Crossing experiments with Philaenus spumarius (Homoptera).

HERE 56-28 CROSSING EXPERIMENTS WITH PHILAENUS SPUMARIUS (HOMOPTERA) By 0. HALKKA, LllSA HEINONEN, M. RAATIKAINEN and A R J A VAS -4 R A1 N E N DEPARTMENT OF GENETICS, UNIVERSITY OF HELSINKI, AND DEPARTMENT OF PEST INVESTIGATION, AGRICULTURAL RESEARCH CENTRE, TIKKURILA, FINLAND (Received September lGth, 1966) INTRODUCTION OLYMORPHISM in field populations of Philaenus spumarius has P been studied by a number of authors, and the frequencies of the different colour forms have been reported in about 20 publications. Although, from results obtained with natural populations, it has been inferred that the colour polymorphism is genically determined, crucial proof from crossing experiments has been wanting. In the USA, C. R. WEAVER has succeeded in obtaining offspring from known females with unknown mating partners, and has found that the forms tend to reproduce themselves (see OWEN and WIEGERT, 1962). Similar experiments were performed by the present authors in the autumn of 1964. In these experiments, females representing the colour forms marginella and leucophthalma were found to produce among their offspring daughters like their mother, in addition to males and females belonging to the colour form typica. Pictures of the colour forms as distinguished by us, have been published by HALKKA (1964). Material and methods All the females and most of the males used for crosses were isolated as larvae. The isolation work was done in the forest and garden area surrounding the "Nurmijarvi A" field, which harbours one of the natural populations studied by the senior author (HALKKA, 1964). The frothy spittle masses produced by the larvae usually contained a single specimen only. Altogether, 49 species of plants were used as food sources by Philaenus larvae in the Nurmijarvi area. A detailed report on the distri- CROSSING EXPERIMENTS WITH PHILAENUS 307 bution of the different colour forms on these plants will be published elsewhere. Here it will suffice to say that the distribution appears to be perfectly random. In July 1965, 60 pairs were placed in cages containing potted Trifolium pratense (“Tammisto” product) and Avena satiua (Swedish “Sol”). The clover was intended to serve as food, the oats mainly as a site for oviposition. The flowerpots were sunk into the soil up to the rim, the lower part of the nylon cage net surrounding the pot. Apart from occasional watering, the cages were untended, until 15 of them were moved into a greenhouse on December 31, 1965, and the remaining 45 on February 26, 1966. The cages were maintained at the Agricultural Research Centre. The winter of 1965-1966 was unusually severe in Finland. At Tikkurila, the mean temperatures of January and February were - 13.9’ C and - 13.5O C, respectively. The cages, however, were fairly well protected by about 110 cm of snow on the ground. In the greenhouse, a temperature range between 18 and 25 degrees centigrade was maintained and humidity was kept around 75 %. High humidity is essential for successful rearing of Philaenus. Of the 60 pairs, 24 produced at least one offspring, while 36 were failures. Most of the failures were not due to inability of Phifaenus eggs to hibernate but to high larval mortality resulting from poor hibernation of the Trifolium used as food plant. Spittle masses were observed in many of the cages which later failed to produce adults. A total of 234 F, offspring were obtained, 118 females and 116 males. RESULTS The crosses, together with their F, offspring, are reported in Table 1. The form frilineata is rare at Nurmijarvi, and the isolations did not produce enough males belonging to this form. In fact, all the trifineata male parents of the successful crosses originated from field collections. The typica and populi males and the different female forms were isolated from altogether 12 species of food plants. At the time of isolation, the male and female parents of crosses 9, 41, 57 and 59 were living together as larvae in a common spittle mass. In Table 1 and in the following report, the crosses are grouped into three sections according to the form of the male parent: 308 HALKKA, HEINONEN, RAATIKAINEN AND VASARAINEN (1) With typica CIS the male parent: In crosses 9 and 57 the female, too, belonged to typica, and in both cases both parents were isolated from the same spittle mass. Only typica offspring were produced. In crosses 5, 23 and 59 the female belonged to trilineata. The pooled result of these crosses is 18 trilineata and 16 typica specimens. In crosses 1 and 30, the female belonged to marginella. In addition to marginella and typica specimens, cross 30 includes in the F, generation four leucophthalma females, two of which bear small white spots on their elytrae and may better be assigned to the form albomaculata. The male parent of this cross obviously bore a gene responsible for the leucophthalma phenotype in one of his chromosomes, although this gene was not expressed in his phenotype. The two albomaculata specimens may have resulted from variation in the expressivity of this gene in the female sex. If this explanation is correct, crosses 1 and 30 demonstrate the existence of two major genes, which are here provisionally named mar and lop. Cross 32, with lateralis as the female parent, produced a female like her mother and a male like his father. In crosses 27 and 50, the female belonged to flauicollis. Altogether 17 F, offspring were obtained, all typica. The failure of these crosses to produce flavicollis may be due to chance, since only 7 females were found in the F, of the two crosses combined. In cross 31, the female belonged to leucocephala. Two females, one orthodox leucocephala and one with two small white spots on both elytrae, were obtained. This latter specimen differs from a true flauicollis phenotypically and is best interpreted as resulting from variation in the expressivity of a major gene responsible for the leucocephala phenotype. This gene is here provisionally named Ice. In crosses 20, 25 and 41, the female belonged to leucophthalma. Only leucophthalma and typica offspring were found in the F, generation. The gene responsible for the totally black leucophthalma phenotype is obviously expressed only in the female sex, a result indicated by the combined information from crosses 30, 20, 25 and 41. If the lop gene really is sex-controlled, the black males encountered priniarily in northern Finland have a different genotype. It is, of course, also possible that the same gene is sex-controlled only in some parts of its distribution area. CROSSING EXPERIMENTS WITH PHILAENUS 309 TABLE 1. The results of crosses between Philaenus colour forms. tri 2 typ 2 - tri 7 typ 6 ~- Cross no. tri 3 tri 2 tri 4 typ 8 tri 3 typ 1 fla 3 tri 2 typ 2 tri 2 typ 1 Female Male parent parent tri 1 typ 2 tri 6 typ 1 tri 1 typ 1 Total F, offspring 3 44 9 57 tY P tri tY P tri tY P mar tYP tri 1 typ mar I tYP _____ 7 2 25 5 23 59 1 30 32 1 14 2 8 9 27 50 31 tYP 2 tYP 5 Ice 1 “fla” 1 I2 1 1 11 3 20 25 41 - lop 1 lop 5 typ 1 tYP 5 1 tYP1 - tYP I POP typ I tri tri I tri tYP 2 1 tYP 1 typ 4 tri 3 I typ 2 tri 5 7 54 14 48 tri 2 typ 1 1 tri 3 typ 1 39 40 mar 1 - tri 3 typ 1 I tYP 1 mar ~~ lat Ila tri fla 1 tri fla tri ]at 3 tri 7 typ 7 I tri 4 typ 6 lat 2 tri 8 tri 2 typ 9 27 21 47 49 19 44 46 7 14 5 234 24 118 I 116 (2) With populi as the mule parent: The offspring from the single cross (cross number 7) with populi as the male parent and typica as the female contained only typica specimens. (3) With trilineata as the male parent: Cross 54, with typica as the female parent, is reciprocal to crosses 5, 23 and 59. Again, as in these crosses, typica and trilineata phenotypes are equally common in both males and females. This fact proves, that tri is an autosomal gene, since Philaenus males are XO as regards sex determination. The combined information from crosses 5, 23, 59, 54 and 310 HALPKA. HEINONEN, RAATIKAINEN AND VASARAINEN 48 shows, that the tri gene is dominant in both sexes. In cross 48, trilineata parents produced both frilineata and typica offspring. The offspring of crosses 39 and 40, between a marginella female and a trilineata male, shows the recessiveness of the typica condition towards the effect of the mar and tri genes. The two crosses, 47 and 49, both produced frequent offspring, including, in addition to the parental types lateralis and frilineata, a number of typica specimens. The offspring from crosses 47 and 49, together with the result of cross 32, prove the existence of a gene, lat, responsible for the lateralis phenotype. Crosses 19, 44 and 46 all produced trilineata (paternal phenotype) and typica females and males. The maternal phenotype flavicollis was obtained in cross 44 only. The existence of a gene for this phenotype, fla, seems unquestionable, but either the gene in question has a low penetrance or the flavicollis phenotype may also be caused by the action of other genes. DISCUSSION The results of the crosses described above show convincingly that each of the distinctly coloured and readily distinguished phenotypes is determined by a particular major gene. Most of the genes are regularly expressed in the females but practically never in the males. This is true of the expression of the major genes mar, lat, Ice and lop. The tri gene, in contrast, is expressed in both sexes. A fifth sex-controlled gene, the expression of which is confined to the female sex, fla, also exists. However, the dependence of the flavicollis phenotype on the presence of the fla gene is perhaps not so clear-cut as in the case of the other four genes and their respective phenotypes. Samples collected from the field reveal the extreme variability and obvious heterogeneity of the basic colour form, typica. Obviously a great number of modifying colour genes, the effect of some of which is probably further modifiable by external factors, combine to create the long array of phenotypes from uniformly light to uniformly dark forms. Many of these modifying genes are not allelomorphs of the major colour pattern genes. Although it is likely that the fri, mar, lat, fla, Ice and lop genes are allelomorphs or closely linked loci, and that they are all autosomal, even this has not been conclusively established. For this reason, neutral and noncommittal symbols for these loci or genes are used throughout this paper. CROSSING EXPERIMENTS WITH PHILAENUS 311 Provided that the penetrance of the colour genes is complete, the dominance of the tri, mar, lat, fla, Ice and lop genes makes estimation of 2pq frequencies fairly easy in populations in which the phenotypes are sharply demarcated. For these genes, the 2pq frequencies vary between 1 % and 7 % in most populations in Finland. The homozygotes thus contribute insignificantly to the gene frequencies in these populations. At the present phase of genetical work on Philaenus it is not known whether some of the genes are possibly lethal when homozygous. We also know nothing of the order of dominance or epistasis in this array of colour genes. The experimental program for obtaining F, is being extended by us and steps for producing Fa have been taken. In many respects, colour polymorphism in Philaenus affords an interesting comparison with what is known of balanced polymorphisms in the Lepidoptera. As with the butterflies, polymorphism in the meadow spittlebug is largely sex-controlled. It seems that many of the rules governing polymorphism in the Lepidoptera may also be valid in this Homopteran. After a long period of persistent and painstaking work, both the genetics and ecology of certain butterflies are now being investigated at a rather advanced stage (e.6. SHEPPARD, 1961). With Philaenus, ecological work synchronized and parallel with genetic analysis has been barely begun by us. Philaenus affords favourable material for studies on sex-controlled inheritance and also for investigations on the evolution of dominance.' Acknowledgements. - The English of the manuscript was checked by Mrs. JEAN MARGARET PERTTUNEN, M.A., to whom our sincere thanks are due. Grants for the study have been received from the University of Helsinki and from the National Research Council for Sciences. SUMMARY The mode of inheritance of the different genes determining the coIour forms of Philaenus spumarius was studied at the F, generation level. Six major genes, each one responsible for a distinct colour pattern, were observed in this work. In a single dose, five of these genes, here provisionally named mar, lat, fla, Ice and lop, always manifest their ' Note added in proof: The first adult specimens belonging to the Fa generation emerged in November 1966. They show dominant transmission of the genes tri and lop and strengthen the view that in the females typ is the bottom recessive in a dominance hierarchy series. A detailed account of the F, progeny will be published in another context. 312 HALEKA, HEINONEN. RAATIEAINEN AND VASARAINEN effects in the females but never in the males. The sixth gene, tri, has a dominant mode of inheritance in both sexes. The flavicollis phenotype may perhaps have three alternative genetic backgrounds: a) the fla gene, b) action of darkening modifiers on the typica genetic condition and c) action of genes nonallelic with flu or Ice and epistatically causing the appearance of two white spots on both elytrae of an individual with a Ice gene in her genotype. The six major genes may be allelomorphic with each other. The expressivity of the six major genes is remarkably independent of external factors, including food plants of the P and F, generations. The possibiIity that the six major genes constitute a dominance hierarchy is being further studied, together with the ecologicd aspects of the sex-controlled inheritance of the colour genes. Literature cited IIALKKA, 0. 1964. Geographical, spatial and temporal variability in the balanced polyOWEN, D. F. and WIEGERT, R. G. 1962. Balanced polymorphism in the meadow SHEPPARD, P. M. 1961. Recent genetical work on polymorphic mimetic Papilios. - morphism of Philaenus spumarius. - Heredity 19: 383-401. spittlebug, Philaenus spumarius. - Amer. Nat. 96: 353-359. Symp. Royal Entom. SOC. London I: 20-29.