Isolation of a Pericentromeric Satellite DNA Family in Chnootriba argus (Henosepilachna argus) with an Unusual Short Repeat Unit (TTAAAA) for Beetles

Abstract

Ladybird beetles (Coccinellidae) are one of the largest groups of beetles. Among them, some species are of economic interest since they can act as a biological control for some agricultural pests whereas other species are phytophagous and can damage crops. Chnootriba argus (Coccinellidae, Epilachnini) has large heterochromatic pericentromeric blocks on all chromosomes, including both sexual chromosomes. Classical digestion of total genomic DNA using restriction endonucleases failed to find the satellite DNA located on these heterochromatic regions. Cloning of C0t-1 DNA resulted in the isolation of a repetitive DNA with a repeat unit of six base pairs, TTAAAA. The amount of TTAAAA repeat in the C. argus genome was about 20%. Fluorescence in situ hybridization (FISH) analysis and digestion of chromosomes with the endonuclease Tru9I revealed that this repetitive DNA could be considered as the putative pericentromeric satellite DNA (satDNA) in this species. The presence of this satellite DNA was tested in other species of the tribe Epilachnini and it is also present in Epilachna paenulata. In both species, the TTAAAA repeat seems to be the main satellite DNA and it is located on the pericentromeric region on all chromosomes. The size of this satDNA, which has only six base pairs is unusual in Coleoptera satellite DNAs, where satDNAs usually have repeat units of a much larger size. Southern hybridization and FISH proved that this satDNA is conserved in some Epilachnini species but not in others. This result is in concordance with the controversial phylogenetic relationships among the genera of the tribe Epilachnini, where the limits between genera are unclear.

Keywords: C0t-1 DNA; Coccinellidae; Coleoptera; bryony ladybird; heterochromatin; in situ hybridization; satellite DNA.

Figure 1

(a) Alignment of the sequences obtained by cloning of the C0t-1 DNA, showing that they were basically comprised of the repetition of the sequence TTAAAA. M1, M2 and M3 indicate the DNA sample used for cloning (see details in Materials and methods) (b) Dot-blot hybridization on total genomic DNA from Chnootriba argus, Diekeana admirabilis, Epilachna paenulata, Henosepilachna vigintioctomaculata and Henosepilachna septima. Different amount of genomic DNA (1 µg to 62.5 ng) were loaded into nylon membrane and hybridized with the TTAAAA repeat.

Figure 2

(a) Meiotic chromosomes of Chnootriba argus stained with DAPI and subsequently fluorescence in situ hybridization (FISH) with the TTAAAA probe (b). Chnootriba argus mitotic metaphase plate stained with Giemsa (c), DAPI (d) and digested with Tru9I and stained with propidium iodide (e). (f) Meiotic chromosomes of Epilachna paenulata stained with DAPI and subsequently, FISH with the TTAAAA probe (g). The arrows indicate the sex chromosomes (X and y).

Figure 3

Phylogenetic tree using concatenated sequences of the ND2 gene and the 28S rDNA. The first number at nodes indicates the bootstrap values obtained in the maximum-likelihood analysis (only when higher than 70%) and the second indicates the posterior probability values in the Bayesian inference analysis (only when higher than 0.7). This tree was created using the data of a previous phylogeny [18] by adding the sequences of Chnootriba argus and Epilachna paenulata. All species appear with the name used in Katoh et al. [18], although the recent revision of the tribe Epilachnini places many of them in other genera [19,33]. Well-supported clades—Asian-Australian Henosepilachna, American Epilachna and Asian Epilachna—are highlighted with colors. Species with the TTAAAA satellite DNA are shaded in yellow whereas species shaded in pink lack this satellite DNA.