%) and of 1963 (14%) were interpreted to be partly due to heterotic cis-formed double lethal genes contained in the population (Oshima 1962; Minamori and Saito 1964). The lowering observed in the later period, 1966-1971, was well correlated with the increase in le+ sle frequencies in the same period. This negative correlation appears to suggest that the effective number of the population may be increased during the period. Relation of infection and delta-retention to le+sle frequencies It was reported that flies carrying a lethal chromosome suffer severer damage by infection with the killing agent than flies carrying a semilethal or quasinormal chromosome; i.e., the viability of lethal heterozygotes was reduced by 10% compared to that of semilethal or of quasinormal heterozygotes when infected (Minamori 1967). This finding may predict the reduction in the lethal frequency of an infected population. In fact, the frequency was negatively correlated with the frequency of infected flies during 1964-1966 (Minamori and Tatsukawa 1968). The increasing trend of le+ sle frequency in earlier years turned to a decreasing trend in 1964 when the infection was detected for the first time. Then the lethal frequency began to increase in the fall of 1967 simultaneously with a decrease in the frequency of infected flies. Such a negative correlation between the two frequencies was also distinct in the fall of 1968 and of 1969, and in the summer of 1971. Although the lethal frequency was reduced in 1964-1968, the le+ sle frequency appeared to remain at the same level during this period. This plateau might be maintained by an increase of semilethal chromosomes. Assuming there was no infection, the le+ sle frequency in this population might show a greater increase without the plateau. As noted earlier, delta has a mutagenic action. It induces frequent recessive lethals in specific regions of second chromosomes (Minamori and Ito 1971). Accordingly, it is expected that the frequency of both lethal and allelism frequencies of the population would become higher when the frequency of flies carrying delta is higher. This expectation was examined in a previous study (Minamori et al. 1970) ; however, no conclusive evidence was obtained, although the lethal frequency of the S and ID chromosomes (the delta-retaining chromosomes) was higher than that of the population. The frequency of S+ ID chromosomes which retain or are able to retain delta was 8% in the fall of 1967, then it suddenly increased to about 50% in the summer of 1968, and then decreased continuously every year, dropping to 22% in 1971. The causes of

INCREASE IN LETHAL FREQUENCY 47 the sudden increase and the directional decrease in the frequency remain unknown. At any rate, the decrease happened simultaneously with the increase in le+ sle frequencies, hence, delta may not contribute to the increase of the le + sle frequency in the 1968-1971 period. This conclusion appears to be supported by the finding that the allelism frequency decreased during the period, in contrast, delta was expected to elevate the frequency by inducing lethals repeatedly in specific regions of the chromosome. Climatic factors and le+ sle frequencies As mentioned earlier, Ives and Band (loc. cit.) pointed out that the le+sle frequency of the Amherst population is correlated 1) negatively with the temperature range of the week prior to collections, and 2) positively with rainfall in the summer months. Fig. 2. Changes in climatic factors in Hiroshima City in the 1961-1971 period. Upper, temperatures; below, rainfall. maximum temperature; --- mean temperature; AA minimum temperature; - - daily temperature range of the week prior to collection. The Hiroshima population was also examined for such correlations by employing climatic data reported by the Hiroshima Meteorological Observatory which is located about 4 km from the Tera Station, the collection site. Changes in the mean, maximum and minimum temperatures in each year, average daily temperature of the week prior to collection, and rainfall during the 11 years are presented in Fig. 2. The mean temperature in each year fluctuated between 14-16 C, the maximum temperature between 19-20 C and the minimum temperature between 10-11 C. Although these three temperatures appear to be slightly higher in earlier periods, they were maintained at a similar level, respectively; hence they may not be directly correlated with the increasing trend in the le+ sle frequency. The temperature range fluctuated between 5-11 C, an average of 8 C, and the correlation with the le+ sle frequency was not so evident as in the survey made

48 S. MINAMORI, K. ITO, A, NAKAMURA, Y. ANDO AND H. SHIOMI by Ives and Band, though a negative correlation was observed in the last three years. Rainfall fluctuated widely from 1,200 to 2,100 mm, an average of 1,600 mm during the 11 years. As shown in the figure, the change in rainfall also does not appear to affects the le+ sle frequency. Therefore, it may be concluded that the effects of these climatic factors on the le+sle frequency are not demonstrable in this study. The le+sle frequency and population size A number of reports have been published on le+sle and allelism frequencies of natural populations in D. melanogaster from various regions of the world. Ives (1945, 1954) found an intimate relationship between the le+sle frequency and geographical distribution which shows that the frequency decreases from south to north. In a review of data published before 1961, Minamori et al. (1962) pointed out that American populations are generally higher in le+sle frequencies and lower in allelism frequencies when compared with those of Eurasian populations (Japan, Korea, Israel, Mediterranean, USSR). The same indication was given by Crumpacker (1967). It has been recognized that lethal and semilethal frequencies may be reduced and the allelism frequency may become higher in a small population, due to genetic drift and/or inbreeding. The average le+sle frequency in the Hiroshima and Shizuoka populations (Oshima and Kitagawa 1961; Oshima 1962) surveyed in the 1959-1962 period was 14.5%. Thus, Japanese populations in that period were considered to be small in population size. However, the frequency in the Hiroshima population increased, as noted in the foregoing section, about three times during the 11 years. The frequency in the last year appeared to be close to those in the Amherst population surveyed by Ives (1970) in 1966-1970. It may be stressed that the le+sle frequency in the Hiroshima population has increased simultaneously with the decrease in allelism frequency. Such increase in le+sle frequencies was also observed by Oshima and Choo (1972) in natural populations from central Japan. This parallelism seems to suggest that some common cause might induce the structural change of these Japanese populations. The directional changes and environmental changes As noted earlier, the climatic changes, infection and the presence of delta in the population do not appear to be directly related with the increasing trend in le+sle frequencies. It would be reasonable to search for the causal factors in various environmental changes of the fly in Japan during this decade, especially a change which allows the growth of population size. As is well known Japan has rapidly developed in its economical field; the Gross National Product has increased about 3 times during this period. Needless to say, such rapid development has brought about environmental pollution and may also cause changes in the environment of domestic and wild life. Although we are ignorance at present of the environmental factors causing the change of population structure, as possible factors, the increase in consumption of fuel used for heating and the progress in motorization are discussed below.

INCREASE IN LETHAL FREQUENCY 49 The effective number of a population which repeats a seasonal change in its size depend upon its minimum number rather than its maximum number during the year (Dubinin 1946). The size of Japanese populations reaches a minimum in winter and grows from spring to autumn; hence, the effective number may strongly depend upon the number of overwintering flies. For the overwintering, the most important environmental factor may be temperatures at sheltering sites of flies. With regard to this aspect, it may be pointed out that the mode of heating a Japanese household has changed this decade; more gas and kerosene are used as fuel instead of charcoal. The consumption of gas increased 2 times and that of kerosene increased 5 times in this decade. Such a change might elevate the temperatures surrounding the microhabitat of the fly, and favor its overwintering. Thus the number of overwintering flies, eventually the effective number of the population, might be increased, even though the warming may be very slight or temporal. Recently, a noxious domestic cockroach has markedly propagated in Japan, and this propagation is generally interpreted to be due to an improvement in winter heating facilities. This view seems to favor the above interpretation. The progress in motorization (the number of cars has increased about 5 times during this decade) may also have some influence on the effective number of population. The increase in transportation media appears to increase the chances of migration of the fly. The migration may be promoted among isolated small populations, and then the effective number may become larger. Thus, we would like to interpret that the environmental changes of the fly caused by man during this period may result in population growth, and further result in the directional increase in lethal and semilethal frequencies in the Hiroshima population. SUMMARY Annual surveys of a natural population of Drosophila melanogaster have been made by setting up a survey station in Hiroshima City from 1961 to 1971. The surveys made through the 11 years disclosed an increasing trend in frequencies of lethal plus semilethal second chromosomes and a decreasing trend in the frequency of allelism between lethals. The lethal plus semilethal frequency increased almost three fold (from 13% to 37%), and the allelism frequency decreased from about 5% to 1%. For this directional change; 1) the infection with a killing agent did not appear to be associated, since the frequency of infected flies was negatively correlated with the lethal and semilethal frequency, 2) the same correlation was observed concerning the frequency of deltaassociating second chromosomes; hence, delta may not be associated, and 3) the effects of weather factors were not evident. The increase in lethal and semilethal frequencies and the decrease in allelism frequencies were interpreted to be due to an increase in population size during this decade, and the environmental change produced by man which might affect the increase was discussed.

50 S. MINAMORI, K. ITO, A. NAKAMURA, Y. ANDO AND H. SHIOMI ACKNOWLEDGMENT We would like to thank Dr. C. Oshima of the National Institute of Genetics for his valuable comments. We are also indebted to Mr. R. Kakigi of the University of California (Berkeley) who kindly polished up the manuscript and to Miss K. Sugimoto for her competent assistance. This work was supported in part by a research grant from the Ministry of Education. LITERATURE CITED Band, H. T., 1972 Minor climatic shifts and genetic changes in a natural populartion of Drosophila melanogaster. Am. Naturalist 106: 102-115. Band, H. T., and P. T. Ives, 1961 Correlated changes in environment and lethal frequency in a natural population of Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 47: 180-185. Band, H. T., and P. T. Ives, 1963 Comparison of lethal+semilethal frequencies in second and third chromosomes from a natural population of Drosophila melanogaster. Canad. J. Genet. Cytol. S: 351-357. Band, H. T., and P. T. Ives, 1968 Genetic structure of populations. IV. Summer environmental variables and lethal and semilethal frequencies in a natural population of Drosophila melanogaster. Evolution 22: 633-641. Crumpacker, D. W., 1967 Genetic loads in maize (Zea mays L.) and other cross-fertilized plants and animals. Evol. Biol. 1: 306-424. Dobzhansky, Th., W. W. Anderson, and 0. Pavlovsky, 1966 Genetics of natural populations. XXX VIII. Continuity and change in populations of Drosophila pseudoobscura in western United States. Evolution 20: 418-427. Dubinin, N. P., 1946 On lethal mutations in natural populations. Genetics 31: 21-38. Ford, E. B., 1968 Ecological Genetics. 3rd ed. Chapman and Hall, London. Ives, P. T., 1945 The genetic structure of American populations of Drosophila melanogaster. Genetics 30: 167-196. Ives, P. T., 1954 Genetic changes in American populations of Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 40: 87-92. Ives, P. T., 1970 Further genetic studies of the South Amherst population of Drosophila melanogaster. Evolution 24: 507-518. Komai, T., 1954 An actual instance of microevolution observed in an insect population. Proc. Japan Acad. 30: 970-975. Minamori, S., 1967 A killing agent in a natural population of Drosophila melanogaster. Japan. J. Genetics 42: 317-326. Minamori, S., 1969 Extrachromosomal element delta in Drosophila melanogaster. I. Gene dependence of killing action and of multiplication. Genetics 62: 583-596. Minamori, S., 1971 Ibid. V. A variant inherited biparentally at 25 C. Japan. J. Genetics 46: 169-180. Minamori, S., N. Fujioka, K. Ito, and M. Ikebuchi, 1970 Ibid. IV. Variation and persistence of delta-associating second chromosomes in a natural population. Evolution 24: 735-744. Minamori, S., T. Ikemoto, and K. Fukaya, 1962 A characteristic of Japanese populations of Drosophila melanogaster concerning the deleterious genes. Bull. Biol. Soc. Hiroshima Univ. No. 29: 1-8. (in Japanese) Minamori, S., and K. Ito, 1971 Extrachromosomal element delta in Drosophila melanogaster. VI. Induction of recurrent lethal mutations in definite regions of second chromosomes. Mutation Res. 13: 361-369. Minamori, S., and Y. Saito, 1964 Local and seasonal variations of lethal frequencies in naturall populations of Drosophila melanogaster. Japan. J. Genetics 38: 290-304.

INCREASE IN LETHAL FREQUENCY 51 Minamori, S., and K. Sugimoto, Extrachromosomal element delta in Drosophila melanogaster. IX. Induction of delta-retaining chromosome lines by mutation and gene mapping. Genetics (in press) Minamori, S., and K. Tatsukawa, 1968 Relation of infection to population structure in Drosophila melanogaster. Evolution 22: 337-351. Oshima, C., 1962 The persistence of some recessive lethal genes in natural populations of Drosophila melanogaster. II. Proc. Japan Acad. 38: 278-283. Oshima, C., and J. K. Choo, 1972 Deleterious and sterility genes in Japanese and Korean natural populations of Drosophila melanogaster. Ann. Rep. Nat. Inst. Genet. Japan 22: 90-91. Oshima, C., and 0. Kitagawa, 1961 The persistence of deleterious genes in natural populations of Drosophila melanogaster. Proc. Japan Acad. 37: 158-162.