CARYOLOGIA
Vol. 51, n.2: 115-121,1998
Neo-XY chromosome sex determination in four species
of the pamphagid grasshoppers
(Orthoptera, Acridoidea, Pamphagidae) from Bulgaria
A. BUGROV and S. GROZEV A *,
Institute of Systematics and Ecology of Animals, Siberian Branch, Russian Academy of Science,Novosibirsk 630091,
and Novosibirsk State University, Dept. NaturalSci., 630090 Novosibirsk, Russia; ;* Institute of Zoology, Bulgarian
Academy of Science, 1000 Sofia, Bulgaria.
In Memory of Prof. Georgy P. Peshev, the famous Bulgarianorthopterist
SUMMARY - The karyotypes and C-banding patterns in the spermatogenesis of thePamphagidae
grasshoppers Paranocaracris bulgaricus (Ebn. et Drenow), Paranocarodes straubei (Fieb.),
Paranocarodes chopardi Peshev and Asiotmethis limbatus (Charp.) from Bulgaria with 2n
= 18
(16AA + neo-XY) are reported. The evolution of theneoXY
/neo-XX
sex determination in
Pamphagidae is discussed.
INTRODUCTION
Among Acridoidea, grasshoppers of the family Pamphagidae are little studied cytogenetically.
Extreme karyotype conservatism has been demonstrated in this group: the main diploid numbers
were 19( ) and (20 ) acrocentric chromosomes. The mechanism of sex determination is XO
/XX
(WHITE 1973; ALICATA et al. 1976; CAMACHO et al. 1981; SOFRADZIJA and
MIKSIC 1982; SANTOS et al. 1983; CABRERO et al. 1985; FU PENG 1989; MANSUETO and
VITTURI 1989; VITTURI et al. 1993; W ARCHALOWSKA-SLIWA et al. 1994). However,
comparative cytogenetic analysis of some Pamphagidae from Kazakhstan, Central Asia, and the
Caucasus do not support the view of uniformkaryotypes in Pamphagidae. At least two types of
chromosome sets have been observed: 1) standard karyotypes with acrocentric chromosomes
only (2n = 19, NF= 19; 2n
=20, NF=20) and 2) karyotypes with 2n = 16 + neo-XY and
2n
= 16 + neo-XX (BUGROV 1986, 1996a, b). For this reason Pamphagidae grasshoppers
from the Balkan Peninsula have been investigatedcytogenetically for a better understanding of
the formation and evolution of the neo-XY/neo-XX sex determination.
116
BUGROV and GROZEVA
.
MATERIALS AND METHODS
Eleven adult male Asiotmethis limbatus (Charp.) were collected near Harmanli in South Rodopy
in June 1994 and 1995. Seventeen adult maleParanocaracris butgaricus (Ebn. et Drenow) were
collected near Trigrad in South Rodopy in June 1994. Six adult male Paranocarodes straubei
(Fieb.) were collected near the village Veselie in the Strandzha Mountains in June 1994. Five
adult male Paranocarodes chopardi Peshev were collected near the village Pelevun in South
Rodopy in June 1995.
The males were injected with 0.1-0.2 ml of 0.1 per centcolchicine; after 1.5-2.0 hrs. the testes
were fixed in an ethanol-acetic acid mixture (3: 1) . The fixed material was washed and kept in 70
per cent ethanol. Dry squashed slide preparations were obtained by C-banding according to
SUMNER (1972) with minor modifications.
RESULTS
Asiotmethis limbatus (Charp.). 2n
= 18 (16AA+neo-X+neo-Y).
Autosomes are acrocentric and can be divided into three size groups: 311 + 4MM + 1SS. The
neo-X chromosome is large and submetacentric originating from a reciprocal translocation
between the initially acrocentric X-chromosome and a large acrocentric autosome. The neo- Y
chromosome is a large acrocentric (Figs. 1, 2), and seems to be still homologous throughout its
entire length with the XR arm of the neo-X. The homology is demonstrated by complete pairing
in the prophase of meiosis and by the formation of one or twochiasmata between neo- Y and the
XR, which are not distally located (Fig. 3). C-heterochromatic blocks are located in
paracentromeric regions of all autosomes. The S8 pair of autosomes was characterized by the
presence of a small C-band located distally (Figs. 1-6). The neo-X chromosome in addition to the
centromeric C-band has a minute telomeric C-band in the XL arm (Figs. 2, 3, 5). The Y chromosome has a small paracentromeric C-band and additionally several very thinintercalary Cbands in its proximal part (Fig. 4).
During meiotic prophase, large bivalents form 2- 3 chiasmata, medium size bivalents form 1-2
chiasmata and small size bivalents form only one chiasma (Figs. 3,6). The mean chiasmata
frequency is 14.04 (±0.16).
In the karyotype of one male two medium sizeheterochromatic acrocentric B-chromosomes were
found (Fig. 5). During prophase of meiosisBchromosomes formed bivalent with single chiasma
(Fig. 6).
Paranocaracris bulgaricus (Ebn. et Drenow). 2n
= 18 (16AA+neo-X+neo-Y).
Autosomes are acrocentric and can be divided into three size groups: 311 + 4MM + 1SS. The
neo-X is a large submetacentric chromosome resulting from a mutualtranslocation between the
initially acrocentric X-chromosome
SEX DETERM!NATION IN FOUR SPECIES OF PAMPHAGID GRASSHOPPERS
117
Figs. 1-6 Asiotmethis limbatus; 1) C-banded metaphase II with the neo- y chromosomes; 2)Cbanded anaphase II
with the neo-X; 3) metaphase I with C-bands; 4) the neo- y in mitotic metaphase with C-bands; 5) B-chromosomes
in mitotic metaphase with C-bands; 6) metaphase I with C-bands.
and a large acrocentric autosome. The neo- y is anacrocentric chromosome distinctly shorter than
XR arm of the neo-X chromosome (Figs. 7-9). Cheterochromatic blocks are located in
paracentromeric regions of all chromosomes. The 11 and L3 pairs of autosomes were
characterized by the presence of small distal C-bands (Fig. 9). The M7 pair ofautosomes has a
large interstitial C-band near the paracentromeric C-block and may be considered amegameric
chromosome (Figs. 7, 8). The neo-X chromosome has in addition to thecentromeric C-band a
minute telomeric C-band in the XL arm (Figs. 7, 8). The y -chromosome is strongly
heterochromatic in its proximal part (Figs. 8, 9). At
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BUGROV and GROZEVA
Figs. 7-9 - Paranocaracris bulgaricus; 7) C-banded metaphase II with the neo-X chromosome, 8) Cbanded
metaphase II with the neo- Y chromosome, 9) metaphase I with C-band.
Figs. 10-12. - Paranocarodes chopardi; 10) C-banded metaphase II with the neo-X chromosome, 11) C-banded
metaphase II with the neo- Y chromosome, 12) metaphase I with C-band.
Fig. 13. Paranocarodes straubei; metaphase I with C-band, ( ) indicates the neo- Y chromosome, ( ) indicates
the neo-X chromosome, (
) indicates neo-XY bivalent, ( ) indicates B-chromosomes, (*) indicates
megameric chromosomes. Bars equal 10 ~m.
SEX DETERMINATION IN FOUR SPECIES OF PAMPHAGID GRASSHOPPERS
119
meiotic prophase, large bivalents form two chiasmata, medium bivalents form 1 or 2 chiasmata,
while small bivalents form only one chiasma. The neo-XY bivalent in the male forms one
chiasma between the XR arm of the neo-X and the neo-Y. Usually thischiasma is located
terminally (Fig. 9). The meanchiasmata frequency is 14.67 (±0.34).
Paranocarodes chopardi Peshev. 2n
= 18 ( 16AA + neo- X + neo- Y) .
Autosomes are acrocentric and can be divided into three size groups: 311 + 4MM + 1SS. The
neo-X chromosome is large and submetacentric, originating from a reciprocal translocation
between the initially acrocentric Xchromosome and a large acrocentric autosome. The neo-Ychromosome is acrocentric and distinctly smaller in size than the XR arm of the neo-X
chromosome (Figs. 10-12). C-heterochromatic blocks are located in paracentromeric regions of
all chromosomes (Figs. 10-12). The M6 pair ofautosomes has in addition to aparacentromeric Cband multiple thin interstitial C-bands and may be consideredmegameric (Figs. 10-12). The neoX chromosome has in addition to the centromeric C-band a very small telomeric C-band in the
XL arm (Figs. 10, 12). The Y -chromosome has a largeparacentromeric and intercalar C-band
(Figs. 11, 12) . At meiotic prophase largebivalents form two chiasmata, medium bivalents form
1-2 chiasmata, and the small bivalent only onechiasma. The neo-XY bivalent in the male forms a
single terminal chiasma between the XR arm of the neo-X and the neo- Y (Fig. 12). The mean
chiasmata frequency is 13.09 (±0.13).
Paranocarodes straubei (Fieb.). 2n
,
= 18 (16AA + neo-X + neo- Y).
The chromosome complement, chiasma frequency, and C-heterochromatin patterns of this
species are more similar to P. chopardi but the neo- Y chromosome has a minute multiple
intercalary C-block in the proximal region (Fig. 13). The meanchiasmata frequency is 12.69
(±0.31).
DISCUSSION
The species belonging to the family Pamphagidae Asiotmetis limbatus , Paranocaracris
bulgaricus, Paranocarodes straubei, and Paranocarodes chopardi have a similar morphology of
their karyotypes: 2n = 18 (16AA + neo-X + neo-Y) . The sex determination neo- XY /neoXX
is the result of a reciprocal translocation between an acrocentric large autosome and
initially acrocentric X -chromosome (X - A fusion) .
Asiotmethis limbatus, belonging to the subfamily Akicerinae, has a karyotype similar to the
earlier studied Asiotmethis heptapotamicus (Zub.). from
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BUGROV and GROZEV A
Kazakhstan with neo-XY sex chromosome determination resulting from acentromere-centromere
fusion of the acrocentric large autosome and the initially acrocentric X-chromosome (BUGROV
1986). In Asiotmethis limbatus and A. heptapotamicus the neo- Y chromosome and its
homologous XR arm of the neoX are almost isomorphic in size and they have complete pairing
during meiotic prophase. A minor difference in C-banding found between XR and neo- Y inA.
limbatus may be considered as the initial stage of theheterochromatinization of the neo- Y .
Paranocaracris bulgaricus, Paranocarodes straubei, and Paranocarodes chopardi belonging to
the subfamily Pamphaginae, displayed a similar level of decrease in pairing of the XR-arm of
neo-X and neo- Y in meiosis, heterochromatinization and reduction in size of the neo- Y .
Structural differences between the originally homologous autosomal part of the neo-Xchromosome (XR-arm) and the Y -chromosome are most evident in the presence of a
heterochromatinization of the paracentromeric and intercalar regions of the Y -chromosome in
this species.
The heteromorphization of the sex chromosomes inPamphaginae grasshoppers conforms to the
classical models (MULLER 1914; WHITE 1973; CHARLESWORTH 1978), which assumed that
recessive mutations connected with loss of function of different genesaccomulated in the Y chromosome, because of the absence of crossing-over, lead to an inactivation and reduction of the
Ychromosome. One may therefore pose a hypothesis of aphylogenetic relationship between the
Bulgarian species of the subfamily Pamphaginae and some Caucasian species such as
Paranocaracris rubripes (F .- W .) and Nocaracris cyanipes (F.-W.) (2n = 16AA+neo-X+neoY) (BUGROV 1991,1996; BUGROV and WARCHALOWSKA-SLIWA 1997). It may be that
this group of species is monophyletic and that they differ from West Mediterranean
Pamphaginae, having extremely uniform chromosome sets(2n
= 19, 2n
= 20) and sex
chromosome mechanism XO
/XX
(CAMACHO et al. 1981; SANTOS et al. 1983;
MANSUETO and VITTURI 1989; VITTURIet al. 1993; WARCHALOWSKA-SLIWA et al.
1994). Probably, the evolutionary formation and divergence of these "Caucasian"taxa, which are
distributed in the East Mediterranean up to the Iranian plateau region, arose on the basis of the
transformation of the X-A karyotype.
It seems that further karyological investigation of thePamphagidae grasshoppers from the Balkan
Peninsula, the Caucasus, and Central Asia will provide other patterns of structural evolution of
neo-XY sex chromosome determination.
AcknouJledgements. - This project was supported by the Bulgarian Ministry of Sciences and Education, the "Young
Scientists» programme (grant YS-11-BE).
The authors would like to express their gratitude to Dr. MarkColwell (Humboldt State University, California) for
English correction of the first version of this paper.
SEX DETERMINATION IN FOUR SPECIES OF PAMPHAGID GRASSHOPPERS
121
REFERENCES
ALICATA P., MESSINA A. and OLIVERI S., 1976. - Frequenza e distribuzione dei chiasmi in Pamphagus
marmoratus Burm., Acinipe calabra (Costa) e Ocneridia canonica (Fish.f (Orthoptera Pamphagidae). Animalia,
3.171-193.
BUGROV A.G., 1986. - Neo-XY sex chromosome determination in the grasshopper Asiotmethis heptapotamicus
heptapotamicus (Zub.) and Atrichotmethis semenovi (Zub.) (Orthoptera, Pamphagidaef Tsitologia, 28.117-119. (In
Russian).
- 1996a - Karyotypes of the short-horned Orthopteran insects (Orthoptera, Caelifera) from Russia, Kazakhstan,
Central Asia, and the Caucasus. Folia Bioi., 44.15-25.
- 1996b. - Evolution of the neo-Y sex chromosome in Pamphagidae grasshoppers. 5-th Intern. Congress of Systematic
and Evolutionary Biology, Budapest. 233.
BUGROV A. and WARCIIALOWSKA-SLIWA E., 1997. -Chromosome numbers and C-banding patterns in some
Pamphagidae grasshoppers (Orthoptera, Acridoidea) from the Caucasus, Central Asia, and Transbaikalia. Folia Bioi.,
45: 133-138.
CABRERO J, CAMACHO J.P.M. and PASCUAL F., 1985.Cytotaxonomic studies on pamphagids genus Eumigus.
Detection of two chromosomal races in E. monticola (Rambur) (Insecta, Orthoptera). Caryologia, 38. 1-12.
CAMACHO J.P.M., CABRERO J. and VISERAS E., 1981. -C-heterochromatin variation in the genus Eumigus
(Orthoptera Pamphagoideaf Genetica, 56. 185-188.
CHARLESWORTH B., 1978. - Model for evolution of Y chromosomes and dosage compensation. Proc. Nat. Acad.
Sci. USA, 75: 5618-5622.
FU PENG, 1989. - Karyotype C-banding staining on two species of the genus Sinotmethis B.-Bienko (Orthoptera,
Pamphagidaef Hereditas (China), 11 (3): 26-28.
MANSUETO C. and VITTURI R., 1989. -NORs location and C-banding pattern in spermatogenesis of Pamphagus
ortolanii (Orthoptera, Acrididae). Caryologia, 42 303-311.
MULLER H.J., 1914, -AgenefortheforthchromosomeofDrosophila.J. Exp. Zool., 17.324-336.
SANTOS J.L., ARANA P. and GIRALDEZ R., 1983. -Chromosome C-banding patterns in Spanish Acridoidea.
Genetica, 61. 65- 74.
SOFRADZIJA A and MIKSIC S., 1980/1981. - Characteristics of the chromosomal set of the species Pamphagus
tunetanus Vosseler, 1902 (Orthoptera, Pamphagidae). Glas Zemaljsk. Mus. BiH. Sarajevu Prir Nauke, 19-20. 87-92.
(In Croatian).
SUMNER A.T., 1972. - A simple technique for demonstrating centromere heterochromatin Exp. Cell Res.,75 303-306.
V!TTURI R., MANSUETO C. and FICARELLA P., 1993. -IIeterochromatin variation in four species of the genus
Pamphagus (Orthoptera, Pamphagidae) analyzed by C-banding. Bioi Zentralbl., 112. 335341.
WARCHALOWSKA-SLIWA E., MARYANSKA-NADACHOWSHA A. and MASSA B., 1994 Some
new data on
C-banding and NORs in three species of Pamphagidae (Orthopteraf Folia Bioi., 42. 13-18.
WHITE M.J .D , 1979. - Animal cytology and evolution. Cambridge Univ. Press, London.
Received 11 November 1997; revision accepted 9 December 1997