Emiliano Mancini

The "far-west" of Anopheles gambiae molecular forms.

The main Afrotropical malaria vector, Anopheles gambiae sensu stricto, is undergoing a process of sympatric ecological diversification leading to at least two incipient species (the M and S molecular forms) showing heterogeneous levels of divergence across the genome. The physically unlinked centromeric regions on all three chromosomes of these closely related taxa contain fixed nucleotide differences which have been found in nearly complete linkage disequilibrium in geographic areas of no or low M-S hybridization. Assays diagnostic for SNP and structural differences between M and S forms in the three centromeric regions were applied in samples from the western extreme of their range of sympatry, the only area where high frequencies of putative M/S hybrids have been reported. The results reveal a level of admixture not observed in the rest of the range. In particular, we found: i) heterozygous genotypes at each marker, although at frequencies lower than expected under panmixia; ii) virtually all possible genotypic combinations between markers on different chromosomes, although genetic association was nevertheless detected; iii) discordant M and S genotypes at two X-linked markers near the centromere, suggestive of introgression and inter-locus recombination. These results could be indicative either of a secondary contact zone between M and S, or of the maintenance of ancestral polymorphisms. This issue and the perspectives opened by these results in the study of the M and S incipient speciation process are discussed.

The EU protected taxon Morimus funereus Mulsant, 1862 (Coleoptera: Cerambycidae) and its western Palaearctic allies: systematics and conservation outcomes

Morimus funereus is a large longhorn beetle included in the European Habitats Directive and in previous releases of the IUCN red list. It represents a flagship species of old-growth forest saproxylic communities in E and SE Europe. The morphologically based taxonomy of W Palaearctic Morimus is rather unstable due to high phenetic intrapopulational and geographic variability and different authors have attempted to recognise one to five different taxa of specific/subspecific rank. No previous molecular data are available for the genus Morimus. Here, for the first time, a molecular approach based on COI and ITS2 gene sequences was applied in European and Anatolian Morimus specimens. The genetic variability among Euro-Anatolian Morimus populations and the geographical structure suggest that they can not be ascribed to the currently accepted five W Palaearctic Morimus species and may actually represent a single, genetically and morphologically variable biological species (M. asper), highlighting the necessity of an extended taxonomical revision. In light of these results, a phylogeographical hypothesis of postglacial colonisation of the central Mediterranean area has been developed and the consequences of this new taxonomic arrangement regarding conservation strategies for “Morimus funereus” and allied taxa in Europe and Turkey are discussed.

Comparative analyses reveal discrepancies among results of commonly used methods for Anopheles gambiae molecular form identification

BackgroundAnopheles gambiae M and S molecular forms, the major malaria vectors in the Afro-tropical region, are ongoing a process of ecological diversification and adaptive lineage splitting, which is affecting malaria transmission and vector control strategies in West Africa. These two incipient species are defined on the basis of single nucleotide differences in the IGS and ITS regions of multicopy rDNA located on the X-chromosome. A number of PCR and PCR-RFLP approaches based on form-specific SNPs in the IGS region are used for M and S identification. Moreover, a PCR-method to detect the M-specific insertion of a short interspersed transposable element (SINE200) has recently been introduced as an alternative identification approach. However, a large-scale comparative analysis of four widely used PCR or PCR-RFLP genotyping methods for M and S identification was never carried out to evaluate whether they could be used interchangeably, as commonly assumed.ResultsThe genotyping of more than 400 A. gambiae specimens from nine African countries, and the sequencing of the IGS-amplicon of 115 of them, highlighted discrepancies among results obtained by the different approaches due to different kinds of biases, which may result in an overestimation of MS putative hybrids, as follows: i) incorrect match of M and S specific primers used in the allele specific-PCR approach; ii) presence of polymorphisms in the recognition sequence of restriction enzymes used in the PCR-RFLP approaches; iii) incomplete cleavage during the restriction reactions; iv) presence of different copy numbers of M and S-specific IGS-arrays in single individuals in areas of secondary contact between the two forms.ConclusionsThe results reveal that the PCR and PCR-RFLP approaches most commonly utilized to identify A. gambiae M and S forms are not fully interchangeable as usually assumed, and highlight limits of the actual definition of the two molecular forms, which might not fully correspond to the two A. gambiae incipient species in their entire geographical range. These limits are discussed and operational suggestions on the choice of the most convenient method for large-scale M- and S-form identification are provided, also taking into consideration technical aspects related to the epidemiological characteristics of different study areas.

Molecular evolution of a gene cluster of serine proteases expressed in the Anopheles gambiae female reproductive tract

BackgroundGenes involved in post-mating processes of multiple mating organisms are known to evolve rapidly due to coevolution driven by sexual conflict among male-female interacting proteins. In the malaria mosquito Anopheles gambiae - a monandrous species in which sexual conflict is expected to be absent or minimal - recent data strongly suggest that proteolytic enzymes specifically expressed in the female lower reproductive tissues are involved in the processing of male products transferred to females during mating. In order to better understand the role of selective forces underlying the evolution of proteins involved in post-mating responses, we analysed a cluster of genes encoding for three serine proteases that are down-regulated after mating, two of which specifically expressed in the atrium and one in the spermatheca of A. gambiae females.ResultsThe analysis of polymorphisms and divergence of these female-expressed proteases in closely related species of the A. gambiae complex revealed a high level of replacement polymorphisms consistent with relaxed evolutionary constraints of duplicated genes, allowing to rapidly fix novel replacements to perform new or more specific functions. Adaptive evolution was detected in several codons of the 3 genes and hints of episodic selection were also found. In addition, the structural modelling of these proteases highlighted some important differences in their substrate specificity, and provided evidence that a number of sites evolving under selective pressures lie relatively close to the catalytic triad and/or on the edge of the specificity pocket, known to be involved in substrate recognition or binding. The observed patterns suggest that these proteases may interact with factors transferred by males during mating (e.g. substrates, inhibitors or pathogens) and that they may have differently evolved in independent A. gambiae lineages.ConclusionsOur results - also examined in light of constraints in the application of selection-inference methods to the closely related species of the A. gambiae complex - reveal an unexpectedly intricate evolutionary scenario. Further experimental analyses are needed to investigate the biological functions of these genes in order to better interpret their molecular evolution and to assess whether they represent possible targets for limiting the fertility of Anopheles mosquitoes in malaria vector control strategies.

HIV Reservoirs and Immune Surveillance Evasion Cause the Failure of Structured Treatment Interruptions: A Computational Study

Continuous antiretroviral therapy is currently the most effective way to treat HIV infection. Unstructured interruptions are quite common due to side effects and toxicity, among others, and cannot be prevented. Several attempts to structure these interruptions failed due to an increased morbidity compared to continuous treatment. The cause of this failure is poorly understood and often attributed to drug resistance. Here we show that structured treatment interruptions would fail regardless of the emergence of drug resistance. Our computational model of the HIV infection dynamics in lymphoid tissue inside lymph nodes, demonstrates that HIV reservoirs and evasion from immune surveillance themselves are sufficient to cause the failure of structured interruptions. We validate our model with data from a clinical trial and show that it is possible to optimize the schedule of interruptions to perform as well as the continuous treatment in the absence of drug resistance. Our methodology enables studying the problem of treatment optimization without having impact on human beings. We anticipate that it is feasible to steer new clinical trials using computational models.

A simulation framework to investigate in vitro viral infection dynamics

Abstract Virus infection is a complex biological phenomenon for which in vitro experiments provide a uniquely concise view where data is often obtained from a single population of cells, under controlled environmental conditions. Nonetheless, data interpretation and real understanding of viral dynamics is still hampered by the sheer complexity of the various intertwined spatio-temporal processes. In this paper we present a tool to address these issues: a cellular automata model describing critical aspects of in vitro viral infections taking into account spatial characteristics of virus spreading within a culture well. The aim of the model is to understand the key mechanisms of SARS-CoV infection dynamics during the first 24h post infection. Using a simulated annealing algorithm we tune free parameters with data from SARS-CoV infection of cultured lung epithelial cells. We also interrogate the model using a Latin Hypercube sensitivity analysis to identify which mechanisms are critical to the observed infection of host cells and the release of measured virus particles.

Modeling HIV-1 intracellular replication: two simulation approaches

Many mathematical and computational models have been developed to investigate the complexity of HIV dynamics, immune response and drug therapy. However, there are not many models which consider the dynamics of virus intracellular replication at a single level. We propose a model of HIV intracellular replication where infected cells undergo a single cycle of virus replication. A cell is modeled as an individual entity with certain states and properties. The model is stochastic and keeps track of the main viral proteins and genetic materials inside the cell. Two simulation approaches are used for implementing the model: rate-based and diffusion-based approaches. The results of the simulation are discussed based on the number of integrated viral cDNA and the number of viral mRNA transcribed after a single round of replication. The model is validated by comparing simulation results with available experimental data. Simulation results give insights about the details of HIV replication dynamics inside the cell at the protein level. Therefore the model can be used for future studies of HIV intracellular replication in vivo and drug treatment.

Molecular and morphological evidence of a new sibling species of Calobius (Coleoptera: Hydraenidae) of the C. quadricollis complex from peninsular Italy

A molecular and morphological analysis was performed to clarify the taxonomic status of Italian members of the Calobius quadricollis complex (Coleoptera, Hydraenidae, Ochthebiinae), including species strictly associated with hypersaline marine rock pools along the Mediterranean and Macaronesian coasts. The analysis was mainly focused on the specific distinction and formal description of a new species, Calobius urbanelliae n. sp., from peninsular Italy. This species is of difficult morphological distinction, but, on the basis of the mitochondrial DNA marker cytochrome c oxidase subunit I, it is highly differentiated from C. quadricollis Mulsant, 1844 (NW Mediterranean) and C. steinbuehleri Baudi, 1864 (NE Mediterranean), which are partially sympatric with it along part of the western and eastern coasts of peninsular Italy. One possible palaeogeographical scenario underlying the specific differentiation of the three species is briefly discussed.

Redescription and natural history of Meligethes longulus Schilsky, 1894, and provisional revision of the M. coracinus species‐complex (Coleoptera, Nitidulidae, Meligethinae)

Abstract The taxonomic identification and a re‐description of the problematic Meligethes longulus Schilsky, 1894 from eastern Turkey are presented. This species, belonging to the M. coracinus complex and known so far on the basis of a single immature holo‐type, was repeatedly confused in the past with other closely related taxa, thus introducing considerable instability in the nomenclature and taxonomy of the whole group, involving several distinct biological species throughout southern Europe and western Asia. The recent re‐discovery of this species in eastern Turkey allowed M. longulus to be definitely identified as being an extremely rare and localized species, with larval stages strictly associated with the host‐plant Matthiola odoratissima (Bieb.) R.Br. (Brassicaceae), in dry exposed terrigenous and sandy hill slopes of eastern Turkey. An updated key to the identification of both described and un‐described members of the M. coracinus complex is also presented, and a rather isolated species of the M. coracinus complex from southern Turkey, M. cristofaroi n. sp., is described as new. A preliminary evolutionary scenario of the whole species‐complex, based also on results from a companion molecular research, is finally discussed.

Computer-aided photographic identification of Rosalia alpina (Coleoptera: Cerambycidae) applied to a mark-recapture study

Assessing the conservation status of protected species needs quantitative population data, generally obtained using Capture‐Mark‐Recapture methods (CMR). The exploitation of natural marking (e.g. individual morphological traits) offers an interesting alternative, based on image analyses, which may result in a less manipulation of protected species compared to the typical artificial marking method. In our 2‐year CMR study, we tested for the first time in the natural setting the feasibility and the application of the computer‐aided photographic identification method of Rosalia alpina using the individual elytral spots as the natural marking. The I3SC software was used for the photographic analysis. Data were collected from populations of two National Parks of central Italy during July–August in 2014 and 2015. We developed a standard procedure in order to optimise the image acquisition in the field and to acquire clear and comparable images, facilitating the I3SC screening process. The results demonstrated that the computer‐aided photographic identification of natural markings can be implemented in a CMR population study of R. alpina. Our image processing approach showed that using only the elytral central spot contours made the tracing contour process less time‐consuming obtaining reliable results. Furthermore, I3SC output scores were used to identify a threshold value for the identification of new individuals or recaptures, facilitating the final identification proposed by operators. Finally, we assessed the possibility of performing the methodology using a Citizen Science approach.