Annalisa Marchi

Karyotype, DNA replication and origin of sex chromosomes in Anopheles atroparvus

Anopheles atroparvus has two pairs of autosomes similar in length and morphology and two sex chromosomes with equal, heterochromatic, late replicating long arms with homologous C-, G-, and Q-bands. The short arm of the Y is shorter than that of the X and both are euchromatic. The mean number of chiasmata per cell in the male is 3.2. During mitosis there is a high grade of somatic pairing but X and Y, which form a heteropycnotic mass in the interphase nucleus, have a differential behaviour. The chronology of DNA replication was studied in spermatogonia and brain cells by autoradiography. It is hypothesized that the present sex chromosomes of A. atroparvus evolved by accumulation of sex determining factors and gene deterioration resulting in heterochromatinization of the long arms, followed by structural rearrangements.—The homology of the two sex chromosomes requires limited dosage compensation which is achieved either as in Drosophila by modifier genes or by accumulation on the short arm of the X, only of female determining factors which do not require dosage compensation.

Inter- and intraspecific heterochromatin variation detected by restriction endonuclease digestion in two sibling species of the Anopheles maculipennis complex

The sibling species Anopheles atroparvus and Anopheles labranchiae are cytogenetically almost indistinguishable. The chromosome complement (2n = 6) consists of two pairs of autosomes and two heteromorphic sex chromosomes with largely homologous heterochromatic long arms. Treatment of chromosome preparations with the restriction endonucleases, Alu I, Hae III, Mbo I, Hpa II, revealed species-specific differences of the sex chromosome banding pattern. These differences involved both amount and location of digested heterochromatin. Heterochromatin heterogeneity and a high level of intraspecific polymorphism, undetected with standard banding techniques, were observed in both species. Quantitative heterochromatin differences between the sex chromosomes did not inhibit their pairing and chiasmata formation. The endonuclease Msp I, which cleaves the same target sequence as Hpa II, did not digest heterochromatic as well as euchromatic regions in both species: inhibition of cleavage by methylation of the target sequence or limited access of the enzyme to the target could be involved in this response.

Restriction endonuclease digestion and chromosome banding in the mosquito, Culiseta longiareolata (Diptera: Culicidae).

Fixed chromosomes of the mosquito, Culiseta longiareolata (2n = 6) were treated in situ with nine restriction endonucleases and stained with ethidium bromide or Giemsa. All the heterochromatic regions were apparently protected from digestion by all enzymes except Mbo I. This enzyme selectively digested one of the three types of heterochromatin present in the species. Staining with the fluorochrome quinacrine after enzyme treatment produced a standard Q-banding pattern or a Hoechst 33258-like pattern, depending on the enzyme. These results confirmed: (a) the presence of three types of heterochromatic containing different DNA fractions in the chromosomes of this species, (b) restriction enzymes accessibility to the DNA of heterochromatin regions, and (c) the selective cleavage of particular DNA fractions without DNA removal. Moreover, quinacrine staining after enzyme digestion proved useful in detecting differential activity among enzymes which produced the same banding pattern with standard dyes.

The mosquitoes of Sardinia: species records 35 years after the malaria eradication campaign

ABSTRACT. 1. Thirty‐five years ago malaria was eradicated from Sardinia by massive application of DDT directed against the mosquito vector species, Anopheles labranchiae, but the long‐term effects on the mosquito fauna have not been investigated previously.

The longitudinal differentiation produced by photo-oxidation and acridine-orange staining in eukaryote chromosomes: role and involvement of DNA base composition

Photo-oxidation and staining with acridine-orange result in a longitudinal green/orange-red/brown-red color differentiation in fixed chromosomes of Drosophila melanogaster, D. virilis, Culiseta longiareolata, and Mus musculus. Such a result has been correlated with specific DNA base sequences situated in specific chromosomal areas. In fact, given the known base composition of satellite DNAs in three of the species we studied, a correspondence seems to exist between molecular hybridization in situ of AT-rich satellite DNAs and the location of specific stain reactions in specific chromosome regions. Moreover, our results support previous findings suggesting preferential guanine destruction as a consequence of photo-oxidation.

Genetic structure and population dynamics of the biting midges Culicoides obsoletus and Culicoides scoticus: implications for the transmission and maintenance of bluetongue

Culicoides species belonging to the Obsoletus complex (Diptera: Ceratopogonidae) have been indicated as primary bluetongue (BT) vectors in many European countries and their possible involvement in the maintenance and overwintering of BT viruses has been suggested, even in regions where Culicoides imicola Keiffer is the main vector. The Obsoletus complex includes two predominant taxa, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle. However, the role played by each species in the epidemiology of BT is still unknown. Taxonomic identification is mainly based on the morphology of male genitalia and the lack of other reliable diagnostic features makes the screening of trap‐collected vector populations, mainly females, particularly difficult. Although molecular markers have facilitated species identification, little information is yet available on the biology, abundance and population dynamics of the two taxa. The aim of this work was to investigate the genetic profile and temporal distribution of C. obsoletus and C. scoticus by using isozyme electrophoresis applied to adult midges, collected weekly at two selected farms in southern Sardinia. A total of nine enzyme loci were analysed and five of them provided diagnostic allozyme markers (Hk, Mdh, Pgi, Idh‐1 and Idh‐2). Neis genetic distance between the two taxa was in the range of other well‐separated taxa (D = 1.792), supporting their status as true species. Culicoides scoticus represented almost 61% of the 562 specimens analysed; its genetic structure was characterized by a very low level of intra‐population variation (mean heterozygosity He = 0.019) and higher genetic divergence between populations (FST = 0.0016) than in C. obsoletus. The latter species had significantly more heterozygotes (He = 0.123), a higher percentage of polymorphic loci, and no inter‐population differentiation (FST≅0). We suggest that different biological and ecological constraints, such as breeding habitat requirements, may contribute to shaping the genetic profiles of C. scoticus and C. obsoletus. However, enough gene flow was maintained between populations of each species as no spatial and temporal structuring was sustained by Fishers exact probability test (P > 0.5). The seasonal distributions of C. scoticus and C. obsoletus only partially overlapped: both species were mainly found early in the year, when the main vector, C. imicola, was present in low numbers, and peaked in abundance in April and May. Culicoides scoticus was predominant until May, decreased rapidly in the following months and increased again in winter, whereas C. obsoletus decreased more slowly and was still present in early summer. Consequently, C. scoticus may be a good candidate for playing a role in the transmission and maintenance of BT virus in Sardinia, as well as in other Mediterranean countries, during the months of late winter and early spring when the seroconversion of sentinel animals is still occurring in the absence of the main vector.

Light-induced banding (LIB) in metaphase chromosomes of Culiseta longiareolata (Diptera: Culicidae)

A technique already used on Drosophila polytene chromosomes was employed on metaphase chromosomes of Culiseta Iongiareolata (Diptera: Culicidae). The coincidence between Q-bands and green fluorescence was pointed out. The centromere regions of all chromosome pairs and one telomere of the Y, although demonstrating uniformly stained C-bands, were shown to differentially stain with Coriphosphine-0 after light treatment. A base destruction mechanism, similar to that proposed for the methylene blue-light-chrtimosomal DNA interaction, has been hypothesized for explaining these findings.

Methylene-blue/coriphosphine-O/acridine-orange, chromosomal DNA and visible light: Interaction and cytological effect in Drosophila melanogaster

Metaphase preparations of Drosophila melanogaster were irradiated with visible light in the presence of either Methylene-blue, Coriphosphine-O or Acridine-Orange. After treatment with any of these three compounds, Coriphosphine-O staining shows an identical green/orange-red/brown-red chromosome pattern. These findings are discussed in the light of previous reports and guanine destruction is hypothesized as representing the most plausible mechanism for explaining the cytological results.

Genetics of sterility ofAnopheles atroparvus × Anopheles labranchiae hybrids

SummaryIt is well established that in crosses betweenAnopheles atroparvus andA. labranchiae, F1 males are sterile while the females are fertile. This paper presents results of backcrossing the F1 females and of further crosses which suggest that the genetic situation causing sterility is the simultaneous presence, in a male, of the Y chromosome of one species and of a gene (or genes) from the X chromosome of the other species.The testes of the F1 male show marked developmental retardation, and at the end of the fourth larval instar, an absence of cells undergoing meiotic divisions. Analyses of the egg laying capacity and of larval survival are also presented. In addition to being sterile, F1 males show higher larval mortality relative to females than among the offspring of the other crosses. These results are discussed and compared with similar results in Drosophila.

Chromosome evolution in treehole breeding Anopheles (Diptera, Culicidae)

Abstract The cytogenetic profile of two Nearctic and one Paleartic treehole mosquitoes belonging to the Coelodiazesis group, Anopheles barberi, Anopheles judithae, and Anopheles plumbeus, was outlined by means of standard banding techniques and in situ hybridization. Results indicate that, during the evolution of this species group, several rearrangements have occurred including inversions, translocations, and loss of heterochromatic material and rRNA genes copy number. The karyotype of A. barberi is markedly different from the other two species, in spite of morphological similarities with the other North American species, A. judithae. This latter species has retained part of a more ancestral karyotype still shared with the Paleartic A. plumbeus. Chromosomal repat‐terning in A. barberi has probably facilitated isolation and divergence of the two Nearctic species, impairing introgressive hybridization with A. judithae.