Tuatara: Volume 18, Issue 1, July 1970
The Use of Grasshopper — Chromosomes to Demonstrate — Meiosis
The Use of Grasshopper
Chromosomes to Demonstrate
Since the beginning of this century the chromosomes of the shorthorned grasshoppers (Family Acrididae) have been used for a vast number of cytological studies. These chromosomes present a number of advantages to the cytologist.
They are large and relatively few in number.
The range of chromosome lengths in the complement is such that each bivalent formed at meiosis can usually be individually identified according to its length.
Chiasmata are very clear during diplotene and diakinesis thus allowing analyses of their structure, frequency, distribution and movement.
Often the position of the centromere is marked by relatively denser staining (precocious condensation) at early diplotene.
Besides these cytological advantages, the techniques involved in the preparation of slides of this material are quick and simple and therefore it is ideal for demonstrating the stages of meiosis to students.
The purpose of this article is to suggest suitable New Zealand species of grasshoppers, outline dissection and cytological techniques, and to give a brief description of the chromosomes and the stages of meiosis.
The New Zealand Acrididae
The family is represented in New Zealand by fifteen species (Bigelow, 1967) of which fourteen are endemic and one is the worldwide species. Locusta migratoria.page 2
From the point of view of availability, P. marginale is the best species to choose. However a comparison of the cytogenetics of several New Zealand species (Martin, 1970) showed the chromosomes of the alpine species to be slightly longer, with a higher number of chiasmata per cell, than P. marginale. For this reason the alpine grasshoppers are recommended if they are available.
Preparation of Material and Cytological Techniques
If P. marginale is being used it can be distinguished from another lowland grasshopper, the long-horned grasshopper (Family Tettigoniidae), by the fact that the antennae of the former are only about 4 mm. in length whereas those of the Tettigoniidae are about 25 mm. long. The alpine short-horned species are the only grasshoppers found at high altitudes.page 4
Young adult or last instar males (females are not suitable for studying meiosis) provide the best testis material. The males and females can be distinguished by the morphological differences shown in figure 2 and also by the fact that the males are smaller than the females. The grasshoppers can be caught by hand or with the aid of a small net. They are available from late November to early April during which time the spermatogonia are dividing rapidly.
The insects are chloroformed or etherised and then dissected in insect saline (see below). The testes lie in a dorsal position in the anterior half of the abdomen and can be easily located by making a dorsal, longitudinal, abdominal cut. They can be identified by the orange-yellow fatty tissue that cover them. Once this is removed with dissecting needles (while still in the insect saline) each testis can be seen to consist of many follicles.
The best fixative for these preparations is 1 : 3 acetic alcohol (see below). The testis material is left in the fixative for at least five minutes before one of the staining procedures described below is carried out. The material can also be stored before being stained either in the fixative or by transferring it to 70 per cent ethyl alcohol after fixation. It will keep satisfactorily for at least a year either at room temperature or, preferably, in a refrigerator.
Place a small drop (about 5 mm. in diameter) of the stain in the middle of a clean slide.
Take three or four testis follicles from the fixative (or alcohol), drain off excess moisture on a piece of blotting paper, and leave in the stain for three to five minutes.
After this time the follicles are broken up by firmly tapping them with a metal or glass rod until there is only a suspension of small particles in the stain.
After any remaining large pieces of material have been removed, a clean cover-slip can be applied.
Heating the slide gently over the flame of a spirit lamp at this stage will flatten and spread the chromosomes. The stain must not boil.
The slide should then be placed between two pieces of blotting paper which is firstly pressed down lightly so that excess stain around the edges of the cover-slip is absorbed, and then the preparation is squashed by firm, vertical, thumb pressure. Avoid any sideways movement.
If the cover-slip is ringed with petroleum jelly the temporary squash preparation will keep for at least ten days.
Snow's HCl carmine: Using this stain, the material must be pre-stained and then stored in 70 per cent ethyl alcohol until required.
The material is transferred from the fixative or alcohol to the stain (in a small stoppered vial) for 24 hours.
After this time the material should be transferred to 70 per cent alcohol and stored in the refrigerator until needed.
Preparation of the slide is as above except that the material is squashed in a drop of 45 per cent acetic acid.
If desired, temporary squashes can be made permanent using a method similar to that of Conger and Fairchild (1953).
Freeze the preparation by inverting the slide on a flat piece of dry ice for 30 seconds to one minute.page 6
Without letting the preparation thaw, prise the cover-slip off with a scalpel or razor blade. The material should remain attached to the slide but if some does remain on the cover-slip this can also be remounted on a clean slide by the same procedure.
Immerse the slide, while the material is still frozen, in absolute ethyl alcohol for ten minutes.
Place the slide in fresh absolute alcohol for at least another ten minutes.
Put a small drop of euparal in the middle of the squashed material and apply a clean cover-slip.
Heat the slide gently to remove any large air bubbles and then leave it in a dust-free place to harden.
Fig. 4 a-o: Meiosis in Brachaspis collinus. (All photographs are of acetic-orcein preparations, x 1000.) (a.) Leptotene: The chromosomes appear as a mass of single threads. The X-chromosome is positively heteropycnotic (arrow). (b.) Zygotene: The nucleus has increased in volume and homologous chromosomes have paired. The X-chromosome is still positively heterochromatic (arrow). (c.) Pachytene: Homologues are separating and each can be seen to consist of two chromatids which are joined at chiasmata, the points of cross-over between non-sister chromatids. (c′.) Interpretation of 4c. Each line represents a chromatid. The eleven bivalents can be individually identified (classification is given). (d.) Mid-diplotene: The bivalents have contracted somewhat and chiasmata are more obvious. (d′.) Interpretation of 4d. The position of the centromeres is indicated by small black circles and the chiasmata by arrows. (e.) Early diakinesis: The bivalents have contracted further and some are beginning to become heterochromatic. (e′.) Interpretation of 4e. (f.) Metaphase I: The bivalents have contracted fully and the autosomes are now heterochromatic. The X is negatively heteropycnotic (arrow). (g.) Anaphase I: Separation of the homologous chromosomes each of which consists of two chromatids joined by the terminal centromere giving a ‘v’ appearance. The X-chromosome has segregated undivided to one pole (arrow). (h.) Telophase I: The ‘v-’ shape of the chromosomes is still apparent. The chromosome number at each pole is n = 11 plus X at one of them. (i.) Interkinesis: A short stage between the two divisions of meiosis. Note that only one cell has an X-chromosome (arrow). (j.) Prophase II: The chromosomes are distinguishable once more and are heterochromatic. (The photographs of meiosis II show the division of only one of the cells formed after meiosis I). (k.) Metaphase II: Chromatids of each chromosome are distinguishable and are still joined at their centromeres (arrows). The chromosome number is n = 11 + X. (l.) Anaphase II: The centromeres have divided and the chromatids have separated to opposite poles. The X-chromosome has also divided. (Some of the chromosomes in this photograph are slightly out of focus.) (m-o.) Spermiogenesis: Differentiation of spermatid nuclei (4m) into spermatids (4n-o). Spermatid nuclei with (arrows) and without an X-chromosome can be seen.
Fig. 5a-c: B-chromosomes in Phaulacridium marginale. (All photographs are of acetic-orcein preparations, x 1000.) (a.) Pachytene: The B-chromosome can be seen to consist of a large block of heterochromatin, a section of euchromatin, and a small, terminal heterochromatic segment. (Heterochromatin is indicated by h, and the euchromatin by e.) (b.) Diplotene: Although contraction of the bivalents has occurred, the three elements of the B-chromosome are still obvious (arrow). (c.) Metaphase I: The cell has two B-chromosomes which are segregating to the same pole.
Insect saline, fixative and stains
Insect saline: 7.5 gms. NaCl per litre of water.
Fixative: 1 part glacial acetic acid: 3 parts absolute ethyl alcohol.
Acetic-orcein: 2 gms. synthetic orcein.
45 ml. glacial acetic acid.
15 ml. distilled water
Boil gently until the orcein has dissolved. Cool and then filter.
Snow's HCI carmine: 4 gms. carmine.
15 ml distilled waterpage 11
1 ml. concentrated HC1.
Boil until the carmine dissolves. Cool, add 95 ml. 85 per cent ethyl alcohol, and then filter.
A recent investigation on the cytogenetics of some New Zealand grasshoppers (Martin, loc. cit.) showed that in five species (P. marginale, S. piliferus, Brachaspis collinus, B. nivalis and Alpinacris crassicauda) the male diploid chromosome number is 22 + X. L. migratoria and Paprides nitidus also have this number. This is typical of the majority of acridids and it is highly likely that the other New Zealand species also have the same number of chromosomes.
The sex chromosome
In the Acrididae the sex determining mechanism is an XO system. Therefore the females have two, and the males one X-chromosome. The X-chromosome (in the male) segregates randomly to either pole at anaphase I and at anaphase II its chromatids separate normally to opposite poles. Thus the meiotic division of one spermatogonium results in four spermatid nuclei, of which two have no X-chromosome (see figure 3). The XO system is basically similar to the XY system found in man except that in the latter, of the four spermatids resulting from meiosis, two have an X-chromosome each and two have a Y-chromosome each. Thus in figure 3 the Y-chromosome would be substituted for O.
Up to metaphase I the X-chromosome is densely stained, or heterochromatic, while the autosomes are lightly stained, or euchromatic (figures 4a-e). At metaphase I it becomes lightly stained but returns to its former state during meiosis II (figures 4f-l). If a chromosome has the ability to change from heterochromatic to euchromatic in this way it is described as being heteropycnotic. Thus the X-chromosome is positively heteropycnotic up to metaphase I when it becomes negatively heteropycnotic.
There are 22 autosomes all of which are telocentric (i.e. they have a terminal centromere). Therefore 11 bivalents can be recognised at the beginning of meiosis. The bivalents can be classified into three general size classes: long, which includes the three longest bivalents; medium, consisting of the next five longest bivalents; and short, which consists of the three shortest bivalents. The individual bivalents are enumerated as L1, L2, L3, M4, M5, etc. The S9 bivalent is usually easily recognised by the large heterochromatic blocks at the centromeric ends (figures 4c-e).
Some individuals of P. marginale possess one, and sometimes two extra chromosomes. These are supernumerary or B-chromosomes. The origin of these chromosomes is hard to determine but they have probably resulted from some form of mis-division of an autosome.
The B-chromosomes are very similar to X-chromosomes in appearance and behaviour. The type found in P. marginale are mainly heterochromatic but they also have a segment of euchromatin which is terminated by a very small segment of heterochromatin (figure 5a-b.) At anaphase I they segregate randomly, and independently of the X-chromosome and each other (if there are two), to either pole. At anaphase II the chromatids separate and segregate normally.
In grasshoppers these extra chromosomes do not appear to have any phenotypic effect but it is probable that they do influence other members of the chromosome complement e.g. by affecting the chiasma frequency of a nucleus.
I wish to thank Mr. G. K. Rickards for critically reading the manuscript.
Bigelow, R. S., 1967. The Grasshoppers (Acrididae) of New Zealand. University of Canterbury Publications.
Conger, A. D., and L. M. Fairchild, 1953. A quick-freeze method of making smear slides permanent. Stain Tech. 28: 281-283.
Martin, J. M., 1970. The cytogenetics of some New Zealand grasshoppers (Acrididae). Unpub. Thesis, Victoria University.