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. 2020 Mar 19;11(3):327.
doi: 10.3390/genes11030327.

Structural Variation of the X Chromosome Heterochromatin in the Anopheles gambiae Complex

Atashi Sharma  1 , Nicholas A Kinney  2 , Vladimir A Timoshevskiy  1 , Maria V Sharakhova  1   3   4 , Igor V Sharakhov  1   2   3   5
Affiliations

Structural Variation of the X Chromosome Heterochromatin in the Anopheles gambiae Complex

Atashi Sharma et al. Genes (Basel). .

Abstract

Heterochromatin is identified as a potential factor driving diversification of species. To understand the magnitude of heterochromatin variation within the Anopheles gambiae complex of malaria mosquitoes, we analyzed metaphase chromosomes in An. arabiensis, An. coluzzii, An. gambiae, An. merus, and An. quadriannulatus. Using fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA), a highly repetitive fraction of DNA, and heterochromatic Bacterial Artificial Chromosome (BAC) clones, we established the correspondence of pericentric heterochromatin between the metaphase and polytene X chromosomes of An. gambiae. We then developed chromosome idiograms and demonstrated that the X chromosomes exhibit qualitative differences in their pattern of heterochromatic bands and position of satellite DNA (satDNA) repeats among the sibling species with postzygotic isolation, An. arabiensis, An. merus, An. quadriannulatus, and An. coluzzii or An. gambiae. The identified differences in the size and structure of the X chromosome heterochromatin point to a possible role of repetitive DNA in speciation of mosquitoes. We found that An. coluzzii and An. gambiae, incipient species with prezygotic isolation, share variations in the relative positions of the satDNA repeats and the proximal heterochromatin band on the X chromosomes. This previously unknown genetic polymorphism in malaria mosquitoes may be caused by a differential amplification of DNA repeats or an inversion in the sex chromosome heterochromatin.

Keywords: Anopheles; X chromosome; heterochromatin; mitotic chromosome; mosquito; satellite DNA; sex chromosome.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Comparison of the X heterochromatin structure between mitotic and polytene chromosomes. (A) Multicolor fluorescence in situ hybridization (FISH) of a C0t1 fraction of repetitive DNA and 18S rDNA with mitotic chromosomes of An. gambiae PIMPERENA; scale bar is 2 µm. (B) FISH mapping of BAC clones 05F01 (yellow), 179F22 (red), and 01K23 (green) to the distal heterochromatin block on the X chromosome of the An. coluzzii MOPTI strain. Chromosomes are counter-stained with DAPI. (C) Relative positions of heterochromatic blocks and DNA probes of the polytene and mitotic X chromosome idiograms. rDNA = 18S rDNA probe. Repetitive DNA = C0t1 fraction of repetitive DNA. C = putative centromere. (D) Relative sizes of mitotic and polytene chromosomes in the KISUMU strain of An. gambiae. Chromosomes are counter-stained with YOYO-1. Polytene chromosome physical map is from [38].
Figure 2
Figure 2
Geographical distribution of species and phylogeny of the An. gambiae complex. (A) A map of Africa with approximal distribution of species and places of origin of the laboratory strains. (B) Species phylogeny based on the X chromosome genomic sequences (redrawn from [18]). Times of species divergence in million years (Myrs) are shown at the tree nodes.
Figure 3
Figure 3
Metaphase karyotypes of females in species from the An. gambiae complex. (A) An. gambiae strains. (B) An. coluzzii strains. (C) An. arabiensis, An. quadriannulatus, and An. merus strains. Black and dark gray blocks correspond to compact and diffuse heterochromatin, respectively. X chromosomes are labeled. Species names are indicated in italics and strain names are capitalized.
Figure 4
Figure 4
Mapping of repetitive DNA elements to mitotic chromosomes of species from the An. gambiae complex. (A) FISH of AgY477–AgY53B (green) and 18S rDNA (red) in An. gambiae PIMPERENA. (B) FISH of satellite AgY53A (green) and 18S rDNA (red) in An. gambiae PIMPERENA. (C) FISH of AgY477–AgY53B (green) and 18D rDNA (red) in An. merus. (D) FISH of AgY477–AgY53B (green) and 18S rDNA (red) in An. coluzzii SUA. (E) FISH of satellite Ag53C (yellow) and 18D rDNA (red) in An. coluzzii MOPTI. (F) FISH of 18S rDNA (red) in An. quadriannulatus. (G) FISH of satellite AgY477 (red) and AgY477–AgY53B (green) in An. arabiensis. (H) FISH of satellite AgY477 (red) and 18D rDNA (green) in An. arabiensis. (I) FISH of 18S rDNA (green) and AgY477–AgY53B (red) in An. arabiensis. Scale bar = 2 µm.
Figure 5
Figure 5
Variation in the pattern of heterochromatin blocks, rDNA loci, and satDNA location on the X chromosomes in F1 female hybrids between An. gambiae and An. coluzzii. (A) FISH of AgY477–AgY53B (green) and AgY477 (red) in F1 ♀An. gambiae PIMPERENA × ♂An. coluzzii MOPTI. (B) FISH of AgY477 (red) and 18S rDNA (green) in F1 ♀An. gambiae ZANU × ♂An. coluzzii MALI. (C) FISH of AgY477 (red) and 18S rDNA (green) in F1 ♀An. coluzzii MOPTI × ♂An. gambiae KISUMU. Scale bar = 2 µm.
Figure 6
Figure 6
Polymorphism of the X heterochromatin structure among the An. gambiae and An. coluzzii strains. (A) Distance and order frequency of FISH signal and DAPI band intensity peaks in An. gambiae PIMPERENA and An. coluzzii MOPTI. (B) Distribution patterns of signal intensities for satellites and DAPI along the X chromosomes in An. gambiae PIMPERENA and An. coluzzii MOPTI. (C) Relative positions of satellite peak distance and width with respect to the proximal heterochromatin band for six strains of An. gambiae and An. coluzzii. (D) Clustering of the An. gambiae and An. coluzzii strains based on the relative positions of satellite peak distance and width with respect to the proximal heterochromatin band.
Figure 7
Figure 7
Lengths of the metaphase chromosomes in species from the An. gambiae complex.
Figure 8
Figure 8
Idiograms of metaphase karyotypes of species from the An. gambiae complex. (A) An idiogram of metaphase karyotypes of An. gambiae and An. coluzzii. (B) Comparison of the X chromosome idiograms among species from the An. gambiae complex. Left and right arms are labeled with L and R, respectively. Black bands correspond to condensed heterochromatin. Dark gray bands correspond to diffuse heterochromatin. White areas represent the rDNA locus. Light gray areas show euchromatin. The area of constriction represents the putative centromere. Mapping of the BAC clones and satDNA repeats AgY53A and AgY53C were performed only with An. coluzzii or An. gambiae. Two different positions of AgY477–AgY53B on the X chromosome of An. gambiae and An. coluzzii represent polymorphism among strains.
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