CYTOLOGY AND CYTOTAXONOMY OF ACRIDIDAE : A SUMMARY

A chromosome contains a linked group of genes, each consisting of a unique number of nucleotide pairs specifying a particular gene, arranged in a precise sequence. Functionally, the genome in a species is a stable integrated system and therefore the structure of chromosomes has to be conservative in order to maintain the integrity. Speciation is a change from one stable system to another and therefore, must involve changes in the genes and their subsequent integration either at the molecular or at the structural levels or both. The taxonomists in determining the evolutionary relationships, invariably emphasize the comparison of structures, which are more conservative in evolution and have empirically determined different sets of characters in different groups of organisms for the purpose. The chromosome complement of a species is a conservative set of morphological features and structural changes in them could be a basis for the study of evolutionary relationships within and between species. In ultimate analysis, all evolutionary changes must originate in changes in the genome, but most of them are at the molecular level and therefore, not so easily accessible for studies. In the mean time cytotaxonomists have gathered a lot of useful information at the microscopic levels of changes in the chromosome structure, which are mediators of evolution.

Since then, grasshopper remained the classical animal for the learning of the grammar of chromosome biology.Their availability in most of the places and their easy maintenance in laboratory, relatively short life cycle, large size of chromosomes with low numbers in their complements made them the material of choice to animal cytologists.The ease with which meiosis can be studied and aberrations can be scored made the investigators use them as good experimental systems.
Earlier workers believed that survey of karyotypes of grasshoppers might be a useful endeavour.They had two purposes.The first was to compare closely related species or genera to discover the karyotypic variability and some trends of karyological evolution where data might also help orthopterists as an additional set of characteristics for distinguishing species and other taxononomic categories.Their second purpose was to discover material useful for cytological research, such as species with low diploid number and novel sex chromosome mechanisms.
They deferred presenting a summary of phylogenetic studies because interpretations based on gross chromosome morphology alone had led to erroneous conclusions.Related species possessed nearly identical chromosome constitutions and thus have not been able to revise taxonomy.
The situation has now changed with the advent of various banding techniques.Grasshoppers by virtue of having 'large' chromosomes are most useful, as banding minute differences that can be located and specified.In the present studies 'C' 'G' and Hoechst 33258 fluorescence banding techniques have been utilized for the study of karyotypes, meiotic patterns and species differentiation.35 species under 32 genera and 11 sub-families have been investigated.
(A survey of literature on the cytology and cytotaxonomy of Indian Acridid and Pyrgomorphid grasshoppers has been published by the author (Singh, 2002)

Techniques used in Chromosome Preparations :
Selected male and female grasshoppers were injected with 0.05% colchicine (0.02 to 0.04 ml) depending upon the size of the insect (colchicine is injected prior to dissection to arrest metaphase).Four to six hours after the injection, the testes and hepatic caecae were dissected out in 0.67% solution of insect saline from the males, while females were preferred to be dissected after 10-12 hours with a possibility to accumulate more metaphases in their somatic cells.The tissues thus dissected out were cleaned in the same medium.After cleaning, the tissues were transferred for a hypotonic treatment in a solution of 0.9% sodium citrate for 45-60 minutes.After that, the tissues were fixed instantly in a freshly prepared fixative of acetic acid and methanol (1 : 3) for 45-60 minutes.A slightly more hypotonic treatment and fixation were preferred for the somatic cells for better chromosomal preparation.The fixed tissues were transferred in small tubes and stored preferably at 4°C.The slides were prepared by the air dry technique.The fixed material were squashed in 50% acetic acid on the slides cleaned in dichromate solution, and stored in the vapours of 50% acetic acid at 4°e for over night.Next morning the slides were brought at room temperature and immersed in 1 : 3 acetic acid methanol mixture for an hour.The cover slips were removed with the help of a sharp blade in the immersed condition and dried at room temperature in a dust proof chamber.After a preliminary scanning of the unstained slides, the selected ones were stained in 2-3% solution of Giemsa [E.Merck (India) Private Ltd.] in phosphate buffer at pH 6.8 to 6.9.Staining was checked until appropriate contrast was obtained, they were then immediately rinsed in distilled water (two changes) and rapidly air dried under a lamp.After scanning, the slides were soaked in Xylene and mounted in DPX.a. C-banding e-banding was carried out according to the method by Sumner (1972) with some minor modifications.The air dried slides were treated with 0.2 N Hydrochloric acid for 30-60 minutes at room temperature, rinsed in distilled water and dried.These slides were dipped in a freshly prepared 5% aqueous solution of Barium hydroxide octahydrate at 50 0 e for 1-10 minutes.The treatment time depends on the ageing of slides; the stored slides produce sharp bands after comparatively longer treatment.After thorough rinsing with several changes in distilled water, they were incubated for 1 hour at 60 0 e in 2 x sse (0.3 M sodium chloride and 0.03 M tri-sodium citrate at pH 7), rinsed in distilled water and dried.These slides were stained for 30 to 90 minutes in Giemsa stain; 2.5 ml of stock solution added to 50 ml of buffer (at pH 6.8).Finally, the slides were again rinsed briefly in distilled water, blotted, allowed to be dried thoroughly under lamp, soaked in Xylene and finally mounted in DPX.

b. Fluorescence banding with Hoechst 33258
Staining of chromosomes with Hoechst 33258 has provided yet another valuable technique of chromosome banding (Hilwig and Gropp, 1972) which is popularly called Hoechst 33258 for staining heterochromatin in mouse chromosomes.A simple direct and slightly modified staining procedure given here was followed throughout.The air dried slides were first soaked in McIlvain's buffer at pH 5.4 for 10 minutes, then stained with freshly prepared Hoechst solution (0.05 mg/ml or 0.1 mg/ml) in the same buffer at pH 5.4 for 15 to 20 minutes at room temperature.After staining, the slides were rinsed in the same buffer (two changes) and mounted either in buffer or in a glycerol buffer mixture.The slides were observed immediately with the help of fluorescence microscope or were stored for some days (up to a couple of weeks) in a refrigerator prior to observation.All fluorescence observations were made using Leitz ortholux photomicroscope with its transmitted light fluorescence attachments using appropriate barrier and exciter filters.

c. Photomicrography
Photomicrographs were taken with the help of Leitz ortholux microscope.Planapochromatic objectives of different magnifications were used.Filters of different combinations were used to obtain the best possible contrast.Black and white negative films (panchromatic) ranging from 50 to 120 ASA were used in fluorescence photomicrography.The negatives so obtained were printed on photosensitive Agfa bromide papers of different grades.Fine grain film and paper developer of Agfa-gevaert (A 901 and 902) and Kodak (D 76 and D 163) were used while developing negatives and positive prints.Digital cameras have also been used on several occasions for more precise results.

d. Karyotyping and construction of idiograms
The cells with good chromosome spreads were photomicrographed.The diploid number (2n) were determined by basic or most predominant number observed in the individual.The cut out of individual chromosomes which appeared similar in morphology and staining intensity were paired to construct karyotypes.All the karyotypes that could be prepared were used for the morphometric measurements.In case of C-band karyotypes the sex chromosomes were placed after the last autosomal pair.In some species very few individuals were available for study.In such cases a banding feature that was present on both the homologues of a given chromosome pair was assumed to occur throughout the species.Morphometric measurements of each chromosome were taken from several metaphase plates from either of the sex.Their mean values so obtained were used in calculating the relative length of the chromosome in percent of the total haploid length.These measurements were also used in drawing comparative idiograms.

SPECIES INVESTIGATED
(Taxonomic grouping according to Dirsh, 1961)  The X and the last pair of chromosomes were distinct by their size.C-band was hardly perceptible in the first 2 pairs of autosomes, and in Hoechst staining also, no bright centromeric regions were noticeable.On C-band staining the centronmeric region of the smallest bivalent was prominent.Last 3 pairs were small.All the chromosomes had centromeric band.On the X it was prominent.

Chrotogonus trachypterus
The 7th and 9 th pairs also had a band in their interstitial region.In Hoechst staining chromosomes showed brighter centromeric region.In all the bivalents random distribution of chiasmata was visible.
5 Tristria pulvinata Last 2 pairs were small.First 4 pairs had wider gaps than others.The 1 st pair was distinct on its prominent centromeric band.The X had become marker element on its proximal band.In Hoechst staining, all the chromosomes showed brighter fluorescing centromeric region.The proximal band of the X had not fluoresced prominently.Chiasma was not restricted to any particular region in the bivalents.
6 Gesonula punctifrons (StaI) 18.18 13.60 11.40 9.81 8.30 7.61 6.89 6.41 6.13 5.53 3.59 2.50 The X was largest, and the last 2 pairs were separated as small ones.All the chromosomes had centromeric band.The 5 th pair also had a proximal band.In Hoechst staining, centromeric regions were not distinct as band.Last 2 small pairs had formed chiasma in the distal region.The X was largest.The P\ 2 nd , 3 rd , 4th and the last pair had bigger size difference than remaining ones.All the chromosomes had centromeric band.On the 9 th pair it was hardly perceptible, whereas, proximal half region of the smallest pair was C-band positive.6 t h, 7th, 8 th and 10 th pairs also had distal bands of different magnitude.In Hoechst staining, brighter centromeric ends were not delimited into sharp bands.All the bivalents had shown random distribution of chiasmata.
8 Oxya hyla Serville 16.05 14.17 12.55 10.53 8.89 7.97 7.36 6.84 5.84 4.45 2.92 2.41 The X was the largest.The 1 5 \ 2 nd , 3 rd and 4th autosomal pairs had bigger size difference.The last 2 pairs were separated as small ones.All the chromosomes had centromeric band of equal magnitude.A proximal band was also present on the 2 nd pair.All the chromosomes had brighter fluorescing centromeric band.The 7th, 8 t h, 9 th and 10 th pairs had mostly distal chiasma.
12 Eyprepocnemis rosea Uvarov 13.52 13.20 10.66 9.26 8.19 8.06 7.47 7.28 6.89 5.88 4.81 4.26 The pt pair of autosome and the X of similar size were grouped as large elements.Size difference between other pairs was haphazard.
15 Cyrtacanthacris tatarica (Linnaeus) 16.16 13.72 11.85 10.51 8.79 8.17 7.53 6.81 6.10 4.40 3.43 2.57 The first 3 and the last 3 pairs were grouped as large and small ones in the complement.Size difference was bigger between the pairs in the large size group.In medium and small groups this difference was small and even.
16 Chondracris rosea (deGeer) 15.98 14.52 13.46 11.69 9.93 8.16 6.69 6.04 5.41 3.05 2.70 2.36 The last 3 pairs of similar size were considerably small in the complement.Difference in the size of pairs was large and equal upto the 6 th autosomal pair.The 7th, 8 th and 9 th had small size difference.Grouping of chromosomes as 3 large 5 medium and 3 small sizes was however not distinct.
The 1 s t, 2 nd and 3 rd pairs had large and equal gap in their length.Grouping of chromosomes as 2 large 6 medium and 3 small pairs of auto somes was however not distinct.
21 Stenocatantops splendens (Thunberg) 14.87 12.74 11.51 10.21 9.08 8.15 7.25 6.95 6.46 5.74 4.17 2n = 17(G) consisted of 6 metacentric and 10 acrocentric autosomes and 1 acrocentric X chromosome.Smaller arms of the metacentrics were larger than the last four chromosomes.The X 4th longest in the complement, was half the size of the 3 rd pair.The P\ 2 nd and 3 rd largest metacentric elements were the fusion products of the 2 nd and 4th, 1 st and 8 th and 3 rd and 7th acrocentric chromosomes of the parental complement (i.e.2n = 23 G).
23 Dnopherula (Aulacobothrus) sp.Jago 14.89 13.02 11.05 10.23 9.97 8.74 7.67 6.88 6.41 4.70 3.74 2.75 The first 2 and the last 3 pairs could be grouped as large and small ones in the complement.Large pairs had bigger size gap than the small pairs.Those in between (3 rd to 8 th pairs) had uneven size difference.
24 Acrotylus inficita (Walker) 15.05 12.96 11.59 10.28 9.74 8.50 7.93 7.30 6.20 4.67 3.48 2.35 First 3 pairs of autosomes could be separated as large ones.For other 5 and last 3 pairs medium and small size distinction was not convincing.The last 2 pairs were distinctly small.The P\ 2 nd and 3 rd pairs were large elements.From 4th to 9 th including X (5 th longest) formed medium size group.
29 Acrida exaltata (Walker) 14.54 12.79 10.93 10.17 9.13 8.15 7.76 7.24 6.87 6.21 3.47 2.73 The 1 st and 2 nd pairs were large and the last 2 pairs were very small in the complement.Others with small gap had formed medium size group.All the chromosomes had centromeric band.One small element was the supernumerary chromosome.
30 Sikkimiana darjeelingensis (Bolivar) 13.73 12.45 11.19 10.08 9.92 9.38 8.13 6.84 6.25 5.02 3.98 3.00 Grouping of chromosomes into large, medium or small was not possible.Also, the difference in the size of pairs was not uniform throughout.
32 Phlaeoba infumata Brunner 14.33 12.34 11.01 10.21 9.15 8.34 7.74 7.13 6.36 5.62 2.97 The 1 s t, 2 nd and 3 rd pairs large, and the last pair was small in the complement.Size difference was small between medium pairs.All the chromosomes had centromeric band.On the 9 th pair C-band was prominent.C-band was also present in the distal half region of the 3 rd pair.Brighter fluorescing centromeric region of all the chromosomes was visible in Hoechst staining.

A SUMMARY OF KARYOLOGICAL FINDINGS
The difference in the idiograms of 32 species reveal distinct karyotype for each of them, instead of all having 23G; 24E acrocentric chromosomes.
The number of chromosomes into different size classes, or gradual seriation of the complement were the distinguishing features of the karyotypes .. Position of the X in the karyotypes of different species varied from pt to 6 th and was found to be a valuable cytotaxonomic character.
The finding is encouraging since it was believed by earlier workers that Acridoidea are a group with such uniform karyotypes that their study can throw little light on taxonomic problems.
On chromosomal banding patterns these species are highly distinct from each other.

Meiosis
Meiotic bivalents (ii).Those that stain specific chromosome structures and hence give rise to a restricted number of bands.These include methods which reveal constitutive heterochromatin (C-bands), telomeric bands (T-bands), and nucleolus organizing regions (NORs).
C-band mitotic, meiotic and hoechst 33258 fluorescence band karyotypes-the advent of C-banding has made it possible to differentially stain constitutive heterochromatic regions at condenced stages of a division cycle, and hence, at a time when such regions are normally indistinguishable from euchromatin.Analysis of C-band karyotypes may reveal- (1) interspecific variation, serving to distinguish related species.
(2) it may be intraspecific in which case it may be either Hoechst-33258 fluorescence banding-in grasshoppers it has been found that at early prophase stages only the centromeres of the autosomal bivalents fluoresced brightly whereas the entire X univalent showed bright fluorescence.It has been concluded that H-fluorescence is modulated by chromosome condensation brought about by differential ratios of DNA-protein at different chromosome regions and at different divisional stages.This property helps in distinguishing chromosomes in the karyotypes.
In most sexually reproducing organisms, the doubling of the gametic chromosome number, which accompanies syngamy, is compensated for by a halving of the resulting zygotic chromosome number at some other point during the life cycle.These changes are brought about by a single chromosomal duplication followed by two successive nuclear divisions.The entire process is called meiosis, and it occurs during animal gametogenesis or sporogenesis in plants.
A pairing configuration during the first meiotic division which consists of two completely or partially homologous chromosomes.The number of bivalents per cell (meiocyte) normally corresponds to half the somatic chromosome number of diploid and genome-allopolyploid species.

SINGH:
Cytology and Cytotaxonomy of Acrididae : and 2 small pairs of autosomes were noted in the complement.Large pairs had big gap in their length.SINGH: Cytology and Cytotaxonomy of Acrididae : (a) distinguish different populations of the same species creating polytypism or else (b) it may distinguish different individuals of the same population creating a polymorphism.C-band meiotic karyotypes help in analysis of chiasma localisation in relation to heterochromatin distribution, and morphological alterations among bivalent types.