Background: Aedes albopictusis a very invasive mosquito, which has recently colonized tropical and temperate regions worldwide. Of concern is its role in the spread of emerging or re-emerging mosquito-borne diseases.Ae. albopictusfrom south-western Europe and Brazil were studied to infer genetic and phenetic diversity at intra-individual, intra-population and inter-population levels, and to analyse its spread.Methods: Genotyping was made by rDNA 5.8S-ITS-2 and mtDNAcox1 sequencing to assess haplotype and nucleotide diversity, genetic distances and phylogenetic networks. Male and female phenotyping included combined landmark-and outlined-based geometric morphometrics of wing size and shape.Results: Specimens from seven populations from Spain, France and Brazil provided 12cox1 and 162 5.8S-ITS-2 haplotypes, with great genetic variability difference between both markers (0.9% vs 31.2%). Fivecox1 haplotypes were shared with other countries, mainly Italy, USA and China, but none was shared between Europe and Brazil. The 5.8S-ITS-2 showed 2–7 intra-individual (mean 4.7) and 16–34 intra-/inter-population haplotypes (24.7), including haplotypes shared between Spain, France and Brazil. A 4.3% of ITS-2 haplotypes were shared, mainly with Italy, USA and Thailand, evidencing worldwide spread and introductions from areas where recent outbreaks ofAe. albopictus-transmitted pathogens occurred. Wing size showed sex differences. Wing shape distinguished between Brazilian and European specimens. Both genetic and morphometric markers showed differences between insular Spain and continental Spain, France and Brazil.Conclusions: ITS-2 proves to be a useful marker to assessAe. albopictusspread, providing pronouncedly more information thancox1, including intra-individual, intra-population and inter-population levels, furnishing a complete overview of the evolutionary exchanges followed by this mosquito. Wing morphometry proves to be a useful phenotyping marker, allowing to distinguish different populations at the level of both male and female specimens. Results indicate the need for periodic surveillance monitorings to verify that noAe. albopictuswith high virus transmission capacity is introduced into Europe.Graphical

Species usually develop reproductive isolation mechanisms allowing them to avoid interbreeding. These preventive barriers can act before reproduction, “pre-zygotic barriers”, or after reproduction, “post-zygotic barriers”. Pre-zygotic barriers prevent unfavourable mating, while post-zygotic barriers determine the viability and selective success of the hybrid offspring. Hybridization in parasites and the underlying reproductive isolation mechanisms maintaining their genetic integrity have been overlooked. Using an integrated approach this work aims to quantify the relative importance of pre-zygotic barriers in Schistosoma haematobium x S. bovis crosses. These two co-endemic species cause schistosomiasis, one of the major debilitating parasitic diseases worldwide, and can hybridize naturally. Using mate choice experiments we first tested if a specific mate recognition system exists between both species. Second, using RNA-sequencing we analysed differential gene expression between homo- and hetero-specific pairing in male and female adult parasites. We show that homo- and hetero-specific pairing occurs randomly between these two species, and few genes in both sexes are affected by hetero-specific pairing. This suggests that i) mate choice is not a reproductive isolating factor, and that ii) no pre-zygotic barrier except spatial isolation “by the final vertebrate host” seems to limit interbreeding between these two species. Interestingly, among the few genes affected by the pairing status of the worms, some can be related to pathways affected during male and female interactions and may also present interesting candidates for species isolation mechanisms and hybridization in schistosome parasites.

Schistosomiasis is a Neglected Tropical Disease caused by trematode species of the genus Schistosoma. Both, autochthonous and imported cases of urogenital schistosomiasis have been described in Europe. The present study focuses on eggs, considered pure S. haematobium by genetic characterisation (intergenic ITS region of the rDNA and cox1 mtDNA). A phenotypic characterisation of S. haematobium eggs was made by morphometric comparison with experimental populations of S. bovis and S. mansoni, to help in the diagnosis of S. haematobium populations infecting sub-Saharan migrants in Spain. Analyses were made by Computer Image Analysis System (CIAS) applied on the basis of new standardised measurements and geometric morphometric tools. The principal component analysis (PCA), including seventeen non-redundant measurements, showed three phenotypic patterns in eggs of S. haematobium, S. bovis and S. mansoni. PCA showed that the S. bovis population presented a large egg size range with a pronouncedly larger maximum size. Similarly, S. bovis shows bigger spine values than S. haematobium. Mahalanobis distances between each pair of groups were calculated for each discriminant analysis performed. In general, S. mansoni and S. bovis present larger distances between them than with S. haematobium, i.e. they present the greatest differences. Regarding the spine, S. haematobium and S. mansoni are the most distant species. Results show the usefulness of this methodology for the phenotypic differentiation between eggs from these Schistosoma species, capable of discerning morphologically close eggs, as is the case of the haematobium group. Schistosoma egg phenotyping approaches may be applied to assess not only hybrid forms but also potential influences of a variety of other factors.

Mitochondrial cox 1 and nuclear ribosomal ITSs are commonly combined to distinguish Schistosoma species and identify hybrids in endemic countries but very rarely applied to patients diagnosed in Europe despite the increasing arrival of migrants in southwestern Europe. To assess whether those migrants are carriers of pure or hybrid schistosomes, a complete genetic characterization of Schistosoma entering Spain is performed. A total of 759 eggs (from urine + stools) from 58 patients from 8 African countries were individually processed to describe their mito‐nuclear signature by cox 1 rapid diagnostic multiplex one‐step polymerase chain reaction (RD‐PCR) and ITS‐2/18S sequencing and haplotype identification by means of the complete ITS1‐5.8S‐ITS2 rDNA and cox 1 sequencing. Combined nuclear and mitochondrial DNA markers in sub‐Saharan migrants residing in Spain are described for the first time. Twenty‐two (40.74%) patients were simultaneously carrying pure and hybrid eggs in their urine. S chistosoma haematobium × S. bovis (68.18%) and S. haematobium × S. curassoni (31.82%) hybrid combinations were the most frequent. Six (one pure and five hybrid) and two (pure) mito‐nuclear signatures, in urine and stools, respectively, and 12 nuclear and 61 mitochondrial imported haplotypes were found. This study highlights the genetic complexity of pure and hybrid schistosomes that enter Spain, and consequently Europe, and contributes to the following: correlate the geographical origin of patients with pure and/or hybrid genetic types; detect the presence of hybrids “at distance” (hybrids in migrants from Guinea‐Bissau and Mauritania are first time detected); correlate molecular haplotypes with pathologies, clinical pictures, and treatment responses; and, importantly, warn about possible sources of autochthonous transmission.

Schistosome eggs play a key role in schistosomiasis diagnosis and research. The aim of this work is to morphogenetically study the eggs of Schistosoma haematobium found in sub-Saharan migrants present in Spain, analyzing their morphometric variation in relation to the geographical origin of the parasite (Mali, Mauritania and Senegal). Only eggs considered “pure” S. haematobium by genetic characterization (rDNA ITS-2 and mtDNA cox1) have been used. A total of 162 eggs obtained from 20 migrants from Mali, Mauritania and Senegal were included in the study. Analyses were made by the Computer Image Analysis System (CIAS). Following a previously standardized methodology, seventeen measurements were carried out on each egg. The morphometric analysis of the three morphotypes detected (round, elongated and spindle) and the biometric variations in relation to the country of origin of the parasite on the egg phenotype were carried out by canonical variate analysis. Mahalanobis distances, when all egg measurements were analyzed, showed differences between: (i) Mali-Mauritania, Mali-Senegal and Mauritania-Senegal in the round morphotype; (ii) Mali-Mauritania and Mauritania-Senegal in the elongated morphotype; and (iii) Mauritania-Senegal in the spindle morphotype. Mahalanobis distances, when spine variables were analyzed, showed differences between Mali-Senegal in the round morphotype. In conclusion, this is the first phenotypic study performed on individually genotyped “pure” S. haematobium eggs, allowing the assessment of the intraspecific morphological variations associated with the geographical origin of the schistosome eggs.

Calodium hepaticumis a zoonotic nematode with a worldwide distribution. Although the host range ofC. hepaticumincludes a wide spectrum of mammals (including humans), this parasite is predominantly associated with the families Muridae and Cricetidae. Several Sigmodontinae species from Argentina were found to be infected byC. hepaticum, with a high prevalence inAkodon azarae. The present study focuses onC. hepaticumeggs from natural infection of three species of sigmodontine rodents from Argentina. Eggs were genetically characterized (intergenic 18S rRNA region). The objectives of this work are: (i) to propose a new analytical methodology; and (ii) to morphologically characterizeC. hepaticumeggs, from three Sigmodontinae species (A. azarae,Calomys callidusandOligoryzomys flavescens). Analyses were made by the Computer Image Analysis System based on the new standardized measurements and geometric morphometric tools. The resulting factor maps clearly illustrate global size differences in the parasite eggs from the three Sigmodontinae species analysed. The degree of similarity between egg populations was assessed through pairwise Mahalanobis distances, showing that the largest distances were detected between parasite eggs fromC. callidusandO. flavescens. Herein, the phenotypical plasticity ofC. hepaticumeggs is shown. Significant positive correlations were obtained between each egg parasite principal component 1 and rodent corporal characteristics: weight; liver weight; rodent length; and rodent body condition. The usefulness of the geometric morphometric analysis in studies of the relationship betweenC. hepaticumand its host must be highlighted. The high prevalence observed inA. azarae, associated with the wide size range of the parasite eggs evidenced by principal component analysis, suggestsA. azaraeto be the Sigmodontinae host species that plays the most important role as reservoir host forC. hepaticumin the New World.