Male germ line polysomy in the grasshopper Chorthippus binotatus: extra chromosomes are not transmitted

M. TALAVERA, M. D. LOPEZ-LEON, J. CABRERO, AND J. P. M. CAMACHO Departamento de Biologia Animal, Ecologia y Genetica, Facultad de Ciencias, Universidad de Granada,

18071 Granada, Spain

Corresponding Editor: J. Sybenga

Received August 1 5, 1989

Accepted November 2 1, 1989

TALAVERA, M., LOPEZ-LEON, M. D., CABRERO, J., and CAMACHO, J. P. M. 1990. Male germ line polysomy in the grasshopper Chorthippus binotatus: extra chromosomes are not transmitted. Genome, 33: 384-388.

Some males of the grasshopper Chorthippus binotatus from a natural population in Sierra Nevada (Granada, Spain) were found to be polysomic mosaics for the presence of extra chromosomes (E) in a high proportion of testicular follicles. Transmission analysis of these chromosomes was performed in 21 controlled crosses, 2 of which involved a polysomic parent. While most spermatozoa produced by polysomic males carried E chromosomes, these chromosomes were not transmitted to the progeny, since all 22 embryos descended from a polysomic male parent lacked them.

Key words: transmission, polysomy, grasshopper, Chorthippus binotatus.

TALAVERA, M., LOPEZ-LEON, M. D., CABRERO, J., et CAMACHO, J . P. M. 1990. Male germ line polysomy in the grasshopper Chorthippus binotatus: extra chromosomes are not transmitted. Genome, 33 : 384-388.

Certains miles de Chorthippus binotatus, d'une population naturelle de sauterelles dans la Sierra Nevada (Granada, Espagne), se sont reveles ttre des mosai'ques polysomiques en raison de la presence de chromosomes surnumeraires (E) dans une proportion elevee des follicules testiculaires. Des analyses de transmission de ces chromosomes ont ete faites sur 21 croisements contrbles, dont 2 impliquaient un parent polysomique. Bien que la majorite des spermato- zoi'des produits par les miles etaient porteurs de chromosomes E, ceux-ci n'ont pas ete transmis aux descendants puisque les 22 embryons issus d'un parent mile polysomique en etaient tous depourvus.

Mots clbs : transmission, polysomie, sauterelle, Chorthippus binotatus. [Traduit par la revue]

Introduction Extra (E) chromosomes restricted to the male germ line

have been reported in several species of grasshoppers (Hewitt and John 1968, 1970; John and Hewitt 1969; Gosalvez and Lopez-Fernandez 198 1 ; Peters 198 1 ; Viseras and Camacho 1984). They have been regarded as cases of recurrent polysomy and, in one case, the existence of heritable fac- tors determining the tendency of a particular autosome to nondisjunction during embryogenesis has been demonstrated (Peters 1981). We present here further evidence for this tendency, since extra chromosomes in Chorthippus binotatus are not present in embryos descended from polysomic male parents but are maintained in the natural population. Transmission of E chromosomes was analyzed by means of

females were incubated at 25°C for 10 days and then dissected in insect saline solution. Embryos were removed from the eggs and immersed in 1 mL of 0.05% colchicine in insect saline solution for 2 h, followed by a hypotonic shock treatment effected by adding 1 mL of distilled water. After 15 min the embryos were fixed in ethanol - acetic acid (3:l). Testes were extracted from the males through a small dorsal cut in the third abdominal segment. The males were subsequently injected with 0.05% colchicine in insect saline solution, and 6 h later the gastric caeca were fixed in ethanol - acetic acid (3: 1). Females were injected with 0.05070 col- chicine in insect saline solution for 6 h prior to fixation of the ovarioles and gastric caeca. All materials were analyzed cytologi- cally with the C-banding technique described by Camacho et a/. (1984).

21 controlled crosses. This task was complicated by the fact Results and discussion that egg pods usually contained only six or seven eggs (range 3-10), which obliged us to obtain several pods from each female, with the consequent increase of the probability of death of females before the experiments were completed. Consequently in this species it is very difficult to study the de novo origin of E chromosomes by comparing embryos and adults from the same cross since it is not possible to obtain samples large enough to give significant results. Thus, our investigation was aimed at ascertaining whether E chro- mosomes carried by some adult males are transmitted as physical entities to their embryo descendants.

Materials and met hods

Standard Ch. binotatus specimens possess 2n = 16 + XO 0 / XX 9 chromosomes, with three long metacentric pairs (Ll-L3), four medium-sized subtelocentric pairs (M4-M7), and one short subtelocentric chromosome pair (S8), the X chromosome being shorter than L3 but longer than M4 (Cabrero and Camacho 1987). In addition, six males car- ried extra heterochromatic chromosomes in the testes, similar in size to the X chromosome and likewise showing positive heteropycnosis during the first meiotic prophase (Fig. 1). All males carrying E chromosomes were mosaic in the germ line but lacked E chromosomes in the somatic line (gastric caeca) (Fig. 2). E chromosomes were never found in females (13 caught in 1983 and 21 in 1985).

Thirty-eight last-instar nymph females and a large number P ~ l ~ s o m i c males form a proportion of aberrant gametes, of males of the grasshopper Chorthippus binotatus were collected some of which appeared after meiotic elimination of E chro- at El Navazo (Sierra Nevada, Granada, Spain) to perform con- mosomes. TO quantify this, a large number of spermatic trolled crosses in the laboratory. Egg pods produced by these nuclei were scored (Fig. 3). Although the frequency of sper- Printed in Canada / Imprime au Canada

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TABLE 1. Frequency of spermatic macronuclei and micronuclei

Normal nuclei Macronuclei Micronuclei Total

Follicles with: Number % Number % Number % No.

FIG. 1. Pachytene cell showing one extra chromosome (E). FIG. 2. Mitotic metaphase cell from gastric caeca of a male carrying E chromosomes in the testes. Note the presence of only the 17 standard chromosomes. FIG. 3. Spermatic micronuclei (small arrow) and macronuclei (large arrow) in a polysomic male. FIG. 4. Mitotic metaphase from a male embryo descended from a male parent carrying E chromosomes in the germ line. Note the presence of only 17 standard chromosomes.

matic macronuclei was directly proportional to the number of E chromosomes, an odd-even effect was observed for the frequency of spermatic micronuclei, in that follicles with 1 E or 3 E showed a higher frequency of spermatic micro- nuclei than those with 2 E (Table 1). This odd-even effect may be related to the equational division of E univalents (M. Talavera, M.D. Lopez-Leon, J. Cabrero, and J.P.M. Carnacho, in preparation), since it occurred twice as frequently in follicles with 1 E (8.45%) than in follicles with 2 E (4%). Thus spermatic micronuclei can be assumed to contain equa- tionally divided E chromatids in the first meiotic division,

which then lag and are lost during the second division. On the other hand, spermatic macronuclei were two or four times the size of normal ones, hence they presumably con- tain two or four chromosome sets plus the E chromosomes. In our material we never observed polyploid spermatocytes, thus ruling out a premeiotic origin for the macronuclei observed in Ch. binotatus. Although we have not directly observed cytokinesis failure in telophase cells, the fact that ( i ) these stages are rare in grasshopper meiosis in contrast to prophase I or metaphase I cells and (ii) only two different sizes of macronucleus were found, moves us to believe that

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386 GENOME, VOL. 33, 1990

TABLE 2. Number of fertile eggs in the 21 crosses performed

Cross No. No. No. of Proportion No. of pods of eggs embryos of fertile eggs

macronuclei in Ch. binotatus arise by cytokinesis failure of one or both meiotic divisions. If this failure is induced by the presence of E chromosomes, as suggested by the fact that the frequency of macronuclei is directly proportional to the number of E chromosomes, then macronuclei, like micronuclei, may represent a form of elimination of these chromosomes.

Given these two modes of E-chromosome elimination, macronuclei and micronuclei, together with the observation that all embryos descending from males with E chromo- somes lacked these chromosomes, we calculated the expected frequency of E chromosomes in the sperm of the two polysomic males involved in controlled crossed, to ascer- tain whether the absence of E chromosomes in their off- spring is explainable by elimination of the E elements from macronuclei and micronuclei.

For this analysis, we included only normal spermatic nuclei capable of fertilizing ova. Since macronuclei contain two or four chromosome sets plus E chromosomes, they were accounted for by adding the number of macronuclei multiplied by two or four to the number of normal sper- matic nuclei. Thus we considered each macronucleus as the result of the loss of two or four chromosome sets (the mate- rial contained in each normal spermatic nucleus). We then calculated the frequency of chromosome sets contained in normal spermatic nuclei (transmission not ruled out) in rela- tion to all chromosome sets counted (including those in macronuclei, together with the E chromosomes accompa- nying them; transmission may be ruled out). The resulting frequency was multiplied by the expected transmission rate for 1,2, or 3 E chromosomes (0.5, 1, and 1.5, respectively), which resulted in the frequency of normal spermatic nuclei carrying E chromosomes. Then the frequency of micronuclei was subtrated in each type of follicle with 1,2, or 3 E chro- mosomes, which resulted in the estimated frequency of sperm carrying E chromosomes in each follicle type.

Knowing the frequency of follicles with 0,1,2, or 3 E chro- mosomes, we calculated the frequency of E chromosomes in each male. After estimating the probability that all embryos descending from a male lacked E chromosomes, we could derive the mode of transmission of these chromosomes.

To simplify our analysis, we assumed that all macronuclei observed contained four chromosome sets, which agreed with the size of the macronuclei and gave maximal correc- tion. In follicles with one E we counted 1300 normal sper- matic nuclei versus 33 macronuclei (see Table l) , in a total of 1432 chromosome sets, from which 1300/1432 = 0.9078 were found in normal sperm. The frequency of normal sperm containing E chromosomes, when not lost in micro- nuclei, was 0.9078 x 0.5 = 0.4539. Since many of these normal spermatic nuclei had presumably lost their E chro- mosome in the form of a micronucleus, the actual frequency was ((0.4539 x 1300) - 101)/1300 = 0.3762. By the same logic, from the frequency of normal spermatic nuclei in follicles with2 E, 1700/(1700 + (85 x 4)) = 0.8333, those with E chromosomes were estimated to have a frequency of ((0.8333 x 1 x 1700) - 31)/1700 = 0.8151, and in follicles with 3 E, normal nuclei: 1250/(1250 + (101 x 4)) = 0.7557; with E chromosomes: ((0.7557 x 1.5 x 1250) - 60)/1250 = 1.0856. These calculations demonstrate that E chromosomes must have been well represented in the sperm of mosaic males, despite their partial elimination in the form of spermatic macronuclei and micronuclei. Finally, the mean frequency of E chromosomes contained in sperm produced by a given mosaic male was deduced from the fre- quencies estimated above and the frequency of follicles with 0, 1, 2, or 3 extra chromosomes.

In 2 of 21 crosses analyzed, i.e., crosses 4 and 13, the male parent was polysomic mosaic for the presence of E chromo- somes. In the remaining crosses the male parent was nor- mal, i.e., lacking E chromosomes. All females involved in the 21 crosses, and all other females hitherto analyzed, lacked E chromosomes. All 22 embryo offspring descending from polysomic mosaic males in crosses 4 and 13 lacked E chromosomes (Fig. 4). Table 2 gives the proportion of fertile eggs observed in the 21 crosses. In crosses between normal parents, 126 eggs contained one embryo (62.46%) and 76 were empty, and in crosses between polysomic mosaic males and normal females (crosses 4 and 13), 22 eggs were fertile (64.7%) and 12 were infertile. This similarity in the frequencies of fertile eggs between both types of crosses sug- gests that no differential zygotic mortality can be attributed to the E chromosomes. Thus nontransmission of E chromo- somes must be caused in an earlier stage, before fertiliza- tion. The mean frequency of E chromosome carrying sperm produced by the male parents of crosses 4 and 13 was calculated on the basis of the frequency of follicles with 0, 1, 2, or 3 E chromosomes and the mean frequencies of E chromosomes in 1-E, 2-E, and 3-E follicles (0.3762, 0.8 15 1, and 1.0856, respectively). The estimates were 0.4598 for the male parent of cross 4 (Table 3) and 0.5990 for the male parent of cross 13 (Table 4). Thus, the probability that all 16 embryos from cross 4 lacked E chromosomes was 0.5402'~ = 5.26 x l o p 5 , and the probability that all 6 embryos from cross 13 lacked E chromosomes was 0.4016 = 4.16 x l op3 . These figures rule out chance as the cause of the absence of E chromosomes in these embryos. We therefore postulate that sperm containing

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TABLE 3. Estimated frequency of sperm containing E chromosomes in the male parent of cross 4

Follicles with: No. of

0 E 1 E 2 E follicles Total

Frequency 0.1667 0.5000 0.3333 12 Mean of E 0 0.3762 0.8151 Sperm frequency with E 0 0.1881 0.2717 0.4598

TABLE 4. Estimated frequency of sperm containing E chromosomes in the male parent of cross 13

Follicles with: No. of

0 E 1 E 2 E 3 E follicles Total

Frequency 0.1429 0.2857 0.4762 0.0952 21 Mean of E 0 0.3762 0.8151 1.0856 Sperm frequency with E 0 0.1075 0.3882 0.1033 0.5990

E chromosomes is unfit for fertilization and that all off- spring produced from polysomic males are derived from sperm lacking E chromosomes, which are produced in suf- ficient numbers to fertilize the six or seven eggs produced by the female in each pod.

Although E chromosomes are not transmitted, they were present in the wild population studied in 1983 and 1985. Since these chromosomes are not transmitted through sperm of male carriers, and are not carried by females, their recur- rence may be due to the tendency of a standard chromo- some to nondisjunction during the development of the testes. Obviously this tendency to nondisjunction must have a heritable basis to assure the presence of E chromosomes through successive generations. Genetic variation for heritable factors which affect the formation of E chromo- somes must exist in the wild population, since only a few males develop the polysomy. These genetic factors could be demonstrated if E chromosomes were observed in adult males whose embryo brothers lacked them, indicating the de n o v o origin of E chromosomes. However, in Ch. binotatus, this is made difficult by the low number of eggs in each pod, but it has been demonstrated in Atrac- tomorpha similis, a more favorable species (Peters 1981).

The de novo origin of E chromosomes from some member of the standard complement merits a final comment. In addi- tion to the similarity in size between E chromosomes and the X chromosome, the meiotic behavior of the former shows certain points in common with X univalents, namely, heteropycnosis and, most importantly, autopairing of E univalents during early prophase I, a phenomenon that characterizes the X chromosome of grasshoppers (John and Lewis 1965). If extra chromosomes are actually X chromo- somes, and assuming that sperm with two or more X chro- mosomes are nonfunctional, then the transmission of such elements to progeny would alter the sex ratio in favor of females in embryos descending from polysomic males. The extra females would result from sperm that lack the nor- mal X but contain an extra X. Thus, a comparative anal- ysis of sex ratios in embryos descending from polysomic and nonpolysomic male parents could serve to test both whether

E chromosomes are transmitted by the sperm and whether they are actually X chromosomes. Of 148 embryos analyzed, 126 were the progeny of nonpolysomic males (68 with 2n = 16 + XO and 58 with 2n = 16 + XX), and 22 were the progeny of polysomic males of crosses 4 and 13 (10 with 2n = 16 + XO and 12 with 2n = 16 + XX). The sex ratio in the former embryos was 58/68 = 0.85, while that in embryos descended from polysomic males was 12/10 = 1.2. With the sample sizes analyzed, neither of these sex ratios differs significantly from the expected ratio of 1: 1 (x2 =

0.79, P > 0.50 and x2 = 0.18 P > 0.30, respectively) nor do they differ between one another (contingency X2 = 0.26, P > 0.50). These tests demonstrate that sex ratio does not deviate significantly among the progeny of polysomic male parents. Hence, E chromosomes are either X chromosomes but are not active to modify sex determinism of embryos with one E and without a normal X, or else they are not X chromosomes. Since no embryo was found with more than 18 chromosomes among the progeny of polysomic males, E chromosomes (regardless of whether they are X chromosomes) are not transmitted in the sperm of these males.

Acknowledgements The authors thank Ms. Karen Shashok for revising the

English translation of the manuscript. This research study was supported by the Spanish Direction General de Inves- tigacion Cientifica y Tecnica through program PB87-0886.

CABRERO, J . , and CAMACHO, J.P.M. 1987. Cytogenetic studies in gomphocerine grasshoppers. I. Comparative analysis of chro- mosome C-banding pattern. Heredity, 56: 365-372.

CAMACHO, J.P.M., VISERAS, E., NAVAS, J., and CABRERO, J. 1984. C-heterochromatin content of supernumerary chromosome segments of grasshoppers: detection of an euchromatic extra seg- ment. Heredity, 53: 167-175.

GOSALVEZ, J., and LOPEZ-FERNANDEZ, C. 198 1. Extra hetero- chromatin in natural populations of Gomphocerus sibiricus (Orthoptera: Acrididae). Genetica (The Hague), 56: 197-204.

HEWITT, G.M., and JOHN, B. 1968. Parallel polymorphism for supernumerary segments in Chorthippus parallelus (Zetterstedt).

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388 GENOME, VOL. 33, 1990

I. British populations. Chromosoma, 25: 319-342 toplasmatologia VI F1. Springer-Verlag, Wien. pp. 1-335. 1970. Parellel polymorphism for supernumerary segments PETERS, G.B. 1981. Germ line polysomy in the grasshopper Atrac-

in Chorthippusparallelus (Zetterstedt). IV. Arshurst re-visited. tomorpha similis. Chromosoma, 81: 593-617. Chromosoma, 31: 198-206. VISERAS, E., and CAMACHO, J .P.M. 1984. Polysomy in

JOHN, B., and HEWITT, G.M. 1969. Parellel polymorphism for Omocestus bolivari: endophenotypic effects and suppression of supernumerary segments in Chorthippus parallelus (Zetterstedt). nucleolar organizing region activity in the extra autosomes. Can. 111. The Arshurst population. Chromosoma, 28: 73-84. J . Genet. Cytol. 26: 547-556.

JOHN, B., and LEWIS, X. 1965. The meiotic system. Pro-

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