M. Marinucci

Phylogenetic relationships of seven palearctic members of the maculipennis complex inferred from ITS2 sequence analysis

The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined from seven palearctic mosquitoes species belonging to the Anopheles maculipennis species complex, namely An. atroparvus, An. labranchiae, An. maculipennis, An. messeae, An. melanoon, An. sacharovi and An. martinius. The length of the ITS2 ranged from 280 to 300 bp, with a GC content of 49.4–54.1%. With the exception of An. messeae, negligible levels of intraspecific polymorphism and no intrapopulation variation were observed. The phylogenetic relationships among the members of the maculipennis complex were inferred by maximum‐parsimony analysis of the paup program and the neighbour‐joining and maximun‐likelihood analysis of the phylip program. All of the trees obtained were almost identical in topology, although the relationships among three species, i.e. An. maculipennis, An. messeae and An. melanoon, remained unresolved. The phylogenies were in good agreement with the previous gene–enzyme and polytene chromosome banding pattern studies.

Phylogenetic analysis of Phlebotomus species belonging to the subgenus Larroussius (Diptera, Psychodidae) by ITS2 rDNA sequences

In the genealogy of Phlebotomus (Diptera: Psychodidae), morphological analyses have indicated that the subgenus Larroussius is a monophyletic group which is most closely related to the subgenera Transphlebotomus and Adlerius. We conducted a phylogenetic analysis of the relationships among six representative species of the subgenus Larroussius and one species representatitive of the Phlebotomus subgenus, assessing sequences of the Second Internal Transcribed Spacer (ITS2) of the ribosomal RNA (rRNA). Three of the species (P. perniciosus, P. ariasi and P. perfiliewi perfiliewi) were collected in different parts of the Mediterranean area. The trees estimated from parsimony and neighbour-joining analyses supported the monophyly of the Larroussius subgenus inferred from the morphological analysis. According to our data, P. ariasi may be a sister group to the rest of the Larroussius subgenus, although additional sequence data are needed to confirm this observation. Our results suggest that P. perniciosus and P. longicuspis are distinct species, in spite of the fact that there are only slight morphological differences. The strict congruence between the phylogeny of the Larroussius subgenus inferred from the ITS2 sequences and that based on morphological studies further confirmed the ability of the spacer sequence to identify recently-derived affiliations.

Heterocellular hereditary persistence of fetal hemoglobin (HPFH). Molecular mechanisms of abnormal γ-gene expression in association with β thalassemia and linkage relationship with the β-globin gene cluster

SummaryWe report a study of four families of Italian origin in which heterocellular HPFH is inherited linked to β thalassemia over two or three generations. The HPFH + β thalassemia carriers showed thalassemic blood pictures and elevated HbF and F-cell number without increase in the HbF/F-cell content. Association of this gene complex with a second β thalassemia trait gives rise to a mild clinical picture characterized by 9–12 g/dl of mainly HbF in peripheral blood and no transfusion requirement. In two families, independent segregation of the HPFH or β-thal trait was observed, and in one case the study of the DNA polymorphisms within the γδβ gene cluster indicated that the HPFH mutation lies outside that DNA region. In one family the coexistence of a polymorphic variant of the Aγ chain (the AγT chain) allowed us to demonstrate that the increased γ chain synthesis caused by the heterocellular HPFH determinant is directed by both chromosomes.

Intrapopulation Polymorphism in Anopheles messeae (An. maculipennis Complex) Inferred by Molecular Analysis

Abstract We evaluated the internal transcribed spacer two (ITS2) sequence to detect intraspecific polymorphism in the Palearctic Anopheles maculipennis complex, analyzing 52 populations from 12 countries and representing six species. For An. messeae, two fragments of the cytochrome oxidase I (COI) gene were also evaluated. The results were compared with GenBank sequences and data from the literature. ITS2 analysis revealed evident intraspecific polymorphism for An. messeae and a slightly less evident polymorphism for An. melanoon, whereas for each of the other species, 100% identity was found among populations. ITS2 analysis of An. messeae identified five haplotypes that were consistent with the geographical origin of the populations. ITS2 seems to be a reliable marker of intraspecific polymorphism for this complex, whereas the COI gene is apparently uninformative.

Identification of the sibling species of the Anopheles maculipennis complex by heteroduplex analysis.

The group of anopheline mosquitoes referred to as ‘Anopheles maculipennis complex’ includes the most important malaria vectors of the Palearctic Western region. The species belonging to this complex, however, are difficult or impossible to distinguish by morphological characters. To differentiate sibling palearctic species belonging to this complex, interspecific differences in the ITS2 sequences were used to set up a rapid and sensitive diagnostic tool based on heteroduplex analysis. The relative heteroduplex mobility allowed the following seven species to be readily distinguished: An. atroparvus, An. labranchiae, An. maculipennis s.s., An. martinius, An melanoon, An. messeae and An. sacharovi.

Esterases A5‐B5 in organophosphate‐resistant Culex pipiens from Italy

Abstract. Culex pipiens mosquitoes from Lignano city, Udine province, northeast Italy, were found to carry over‐produced non‐specific esterases Al, A2‐B2 and A4‐B4 or A5‐B5, detected by starch gel electrophoresis, giving multiple resistance to organophosphorus insecticides. In order to differentiate between A4‐B4 and A5‐B5 esterases, the latter known only from Cyprus whereas the former is widespread in Italy and elsewhere, restriction fragment length polymorphism (RFLP) analysis was performed at the esterase B locus. Both B4 and B5 haplotypes were found. This is the first record of A5‐B5 esterase‐mediated resistance in continental Europe.

Haemoglobin Lepore trait: haematological and structural studies on the Italian population.

Summary. Haematological data on 59 heterozygotes for haemoglobin (Hb) Lepore and 10 double heterozygotes for Hb Lepore and β thalassaemia from 36 Italian families are reported. The red cell indices are defined and compared with those of groups of non‐thalassaemic and β thalassaemic subjects of comparable number, age and sex distribution. The relative level of each haemoglobin fraction and the absolute production of single polypeptide chains are calculated in order to compare the expression of the non‐α chain genes in Hb Lepore trait and β thalassaemia. Structural studies demonstrate that the haemoglobin Lepore is of the Boston type (δ87β116) in all subjects, confirming that this type of fusion variant is probably the only one which occurs in Mediterranean populations. The distribution and incidence of the Lepore haemoglobinopathy are discussed.

Possible duplication of the hemoglobin α chain locus in sheep

Only one type of alpha chain has been described so far in the hemoglobins of adult domestic sheep. A variant (Hb D) of the alpha chain, characterized by a substitution glycine leads to aspartic acid at position 15, has been described in Yugoslavian sheep. In this paper we report the identification of a second alpha chain (alpha 2), observed in several sheep when the globin was analyzed by CM-cellulose chromatography or the total hemolysate submitted to isoelectric focusing. The ratio of this chain to the usual one (alpha 1) in the globin of different animals is equal to either 1 : 2 or 1 : 4. The structural difference between alpha 1 and alpha 2 chains consists in the replacement of a leucine residue by an histidine in the position 113 or 114 of the polypeptide chain. Preliminary data on the frequency of the alpha 2 chain in eight domestic breeds indicate that this chain is fairly common, being present in 15 out of 40 animals examined. The results of breeding experiments between sheep of an appropriate alpha chain phenotype suggest the possibility of a duplication of the hemoglobin alpha locus in the Ovinae.

A new abnormal human hemoglobin: Hb prato (α231 (B12) Arg→Ser β2)

Abstract An abnormal human hemoglobin was found in a hemolysate from a 5-year-old healthy child living in Prato (Tuscany, Italy). Structural studies demonstrated a previously unreported amino acid substitution, α31 (B12) Arg → Ser (this is an α 1β1 contact). The new variant has been named Hb Prato. It was unstable in isopropanol and heat-denaturation tests, but has normal functional properties, with respect to whole blood studies. Family studies indicated that the variant had been inherited from the mother, a 39-year-old woman of Sicilian extraction. Hb Prato occurs at 20 and 28% in hemolysates from the boy and woman, respectively.

Hemoglobin Maputo : A New 6-Chain Variant (α2β2 47 (Cd6) Asp→Tyr) in Combination with Hemoglobin S, Identified by High Performance Liquid Chromatography (Hplc)

During a routine hematological investigation, a slowly-moving hemoglobin variant was detected in a 2-year-old child from Maputo (Mozambique) in combination with hemoglobin S. Structural studies carried out by HPLC demonstrated a previously unreported amino acid substitution, β 47 (CD6) Asp→Tyr. The new hemoglobin variant has been named hemoglobin Maputo.