Complementary DNA-DNA hybridization in Drosophila

Summary We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.On leave from the Dipartimento di Biologia, II Università di Roma Tor Vergata, Rome, Italy     

The molecular evolution of four anti-malarial immune genes in the <Emphasis Type="Italic">Anopheles gambiae</Emphasis> species complex

Background  

If the insect innate immune system is to be used as a potential blocking step in transmission of malaria, then it will require targeting one or a few genes with highest relevance and ease of manipulation. The problem is to identify and manipulate those of most importance to malaria infection without the risk of decreasing the mosquito's ability to stave off infections by microbes in general. Molecular evolution methodologies and concepts can help identify such genes. Within the setting of a comparative molecular population genetic and phylogenetic framework, involving six species of the Anopheles gambiae complex, we investigated whether a set of four pre-selected immunity genes (gambicin, NOS, Rel2 and FBN9) might have evolved under selection pressure imposed by the malaria parasite.     

Nondenaturing protein electrotransfer of the esterase activity of lipolytic preparations

Lipolytic enzymes subjected to nondenaturing polyacrylamide gel electrophoresis were electrophoretically transferred under nondenaturing conditions onto a solid-state matrix. Electrotransfer permitted the visualization of hydrolytic activity to the long-chain water insoluble substrate alpha-naphthyl palmitate. Four commercial preparations: a lipase from Candida cylindracea, an esterase from porcine liver, a lipase from Pseudomonas sp., and a cholesterol esterase from Pseudomonas fluorescens were examined.     

Italian neo-endemism in a widespread group of leafhoppers insects: A revision of the Eupteryx aurata group (Auchenorrhyncha: Cicadellidae: Typhlocybinae) using morphology,ecology and genetics

The Eupteryx aurata group is characterised by several features, primarily by colouration and aedeagus morphology. It is distributed mainly in Europe, with some species also in Asia and one species perhaps in North Africa. At least one species is introduced in North America. Until now 12 taxa were known. Two new species, Eupteryx divulsa from central Italy and Eupteryx oscorum from central and southern Italy, are described in this paper. Eupteryx petasitidis Ferrari, 1882, described as Eupteryx carpini petasitidis and later synonymised with Eupteryx atropunctata (Goeze, 1778), is reestablished on species level and redescribed including genital morphology. Ten taxa of the species group are studied in relation to their morphological and molecular characters. The colouration, aedeagus, and pygofer appendage are figured for all of these taxa. Molecular study showed that the E. aurata group is monophyletic. The little or moderate distances in sequences (below 6.0% for mtDNA and up to 2.5% for nuclear ITS2) between the different taxa of the E. aurata group and incongruence of genetic signals from mtDNA and nuclear DNA could be the result of incomplete lineage sorting in young species and/or hybridisation and introgression of DNA among taxa. Of particular importance in this study is material from Italy, a peripheral region in relation to the distribution of the species group and due to its geomorphological features favouring isolation and speciation. A distribution map for the E. aurata species group in Italy and an identification key for all 15 taxa of the aurata group are given. Phylogenetic relationships within the aurata group are discussed in relation to morphological, molecular, ecological, and zoogeographic aspects.     

Lineage fusion in Galápagos giant tortoises

Although many classic radiations on islands are thought to be the result of repeated lineage splitting, the role of past fusion is rarely known because during these events, purebreds are rapidly replaced by a swarm of admixed individuals. Here, we capture lineage fusion in action in a Galápagos giant tortoise species, Chelonoidis becki, from Wolf Volcano (Isabela Island). The long generation time of Galápagos tortoises and dense sampling (841 individuals) of genetic and demographic data were integral in detecting and characterizing this phenomenon. In C. becki, we identified two genetically distinct, morphologically cryptic lineages. Historical reconstructions show that they colonized Wolf Volcano from Santiago Island in two temporally separated events, the first estimated to have occurred ~199 000 years ago. Following arrival of the second wave of colonists, both lineages coexisted for approximately ~53 000 years. Within that time, they began fusing back together, as microsatellite data reveal widespread introgressive hybridization. Interestingly, greater mate selectivity seems to be exhibited by purebred females of one of the lineages. Forward‐in‐time simulations predict rapid extinction of the early arriving lineage. This study provides a rare example of reticulate evolution in action and underscores the power of population genetics for understanding the past, present and future consequences of evolutionary phenomena associated with lineage fusion.     

Spatial and temporal occurrence of Rhabdostyla cf. chironomi Kahl, 1933 (Ciliophora, Peritrichia) as an epibiont on chironomid larvae in a lotic system in the neotropics

In order to show how environmental factors could influence the spatial and temporal distribution of epibiosis, we investigated the occurrence of the ciliate Rhabdostyla cf. chironomi (Ciliophora, Peritrichia) attached to Chironomidae larvae in an urban stream in southeast Brazil. Samples were collected monthly for 1-year period at five sampling stations along the stream. Among the 24 genera analyzed, the genus Chironomus was the most abundant, with 16.84% carrying R. cf. chironomi. This epibiotic relationship was only recorded in sample stations located in a heavily populated urban area of the stream. Statistical analyses demonstrated significant correlations between infestation prevalence and environmental parameters such as rainfall, bacterial density, and host abundance. The occurrence of epibionts on the larvae was spatially and temporally heterogeneous. The results are discussed in terms of possible factors related to the heterogeneous spatial and temporal occurrence. Greater understanding of the ecological factors governing the occurrence of the epibiont–host relationship is essential to establish future use of this relationship as water quality indicator in lotic systems.     

DNA Minor Groove Induced Dimerization of Heterocyclic Cations: Compound Structure, Binding Affinity, and Specificity for a TTAA Site

With the increasing number and variations of genome sequences available, control of gene expression with synthetic, cell-permeable molecules is within reach. The variety of sequence-specific binding agents is, however, still quite limited. Many minor groove binding agents selectivity recognize AT over GC sequences but have less ability to distinguish among different AT sequences. The goal with this article is to develop compounds that can bind selectively to different AT sequences. A number of studies indicate that AATT and TTAA sequences have significantly different physical and interaction properties and different requirements for minor groove recognition. Although it has been difficult to get minor groove binding at TTAA, DB293, a phenyl-furan-benzimidazole diamidine, was found to bind as a strong, cooperative dimer at TTAA but with no selectivity over AATT. In order to improve selectivity, we made modifications to each unit of DB293. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1003, a furan-furan-benzimidazole diamidine, binds strongly to TTAA as a dimer and has selectivity (K_TTAA/K_AATT = 6). CD and DNase I footprinting studies confirmed the preference of this compound for TTAA. In summary, (i) a favorable stacking surface provided by the pi system, (ii) H-bond donors to interact with TA base pairs at the floor of the groove provided by a benzimidazole (or indole) -NH and amidines, and (iii) appropriate curvature of the dimer complex to match the curvature of the minor groove play important roles in differentiating the TTAA and AATT minor grooves.     

Ancient DNA forces reconsideration of evolutionary history of Mediterranean pygmy elephantids

During the Pleistocene pygmy elephantids, some only a quarter of their ancestors' size, were present on Mediterranean islands until about 10,000 years ago (y.a.). Using a new methodology for ancient DNA (aDNA) studies, the whole genomic multiple displacement amplification method, we were able to retrieve cytochrome b (cytb) DNA fragments from 4200 to 800,000 y.a. specimens from island and mainland samples, including pygmy and normal-sized forms. The short DNA sequence (43 bp) retrieved from the 800,000 y.a. sample is one of the oldest DNA fragment ever retrieved. Duplication of the experiments in two laboratories, the occurrence of three diagnostic sites and the results of the phylogenetic analyses strongly support its authenticity. Our results challenge the prevailing view that pygmy elephantids of the eastern Mediterranean originated exclusively from Elephas, suggesting independent histories of dwarfism and the presence of both pygmy mammoths and elephant-like taxa on these islands. Based on our molecular data, the origin of the Tilos and Cyprus elephantids from a lineage within the genus Elephas is confirmed, while the DNA sequence from the Cretan sample falls clearly within the mammoth clade. Thus, the name Mammuthus creticus rather than Elephas creticus, seems to be justified for this form. Our findings also suggest a need to re-evaluate the evolutionary history of the Sicilian/Maltese species, traditionally included in the genus Elephas.     

Urban population genetics of slum‐dwelling rats (Rattus norvegicus) in Salvador,Brazil

Throughout the developing world, urban centres with sprawling slum settlements are rapidly expanding and invading previously forested ecosystems. Slum communities are characterized by untended refuse, open sewers and overgrown vegetation, which promote rodent infestation. Norway rats (Rattus norvegicus) are reservoirs for epidemic transmission of many zoonotic pathogens of public health importance. Understanding the population ecology of R. norvegicus is essential to formulate effective rodent control strategies, as this knowledge aids estimation of the temporal stability and spatial connectivity of populations. We screened for genetic variation, characterized the population genetic structure and evaluated the extent and patterns of gene flow in the urban landscape using 17 microsatellite loci in 146 rats from nine sites in the city of Salvador, Brazil. These sites were divided between three neighbourhoods within the city spaced an average of 2.7 km apart. Surprisingly, we detected very little relatedness among animals trapped at the same site and found high levels of genetic diversity, as well as structuring across small geographical distances. Most F_ST comparisons among sites were statistically significant, including sites <400 m apart. Bayesian analyses grouped the samples in three genetic clusters, each associated with distinct sampling sites from different neighbourhoods or valleys within neighbourhoods. These data indicate the existence of complex genetic structure in R. norvegicus in Salvador, linked to the heterogeneous urban landscape. Future rodent control measures need to take into account the spatial and temporal linkage of rat populations in Salvador, as revealed by genetic data, to develop informed eradication strategies.     

Induced topological changes in DNA complexes: influence of DNA sequences and small molecule structures

Heterocyclic diamidines are compounds with antiparasitic properties that target the minor groove of kinetoplast DNA. The mechanism of action of these compounds is unknown, but topological changes to DNA structures are likely to be involved. In this study, we have developed a polyacrylamide gel electrophoresis-based screening method to determine topological effects of heterocyclic diamidines on four minor groove target sequences: AAAAA, TTTAA, AAATT and ATATA. The AAAAA and AAATT sequences have the largest intrinsic bend, whereas the TTTAA and ATATA sequences are relatively straight. The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA. A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA. All compounds decrease the mobility of the ATATA sequence that is consistent with decreased minor groove width and bending of the relatively straight DNA into the minor groove. Similar, but generally smaller, effects are seen with TTTAA. The intrinsically bent AAAAA and AAATT sequences, which have more narrow minor grooves, have smaller mobility changes on binding that are consistent with increased or decreased bending depending on compound structure.