New Insights into Phasmatodea Chromosomes

Abstract

Currently, approximately 3000 species of stick insects are known; however, chromosome numbers, which range between 21 and 88, are known for only a few of these insects. Also, centromere banding staining (C-banding) patterns were described for fewer than 10 species, and fluorescence in situ hybridization (FISH) was applied exclusively in two Leptynia species. Interestingly, 10-25% of stick insects (Phasmatodea) are obligatory or facultative parthenogenetic. As clonal and/or bisexual reproduction can affect chromosomal evolution, stick insect karyotypes need to be studied more intensely. Chromosome preparation from embryos of five Phasmatodea species (Medauroidea extradentata, Sungaya inexpectata, Sipyloidea sipylus, Phaenopharos khaoyaiensis, and Peruphasma schultei) from four families were studied here by C-banding and FISH applying ribosomal deoxyribonucleic acid (rDNA) and telomeric repeat probes. For three species, data on chromosome numbers and structure were obtained here for the first time, i.e., S. inexpectata, P. khaoyaiensis, and P. schultei. Large C-positive regions enriched with rDNA were identified in all five studied, distantly related species. Some of these C-positive blocks were enriched for telomeric repeats, as well. Chromosomal evolution of stick insects is characterized by variations in chromosome numbers as well as transposition and amplification of repetitive DNA sequences. Here, the first steps were made towards identification of individual chromosomes in Phasmatodea.

Keywords: C-banding; Phasmatodea; fluorescence in situ hybridization; interstitial telomeric sequences; ribosomal deoxyribonucleic acid; stick insects; telomeric repeats.

Figure 1

Metaphase plates of female embryo of Medauroidea extradentata. (a) C-banding: large submetacentrics have in their short arms proximal C-positive regions, indicated by arrows is thepair with the largest C-positive regions; (b) Fluorescence in situ hybridization (FISH) with 18S ribosomal deoxyribonucleic acid (rDNA) (green) and telomeric repeats (red) showed that the in (a) highlighted chromosome pair has rDNA accumulated in C-positive region. Chromosomes were counterstained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). (c) The same metaphase as in (b), but with longer exposure time for green channel, is shown. Other chromosomal regions also gave weak signals using 18S rDNA specific probe. Scale bar indicates 5 μm.

Figure 2

Karyograms from metaphase plates of female embryos of three of the studied species; gonosomes could not be identified. (a) Female M. extradentata have 19 chromosome pairs, i.e., most likely a karyotype 36,XX. (b) For female Sungaya inexpectata no complete metaphases could be obtained; here we show one incomplete metaphase with 53 chromosomes and 32 different chromosome pairs identified—lacking chromosomes are indicated by a V. Still, it is obvious that this species is diploid. (c) Female Sipyloidea sipylus have 22 chromosome pairs, i.e., most likely a karyotype 42,XX.

Figure 3

Metaphase plates of female embryo of S. inexpectata. (a) C-banding: large metacentrics contain C-positive arms (arrows); (b) FISH as in Figure 1b; shown are late metaphase chromosomes (chromatids of many chromosomes were separated); (c) DAPI staining; (d) FISH with 18S rDNA only; (e) FISH with labeled telomeric repeats only. The arrowheads show the regions the regions containing the dispersed telomeric repeats. The arrows show interstitial telomeric sites. Scale bar indicates 5 μm.

Figure 4

Metaphase plates of a female embryo of S. sipylus. (a) C-banding: large metacentrics contain large C-positive regions in one arm (arrows); (b) FISH as in Figure 1b; (c) FISH as in Figure 1b on a partial metaphase plate. Scale bar indicates 5 μm.

Figure 5

Metaphase plates of a female embryo of Phaenopharos khaoyaiensis. (a) C-banding: a large C-positive region is visible (arrow); (b) the C-positive region is highlighted by FISH (18S rDNA, green) and homologue chromosome is also stained. Scale bar indicates 5 μm; (c) Female P. khaoyaiensis have 70 chromosome pairs, i.e., most likely a karyotype 34,XX.

Figure 6

Mitotic and meiotic chromosomes of Peruphasma schultei; (a) C-banding of embryonal metaphase chromosomes. The X chromosomes are labeled with ‘X’. (b) FISH as in Figure 1b. (c) C-banding of adult male derived meiotic chromosomes. (d) FISH on meiotic chromosomes (pachytene), using probes as given in Figure 1b. Scale bar indicates 5 μm; (e) Female P. schultei have 22 chromosome pairs and a karyotype 21,XX.