Karyotypic Studies of Six Species of Grasshopper (Orthoptera: Acrididae) from Kolhapur District, Maharashtra, India

Yogesh Jangonda Koli1, Sunil Madukar Gaikwad1, Dilip Laxman Bharmal2, and Ganesh Parsharam Bhawane1*

1 Department of Zoology, Shivaji University, Kolhapur 416 004. Maharashtra State, India 2 Shri. Panchamkhemraj Mahavidyalaya Savantwadi 416 510. Maharashtra State, India

Received February 27, 2013; accepted August 1, 2013

Summary Chromosome analysis was performed in six grasshopper species of the family Acrididae, viz., Acrida exaltata (2n=23+XO), Phlaeoba antennata (2n=23+XO), Gastrimargus africanus africanus (2n=23+XO), Parahieroglyphus bilineatus (2n=23+XO), Cyrtacanthacris tatarica (2n=23+XO), and Hieroglyphus banian (2n=23+XO), with X chromosome being the largest in the complement. Detailed karyotypic information regarding chromosome length, total complement length of chromosome set, and relative length were measured using the Micromeasure software.

Key words Orthoptera, Acrididae, Chromosome, Karyotype.

The karyology of every species is unique and provides an identity to the species (Channaveerappa and Ranganath 1994). Karyological studies provide important information on the genetic structure, life cycle, and phylogeny of insects (Gokhman and Kuznetsova 2006). Due to great cytogenetic uniformity, the acridids are considered to exemplify “karyotype conservatism” (Aswathanarayana and Ashwath 2006). The karyotype of Orthoptera was ﬁrst reported in the literature late in the 19th century by Asana. According to a review of published data, 393 publications (including 200 abstract papers) have been recorded on Indian grasshoppers (Singh and Singh 2009). The vast majority of them are on the Acrididae. The family Acrididae comprises around 6,000 species under about 1,000 genera distributed worldwide, of which 138 genera and 310 species have been reported from India (Tandon et al. 1991). As stated by Shishodia et al. (2010), the family Acrididae is divided in to 15 subfamilies (Table 2). The family Acrididae of the grasshopper is characterized by a great uniformity in both chromosome number and chromosome morphology; about 90% of the known species have a karyotype consisting of 2n ♂=22+XO chromosomes (Turkoglu and Koca 2002). The number and morphology of the chromosomes are very similar, so it is very difﬁcult to recognize genus and even families of Orthopteran insects based on simple karyotyping (Sandhu and Chadha 2012). In the present study, an attempt has been made to throw light on the chromosome architecture and the karyotypic pattern of the most commonly occurring short-horned grasshopper in Maharashtra.

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.78.255 256 Y. J. Koli et al. Cytologia 78(3)

Table 1. Materials studied.

Specimens Species Localities collected ♂

Acrida exaltata Walker 16 CNP, Ambaiwada, 17°06′54.72″N/73°50′59.77″E Phlaeoba antennata Brunner 07 CNP, Niwale, 17°11′89.25″N/73°80′74.59″E Gastrimargus africanus africanus Saussure 23 CNP, Khundlapur, 17°10′26.88″N/73°52′35.07″E Parahieroglyphus bilineatus Bolivar 07 CNP, Ghotane, 17°05′51.79N/73°46′09.58″E Cyrtacanthacris tatarica Linnaeus 12 CNP, Khundlapur, 17°10′26.88″N/73°52′35.07″E Hieroglyphus banian Fabricius 12 CNP, Ambaiwada, 17°06′54.72″N/73°50′59.77″E

Table 2. Information about the species recorded and cytologically studied from India.

Taxonomically Cytologically Subfamilies recorded studied

Acridinae 36 16 Calliptaminae 11 2 Catantopinae 65 10 Coptacridinae 9 2 Cyrtacanthacridinae 8 6 Eyprepocnemidinae 13 11 Gomphocerinae 44 9 Hemiacridinae 12 4 Melanoplinae 4 – Oedipodinae 52 21 Ommatolampinae 1 – Oxyinae 18 10 Spathosterninae 2 2 Teratodinae 3 1 Tropidopolinae 2 1

Materials and methods

The materials used in this study are listed in Table 1 along with the data on where they were collected. They were brought to the laboratory and injected with 0.02 to 0.04 ml of 0.05% colchicines (depending upon the size of the collected species). They were sacriﬁced 4 to 6 h after the injection, and the testes were dissected out separately in 0.67% Nacl. Then the tissues were ﬁxed in an acetate–methanol mixture (1 : 3). Chromosomes were prepared following the air dry technique of Kacker et al. (1986). Chromosome measurements, including long arm, short arm, and chromosome lengths, total length of chromosome set, and relative chromosome length, were made, and an idiogram was con- structed based on the relative percentage of chromosome. The centromeric index was determined by using the following formula. 100XS I=c C Here, S is the short arm length and C is the total length of the chromosome. The chromosome morphology was determined according to the nomenclature proposed by Levan and Sandberg (1964). The present paper deals with the study of miotic and meiotic chromosomes of Acrida exaltata, Phlaeoba antennata, Gastrimargus africanus africanus, Parahieroglyphus bilineatus, 2013 Karyotypic Studies of Six Species of Grasshopper 257

Fig 1. a, c, e, g, i, k: Spermatogonial metaphase of A. exaltata, P. antennata, G. aﬁcanus africanus, P. bilineatus, C. tatarica, H. banian. b, d, e, h, j, l: Idiogram of A. exaltata, P. antennata, G. aﬁcanus africanus, P. bilineatus, C. tatarica, H. banian.

Cyrtacanthacris tatarica, and Hieroglyphus banian. The karyotype analysis includes the number, morphology, and relative length of chromosomes. The results obtained concerning the number of chromosomes, their structure, and the type of sex determination may be used for establishing and verifying the taxonomic attachment of some species.

Results and Discussion

The six commonly occurring species investigated for the present study were A. exaltata, 258 Y. J. Koli et al. Cytologia 78(3)

Table 3. Mean total complement length and actual and relative chromosome lengths in the short-horned grasshopper.

Total Chromosome length Relative length X chromosome length Species 2n complement length L S L S L S

Acrida exaltata 23 45.27±8.47 4.14±0.75 3.91±0.78 9.14±0.98 8.64±1.01 8.93±1.77 8.59±1.77 Phlaeoba antennata 23 23.37±2.85 2.15±0.26 2.10±0.25 9.20±1.03 8.98±0.96 4.58±0.81 4.52±0.84 Gastrimargus africanus 23 24.28±4.18 2.24±0.37 2.17±0.39 9.22±0.97 8.94±1.03 4.56±0.84 4.28±0.85 africanus Parahieroglyphus bilineatus 23 21.72±4.11 2.02±0.43 1.92±0.32 9.30±1.15 8.84±0.85 4.10±1.18 4.04±1.35 Cyrtacanthacris tatarica 23 26.19±10.78 2.46±1.04 2.31±0.92 9.39±1.07 8.82±0.94 5.71±2.35 5.59±2.18 Hieroglyphus banian 23 52.55±10.78 2.54±0.56 2.25±0.50 4.83±0.53 4.28±0.47 4.10±0.89 3.76±0.89

Table 4. 2n, sex mechanism, position of X chromosome, nature of chromosome, and size of chromosome of six species of grasshoppers.

Sex Position of X Nature of Chromosome size Sr. No Species studied 2n mechanism chromosome chromosome L M S

1 A. exaltata 23 XO Largest Metacentric 3 4 4 2 P. antennata 23 XO Largest Metacentric 3 2 6 3 G. a. africanus 23 XO Largest Metacentric 3 3 5 4 P. bilineatus 23 XO Largest Metacentric 3 4 4 5 C. tatarica 23 XO Largest Metacentric 2 5 4 6 H. banian 23 XO Largest Metacentric 3 4 4

P. antennata, G. africanus africanus, P. bilineatus, C. tatarica, and H. banian. They all show 2n=23 chromosomes (Tables 3 and 4). The males show the XO mechanism of sex determination. All species of the grasshopper presently investigated have metacentric chromosomes in their chromosome complements with a complete absence of acrocentric or telocentric chromosomes. The male A. exaltata in the present study showed 2n=23(22A+XO) chromosomes. The mean actual chromosome length ranged from 3.91 µm to 4.14 µm. The mean total complement length was 45.27 µm. The relative chromosome length was 8.64 µm in the shortest chromosome to 9.14 µm in the longest chromosome, which was measured from 10 metaphasal plates. The sex chromosome was largest in the total complement. The male P. antennata showed 2n=23(22A+XO) chromosomes. In this study, all autosomes and sex chromosome are found to be in a metacentric position. The 11 pairs of autosomes can be easily arranged in three principle groups: 3 pairs of large, 2 pairs of medium, and 6 pairs of small chromosomes. The mean actual chromosome length ranged from 2.10 µm in the shortest to 2.15 µm in the longest chromosome. The relative chromosome length was 8.98 µm in the shortest chromosome and 9.20 µm in the longest chromosome. The mean total complement length was 23.37 µm. The spematogonial metaphase of the male G. africanus africanus comprises 2n=23 (22A+XO). All autosome complements are metacentric and divided into 3 pairs of long, 3 pairs of medium, and 5 pairs of short chromosomes. The X chromosome was easily noticeable because of its large size. The mean actual chromosome length ranged from 2.17 µm in the shortest chromosome to 2.24 µm in the longest chromosome. The mean total complement length was 24.28 µm. The relative chromosome length ranged from 8.94 µm in the shortest chromosome to 9.92 µm in the longest chromosome, which was measured from 10 metaphasal plates. The chromosome number of Parahieroglyphus bilineatus was found to be 2n+23 (22A+XO), and all chromosomes were regular metacentric. Measurements of metaphase plates showed that the actual chromosome length ranged from 1.92 µm in the shortest to 2.02 µm in the longest chromosome. The mean total complement length was 21.72 µm. The relative chromosome length ranged 2013 Karyotypic Studies of Six Species of Grasshopper 259 from 8.84 µm in the shortest to 9.30 µm in the longest chromosome. The karyotype of this species showed gradual decrease in chromosome length. The chromosomes of this taxon were divided into three groups: 3 long, 4 medium, and 4 short chromosomes. The diploid number of the chromosomes of C. tatarica differentiates into 11 pairs of autosomes and sex chromosomes, which are XO in male. X chromosome are metacentric and clearly larger than the ﬁrst autosome pair. The mean actual chromosome length ranged from 2.31 µm in the shortest chromosome to 2.46 µm in the longest chromosome. The mean total complement length was 26.19 µm. The relative chromosome length ranged from 8.82 µm in the shortest chromosome to 9.39 µm in longest chromosome, which was measured from 10 metaphasal plates. The karyotype of the male H. banian is 2n=22+XO. The autosomal complement can be grouped in to 6 large, 2 medium, and 4 small chromosomes. The chromosome morphology was determined to be metacentric for all pairs. Chromosome measurements in the metaphase plate revealed that the actual chromosome length ranged from 2.25 µm in the shortest to 2.54 µm in the longest chromosome. The mean total complement length was 52.55 µm. The relative chromosome length ranged from 4.28 µm in the shortest to 4.83 in the longest chromosome. In the present study, six Acridids have been investigated which belong to the subfamilies Aciridinae, Oedipodinae, Hemiacridinae, and Cyrtacanthacridinae. The chromosome number observed for A. exaltata, P. antennata, G. africanus africanus, P. bilineatus, C. tatarica, and H. banian was 2n=22/XO for males, which is in agreement with the description of variation in chromosome number and in the sex determining system reported by Asana et al. (1938), Sakai and Shigenga (1967), Turkoglu and Koca (2000, 2002), and Rocha et al. (2004) for other species of Acridids. Yadav and Yadav (1986) reported similar results in relation to chromosome number and sex-mechanism among the Haryana population of Acridioideans, while Chadha and Mehta (2011) reported similar results in four species of grasshopper from Punjab. So the Acridid grasshoppers of different localities are showing cytogenetic uniformity regarding chromosome number and sex-determining mechanism. For the species Phlaeoba infumata, Yadav and Yadav (1983 and 1993) gave the distribution pattern of C-banding, and they also tried the trypsin treatment for G-banding but noted very poor response. Rajasekarasetty (1965) reported that in addition to the normal complement of 2n=23, the metaphase I of a few cells had nine bivalent and one X chromosome, indicating the diploid number as 2n=19 in Gastrimargus africanus orientalis. However, later studies by Aswathanarayan et al. (1981) of G. africanus orientalis, and by Fossey and Lichenbery (1991) of four species of Gastrimargus, namely, G. africanus, G. crassicollis, G. vitripennis, and G. transverses, have not revealed a situation similar to the one reported by Rajasekarasetty (1965).

Acknowledgements

The authors are thankful to the Head of the Department of Zoology, Shivaji University, Kolhapur, for providing laboratory facilities. We are thankful to Dr. S. R. Yadav, Dr. Kumar Vinod. C. Gosavi, and Dr. Manoj Lekhak of the Department of Botany, Shivaji University, Kolhapur for providing laboratory facilities and for his constant encouragement during this work. This work was supported by grants from the Department of Science and Technology (DST) of New Delhi.

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