Metagenomic analysis reveals changes of the Drosophila suzukii microbiota in the newly colonized regions.

The spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a highly polyphagous pest of a wide variety of wild or cultivated berry and stone fruit. Originating from Southeast Asia, it has recently invaded a wide range of regions in Europe and North America. It is well known that insect microbiotas may significantly influence several aspects of the host biology and play an important role in invasive species introduction into new areas. However, in spite of the great economic importance of D. suzukii, a limited attention has been given so far to its microbiota. In this study, we present the first in-depth characterization of gut bacterial diversity from field (native and invasive range) and lab-reared populations of this insect. The gut bacterial communities of field insects were dominated, regardless of their origin, by 2 families of the phylum Proteobacteria: Acetobacteraceae and Enterobacteriaceae, while Firmicutes, mainly represented by the family Staphylococcaceae, prevailed in lab-reared population. Locality was the most significant factor in shaping the microbiota of wild flies. Moreover, a negative correlation between diversity and abundance of Enterobacteriaceae and the time elapsed since the establishment of D. suzukii in a new region was observed. Altogether our results indicate that habitat, food resources as well as the colonization phase of a new region contribute to shape the bacterial communities of the invasive species which, in turn, by evolving more quickly, could influence host adaptation in a new environment.

Are Phenacoccus solani Ferris and P. defectus Ferris (Hemiptera: Pseudococcidae) distinct species?

Among the Nearctic species of Phenacoccus (Hemiptera: Pseudococcidae), Phenacoccus solani Ferris and P. defectus Ferris are morphologically similar and it can be difficult to separate them on the basis of microscopic morphological characters of the adult female alone. In order to resolve their identity, a canonical variates morphological analysis of 199 specimens from different geographical origins and host plants and a molecular analysis of the COI and 28S genes were performed. The morphological analysis supported synonymy of the two species, as although the type specimens of the "species" are widely separated from each other in the canonical variates plot, they are all part of a continuous range of variation. The molecular analysis showed that P. solani and P. defectus are grouped in the same clade. On the basis of the morphological and molecular analyses, P. defectus is synonymized under the senior name P. solani, syn. n.

Genetic differentiation of the pine processionary moth at the southern edge of its range: contrasting patterns between mitochondrial and nuclear markers.

The pine processionary moth (Thaumetopoea pityocampa) is an important pest of coniferous forests at the southern edge of its range in Maghreb. Based on mitochondrial markers, a strong genetic differentiation was previously found in this species between western (pityocampa clade) and eastern Maghreb populations (ENA clade), with the contact zone between the clades located in Algeria. We focused on the moth range in Algeria, using both mitochondrial (a 648 bp fragment of the tRNA-cox2) and nuclear (11 microsatellite loci) markers. A further analysis using a shorter mtDNA fragment and the same microsatellite loci was carried out on a transect in the contact zone between the mitochondrial clades. Mitochondrial diversity showed a strong geographical structure and a well-defined contact zone between the two clades. In particular, in the pityocampa clade, two inner subclades were found whereas ENA did not show any further structure. Microsatellite analysis outlined a different pattern of differentiation, with two main groups not overlapping with the mitochondrial clades. The inconsistency between mitochondrial and nuclear markers is probably explained by sex-biased dispersal and recent afforestation efforts that have bridged isolated populations.

High genetic diversity in the Culex pipiens complex from a West Nile Virus epidemic area in Southern Europe.

BACKGROUND: The Culex pipiens complex includes the most widespread mosquito species in the world. Cx. pipiens is the primary vector of the West Nile Virus (WNV) in Europe and North America. Cases of WNV have been recorded in Italy since 1998. In particular, wet areas along the Po River are considered some of the most WNV affected areas in Italy. Here, we analyzed the genetic structure of ten Cx. pipiens populations collected in the last part of the Po River including the Delta area. METHODS: We assessed the genetic variability of two mitochondrial markers, cytochrome oxidase 1 (COI) and 2 (COII), for a total of 1200 bp, and one nuclear marker, a fragment of acetylcholinesterase-2 (ace-2), 502 bp long. The effect of the landscape features was evaluated comparing haplotype and nucleotide diversity with the landscape composition. RESULTS: The analysis showed a high genetic diversity in both COI and COII gene fragments mainly shared by the populations in the Delta area. The COI-COII network showed that the set of haplotypes found was grouped into three main supported lineages with the higher genetic variability gathered in two of the three lineages. By contrast, ace-2 fragment did not show the same differentiation, displaying alleles grouped in a single clade. Finally, a positive correlation between mitochondrial diversity and natural wetland areas was found. CONCLUSIONS: The high mitochondrial genetic diversity found in Cx. pipiens populations from the Po River Delta contrasts with the low variability of inland populations. The different patterns of genetic diversity found comparing mitochondrial and nuclear markers could be explained by factors such as differences in effective population size between markers, sex biased dispersal or lower fitness of dispersing females. Moreover, the correlation between genetic diversity and wetland areas is consistent with ecosystem stability and lack of insecticide pressure characteristic of this habitat. The mtDNA polymorphism found in the Po River Delta is even more interesting due to possible linkages between the mitochondrial lineages and different biting behaviors of the mosquitoes influencing their vector ability of arboviral infections.

A unique midgut-associated bacterial community hosted by the cave beetle Cansiliella servadeii (Coleoptera: Leptodirini) reveals parallel phylogenetic divergences from universal gut-specific ancestors.

BACKGROUND: Cansiliella servadeii (Coleoptera) is an endemic troglobite living in deep carbonate caves in North-Eastern Italy. The beetle constantly moves and browses in its preferred habitat (consisting in flowing water and moonmilk, a soft speleothem colonized by microorganisms) self-preens to convey material from elytra, legs, and antennae towards the mouth. We investigated its inner and outer microbiota using microscopy and DNA-based approaches. RESULTS: Abundant microbial cell masses were observed on the external appendages. Cansiliella's midgut is fully colonized by live microbes and culture-independent analyses yielded nearly 30 different 16S phylotypes that have no overlap with the community composition of the moonmilk. Many of the lineages, dominated by Gram positive groups, share very low similarity to database sequences. However for most cases, notwithstanding their very limited relatedness with existing records, phylotypes could be assigned to bacterial clades that had been retrieved from insect or other animals' digestive traits. CONCLUSIONS: Results suggest a history of remote separation from a common ancestor that harboured a set of gut-specific bacteria whose functions are supposedly critical for host physiology. The phylogenetic and coevolutionary implications of the parallel occurrences of these prokaryotic guilds appear to apply throughout a broad spectrum of animal diversity. Their persistence and conservation underlies a possibly critical role of precise bacterial assemblages in animal-bacteria interactions.

Host and phenology shifts in the evolution of the social moth genus Thaumetopoea.

The genus Thaumetopoea contains the processionary moths, a group of lepidopteran associated with forest trees, well known for the social behaviour of the larvae and for carrying urticating setae. The taxonomy of the genus is partly unresolved and a phylogenetic approach is lacking. The goal of this work is to produce a phylogeny for Thaumetopoea and to identify the main traits driving the evolution of this group. Eighteen mitochondrial and three nuclear genes were fully/partly sequenced. Markers were aligned and analysed singularly or in various combinations. Phylogenetic analyses were performed according to maximum likelihood and Bayesian inference methods. Trees obtained from largest data sets provided identical topologies that received strong statistical support. Three main clades were identified within Thaumetopoea and were further supported by several signatures located in the mitochondrial tRNAs and intergenic spacers. The reference topology was used to investigate the evolution of life history traits related to biogeography, host plant, ecology, and morphology. A multigenic approach allowed to produce a robust phylogenetic analysis of the genus Thaumetopoea, with the identification of three major clades linked to different ecological and life history traits. The first clade is associated with Angiosperm host plants and has a fast spring development of larvae on young foliage. The other clades have originated by one event of host plant shift to Gymnosperm Pinaceae, which implied a longer larval developmental time due to the lower nutritional quality of leaves. These clades showed different adaptations to such a constraint, the first with a switch of larval feeding to cold season (winter pine processionary moths), and the second with a retraction to high altitude and latitude and a development cycle extended over two years (summer pine processionary moths). Recent global warming is affecting all species and seems able to further shape the evolution of the group.

Evidence of two lineages of the symbiont 'Candidatus Erwinia dacicola' in Italian populations of Bactrocera oleae (Rossi) based on 16S rRNA gene sequences.

The close association between the olive fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) and bacteria has been known for more than a century. Recently, the presence of a host-specific, hereditary, unculturable symbiotic bacterium, designated 'Candidatus Erwinia dacicola', has been described inside the cephalic organ of the fly, called the oesophageal bulb. In the present study, the 16S rRNA gene sequence variability of 'Ca. E. dacicola' was examined within and between 26 Italian olive fly populations sampled across areas where olive trees occur in the wild and areas where cultivated olive trees have been introduced through history. The bacterial contents of the oesophageal bulbs of 314 olive flies were analysed and a minimum of 781 bp of the 16S rRNA gene was sequenced. The corresponding host fly genotype was assessed by sequencing a 776 bp portion of the mitochondrial genome. Two 'Ca. E. dacicola' haplotypes were found (htA and htB), one being slightly more prevalent than the other (57%). The two haplotypes did not co-exist in the same individuals, as confirmed by cloning. Interestingly, the olive fly populations of the two main Italian islands, Sicily and Sardinia, appeared to be represented exclusively by the htB and htA haplotypes, respectively, while peninsular populations showed both bacterial haplotypes in different proportions. No significant correlation emerged between the two symbiont haplotypes and the 16 host fly haplotypes observed, suggesting evidence for a mixed model of vertical and horizontal transmission of the symbiont during the fly life cycle.

Restricted gene flow at the micro- and macro-geographical scale in marble trout based on mtDNA and microsatellite polymorphism.

BACKGROUND: The genetic structure of the marble trout Salmo trutta marmoratus, an endemic salmonid of northern Italy and the Balkan peninsula, was explored at the macro- and micro-scale level using a combination of mitochondrial DNA (mtDNA) and microsatellite data. RESULTS: Sequence variation in the mitochondrial control region showed the presence of nonindigenous haplotypes indicative of introgression from brown trout into marble trout. This was confirmed using microsatellite markers, which showed a higher introgression at nuclear level. Microsatellite loci revealed a strong genetic differentiation across the geographical range of marble trout, which suggests restricted gene flow both at the micro-geographic (within rivers) and macro-geographic (among river systems) scale. A pattern of Isolation-by-Distance was found, in which genetic samples were correlated with hydrographic distances. A general West-to-East partition of the microsatellite polymorphism was observed, which was supported by the geographic distribution of mitochondrial haplotypes. CONCLUSION: While introgression at both mitochondrial and nuclear level is unlikely to result from natural migration and might be the consequence of current restocking practices, the pattern of genetic substructuring found at microsatellites has been likely shaped by historical colonization patterns determined by the geological evolution of the hydrographic networks.

Phylogenetic relationships between flies of the Tephritinae subfamily (Diptera, Tephritidae) and their symbiotic bacteria.

The Tephritinae is considered the most specialized subfamily of fruit flies, predominantly infesting flowerheads of Asteraceae. Some species are known to host specific non-culturable symbiont bacteria ("Candidatus Stammerula spp.") in the midgut. In this work we (i) examined the phylogenetic relationships among the insect hosts, (ii) investigated the presence of bacteria in other hitherto unexamined species, and (iii) evaluated the phylogenetic congruence between insects and symbionts. A total of 33 Tephritinae species in 17 different genera were analyzed. Two regions of the mitochondrial DNA (16S rDNA and COI-tRNALeu-COII) were examined in the insect host, while the 16S was analyzed in the bacteria. From the phylogenetic trees, four of the five tribes considered were statistically supported by each of the clustering methods used. Species belonging to the tribe Noeetini never clustered at significant levels. The phylogenetic COI-tRNALeu-COII tree showed internal nodes more highly supported than the 16S phylogeny. The analysis of the distribution of symbiosis across the subfamily has highlighted the presence of bacteria only in the tribe Tephritini and in the genus Noeeta from the tribe Noeetini. A cophylogenetic analysis revealed a substantial congruence between hosts and symbionts. The interesting exceptions can be justified by events like losses, duplications and hosts switching opportunities, which are likely to arise during the biological cycle of the fly in consideration of the extracellular status of these symbionts.

Quaternary history and contemporary patterns in a currently expanding species.

BACKGROUND: Quaternary climatic oscillations had dramatic effects on species evolution. In northern latitudes, populations had to survive the coldest periods in refugial areas and recurrently colonized northern regions during interglacials. Such a history usually results in a loss of genetic diversity. Populations that did not experience glaciations, in contrast, probably maintained most of their ancestral genetic diversity. These characteristics dramatically affected the present-day distribution of genetic diversity and may influence the ability of species to cope with the current global changes. We conducted a range-wide study of mitochondrial genetic diversity in the pine processionary moth (Thaumetopoea pityocampa/T. wilkinsoni complex, Notodontidae), a forest pest occurring around the Mediterranean Basin and in southern Europe. This species is responding to the current climate change by rapid natural range expansion and can also be accidentally transported by humans. Our aim was to assess if Quaternary climatic oscillations had a different effect across the species' range and to determine if genetic footprints of contemporary processes can be identified in areas of recent introduction. RESULTS: We identified three main clades that were spatially structured. In most of Europe, the genetic diversity pattern was typical for species that experienced marked glaciation cycles. Except in refugia, European populations were characterized by the occurrence of one main haplotype and by a strong reduction in genetic diversity, which is expected in regions that were rapidly re-colonized when climatic conditions improved. In contrast, all other sub-clades around the Mediterranean Basin occurred in limited parts of the range and were strongly structured in space, as is expected in regions in which the impact of glaciations was limited. In such places, genetic diversity was retained in most populations, and almost all haplotypes were endemic. This pattern was extreme on remote Mediterranean islands (Crete, Cyprus, Corsica) where highly differentiated, endemic haplotypes were found. Recent introductions were typified by the existence of closely-related haplotypes in geographically distant populations, which is difficult to detect in most of Europe because of a lack of overall genetic structure. CONCLUSION: In regions that were not prone to marked glaciations, recent moth introductions/expansions could be detected due to the existence of a strong spatial genetic structure. In contrast, in regions that experienced the most intense Quaternary climatic oscillations, the natural populations are not genetically structured, and contemporary patterns of population expansion remain undetected.

Characterization and evolution of two bacteriome-inhabiting symbionts in cixiid planthoppers (Hemiptera: Fulgoromorpha: Pentastirini).

Like other plant sap-sucking insects, planthoppers within the family Cixiidae (Insecta: Hemiptera: Fulgoromorpha) host a diversified microbiota. We report the identification and first molecular characterization of symbiotic bacteria in cixiid planthoppers (tribe: Pentastirini). Using universal eubacterial primers we first screened the eubacterial 16S rRNA sequences in Pentastiridius leporinus (Linnaeus) with PCR amplification, cloning, and restriction fragment analysis. We identified three main 16S rRNA sequences that corresponded to a Wolbachia bacterium, a plant pathogenic bacterium, and a novel gammaproteobacterial symbiont. A fourth bacterial species affiliated with 'Candidatus Sulcia muelleri' was detected in PCR assays using primers specific for the Bacteroidetes. Within females of two selected cixiid planthoppers, P. leporinus and Oliarus filicicola, fluorescence In situ hybridization analysis and transmission electron microscopy observations showed that 'Ca. Sulcia muelleri' and the novel gammaproteobacterial symbiont were housed in separate bacteriomes. Phylogenetic analysis revealed that both of these symbionts occurred in at least four insect genera within the tribe Pentastirini. 'Candidatus Purcelliella pentastirinorum' was proposed as the novel gammaproteobacterial symbiont.

The complete mitochondrial genome of the bag-shelter moth Ochrogaster lunifer (Lepidoptera, Notodontidae).

BACKGROUND: Knowledge of animal mitochondrial genomes is very important to understand their molecular evolution as well as for phylogenetic and population genetic studies. The Lepidoptera encompasses more than 160,000 described species and is one of the largest insect orders. To date only nine lepidopteran mitochondrial DNAs have been fully and two others partly sequenced. Furthermore the taxon sampling is very scant. Thus advance of lepidopteran mitogenomics deeply requires new genomes derived from a broad taxon sampling. In present work we describe the mitochondrial genome of the moth Ochrogaster lunifer. RESULTS: The mitochondrial genome of O. lunifer is a circular molecule 15593 bp long. It includes the entire set of 37 genes usually present in animal mitochondrial genomes. It contains also 7 intergenic spacers. The gene order of the newly sequenced genome is that typical for Lepidoptera and differs from the insect ancestral type for the placement of trnM. The 77.84% A+T content of its alpha strand is the lowest among known lepidopteran genomes. The mitochondrial genome of O. lunifer exhibits one of the most marked C-skew among available insect Pterygota genomes. The protein-coding genes have typical mitochondrial start codons except for cox1 that present an unusual CGA. The O. lunifer genome exhibits the less biased synonymous codon usage among lepidopterans. Comparative genomics analysis study identified atp6, cox1, cox2 as cox3, cob, nad1, nad2, nad4, and nad5 as potential markers for population genetics/phylogenetics studies. A peculiar feature of O. lunifer mitochondrial genome it that the intergenic spacers are mostly made by repetitive sequences. CONCLUSION: The mitochondrial genome of O. lunifer is the first representative of superfamily Noctuoidea that account for about 40% of all described Lepidoptera. New genome shares many features with other known lepidopteran genomes. It differs however for its low A+T content and marked C-skew. Compared to other lepidopteran genomes it is less biased in synonymous codon usage. Comparative evolutionary analysis of lepidopteran mitochondrial genomes allowed the identification of previously neglected coding genes as potential phylogenetic markers. Presence of repetitive elements in intergenic spacers of O. lunifer genome supports the role of DNA slippage as possible mechanism to produce spacers during replication.

Presence of specific symbiotic bacteria in flies of the subfamily Tephritinae (Diptera Tephritidae) and their phylogenetic relationships: proposal of 'Candidatus Stammerula tephritidis'.

The presence of symbiotic bacteria in flies belonging to the subfamily Tephritinae, which predominantly infest the flower heads of composite flowers (Asteraceae), was investigated. Twenty-five species of flies, collected mainly in northern Italy, were examined. The bacteria adhered to the midgut epithelium in a space external to the peritrophic membrane and therefore not in direct contact with the gut contents. Specific, unique and live, but unculturable bacteria were consistently found in the majority of the fly species and their presence was also shown to be persistent in flies reared under microbiologically controlled conditions and devoid of any residual culturable intestinal bacteria. Sequencing of the small subunit rRNA gene from the novel bacteria indicated that they belonged to the family Enterobacteriaceae. Three main strongly supported clades were delineated by phylogenetic trees, the first of which featured a coherent set of sequences displaying gene sequence similarities lower than 96 % compared with recognized taxa. The second and third clades featured cases with higher gene sequence similarities to culturable bacteria, including Erwinia persicina and Ewingella americana, respectively. Relative rate tests were supportive of a fast genetic evolution for the majority of the bacterial symbionts of the subfamily Tephritinae. In agreement with the interpretation suggested in 1929 after pioneering observations made by H. J. Stammer, a symbiotic relationship between the novel bacteria and the tephritid flies is postulated. The origin of this apparently polyphyletic relationship is discussed and a novel candidate organism is proposed for the first clade under the designation 'Candidatus Stammerula tephritidis'.

The INSL3-LGR8/GREAT ligand-receptor pair in human cryptorchidism.

Testicular descent is a complex multistep embryonic process requiring the interaction between anatomical and hormonal factors. Failure in any of these steps results in cryptorchidism, the most frequent congenital anomaly of the urogenital tract in human males. Evidence for a genetic cause for cryptorchidism is numerous and supported by animal models. In particular, INSL3 and LGR8/GREAT proteins seem to act as ligand and receptor, respectively, and to have a role in gubernaculum development involved in testicular descent. In a cohort of 87 ex-cryptorchid patients and 80 controls, we looked for mutations in INSL3 and LGR8/GREAT genes by sequencing. Patients were classified on the basis of seminal, hormonal, and testicular cytological analyses. We found three mutations in the INSL3 gene in four patients and one LGR8/GREAT mutation in four patients (8 of 87, 9.2%). The eight patients show different phenotypes, ranging from normozoospermia to complete azoospermia, and from bilateral cryptorchidism to retractile testes. Furthermore, the endocrine function of the testis appears normal in all subjects. The findings of our study demonstrate that INSL3-LGR8/GREAT mutations are frequently associated with human cryptorchidism and are maternally inherited. The only clinical consequence of alterations of the INSL3-LGR8/GREAT system seems to be failure of the testis to normally descend in the scrotum during embryonic development, without affecting the spermatogenic and endocrine components of the testis itself.