Characterisation of the RNA Virome of Nine Ochlerotatus Species in Finland.

RNA viromes of nine commonly encountered Ochlerotatus mosquito species collected around Finland in 2015 and 2017 were studied using next-generation sequencing. Mosquito homogenates were sequenced from 91 pools comprising 16-60 morphologically identified adult females of Oc. cantans, Oc. caspius, Oc. communis, Oc. diantaeus, Oc. excrucians, Oc. hexodontus, Oc. intrudens, Oc. pullatus and Oc. punctor/punctodes. In total 514 viral Reverse dependent RNA polymerase (RdRp) sequences of 159 virus species were recovered, belonging to 25 families or equivalent rank, as follows: Aliusviridae, Aspiviridae, Botybirnavirus, Chrysoviridae, Chuviridae, Endornaviridae, Flaviviridae, Iflaviridae, Negevirus, Partitiviridae, Permutotetraviridae, Phasmaviridae, Phenuiviridae, Picornaviridae, Qinviridae, Quenyavirus, Rhabdoviridae, Sedoreoviridae, Solemoviridae, Spinareoviridae, Togaviridae, Totiviridae, Virgaviridae, Xinmoviridae and Yueviridae. Of these, 147 are tentatively novel viruses. One sequence of Sindbis virus, which causes Pogosta disease in humans, was detected from Oc. communis from Pohjois-Karjala. This study greatly increases the number of mosquito-associated viruses known from Finland and presents the northern-most mosquito-associated viruses in Europe to date.

Predicting Spatial Patterns of Sindbis Virus (SINV) Infection Risk in Finland Using Vector, Host and Environmental Data.

Pogosta disease is a mosquito-borne infection, caused by Sindbis virus (SINV), which causes epidemics of febrile rash and arthritis in Northern Europe and South Africa. Resident grouse and migratory birds play a significant role as amplifying hosts and various mosquito species, including Aedes cinereus, Culex pipiens, Cx. torrentium and Culiseta morsitans are documented vectors. As specific treatments are not available for SINV infections, and joint symptoms may persist, the public health burden is considerable in endemic areas. To predict the environmental suitability for SINV infections in Finland, we applied a suite of geospatial and statistical modeling techniques to disease occurrence data. Using an ensemble approach, we first produced environmental suitability maps for potential SINV vectors in Finland. These suitability maps were then combined with grouse densities and environmental data to identify the influential determinants for SINV infections and to predict the risk of Pogosta disease in Finnish municipalities. Our predictions suggest that both the environmental suitability for vectors and the high risk of Pogosta disease are focused in geographically restricted areas. This provides evidence that the presence of both SINV vector species and grouse densities can predict the occurrence of the disease. The results support material for public-health officials when determining area-specific recommendations and deliver information to health care personnel to raise awareness of the disease among physicians.

The mosquitoes of Finland: updated distributions and bionomics.

Mosquitoes (Diptera: Culicidae) were collected in Finland between 2012 and 2018 to determine the species present and their distributions. In total, 52 466 specimens from 1031 collections formed the basis for the preparation of distribution maps for each of the 40 species that were collected. Anopheles maculipennis s.s., An. claviger, Aedes geminus and Ochlerotatus sticticus are confirmed on mainland Finland after previous records were uncertain or absent. Coquillettidia richiardii, Culiseta morsitans, Cs. ochroptera, Culex territans, Cx. torrentium, Oc. leucomelas, Oc. nigrinus, Oc. pullatus and Oc. punctodes occur more widely than previously reported. Three species, Ae. rossicus, Cs. subochrea and Oc. cyprius were not collected, although Ae. rossicus was subsequently found in Lapland by another researcher. No invasive species were collected. Ochlerotatus communis, an aggressive biter, was the most commonly encountered species. Larval collection data suggest that several species may have up to three generations per year in Finland, with Cx. torrentium and Cx. pipiens having at least two, and Oc. communis and Oc. punctor regularly found as larvae across the summer. These data, especially when coupled with historical records, are vital for monitoring species which have significant vector potential, particularly when faced with a warming climate.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

An amendment to this paper has been published and can be accessed via the original article.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

Anopheles daciae, a new country record for Finland.

Mosquitoes (Diptera: Culicidae) were collected throughout Finland between 2013 and 2018 to determine species distributions. During the course of molecular identifications of specimens belonging to the Anopheles maculipennis complex, ribosomal internal transcribed spacer 2 sequences and link-reared specimens revealed the presence of Anopheles daciae Linton, Nicolescu & Harbach (n = 37), a new country record, as well as Anopheles messeae Falleroni (n = 19) in the collections. Although the sample size is low, distinctions are apparent in the distributions of these two species, with An. daciae present in south-eastern and central Finland, including the regions of Kanta-Häme, Pirkanmaa, Pohjois-Pohjanmaa, Päijät-Häme and Satakunta, and An. messeae present in the southern and south-eastern regions of Åland (Ahvenanmaa), Etelä-Savo, Kanta-Häme, Kymenlaakso, Päijät-Häme, Satakunta, Uusimaa and Varsinais-Suomi. All reports of An. messeae in Finland prior to 2018 should therefore be recognized as potentially being either An. messeae or An. daciae. Because these species are potential vectors of malarial protozoa, it is important to have full knowledge of their distributions across Europe, particularly in the face of climate warming.

A report on the mosquitoes of mainland Åland, southwestern Finland and revised list of Finnish mosquitoes.

To successfully implement surveillance or control strategies for mosquitoes, up-to-date knowledge of regional species composition is vital. The last report regarding mosquitoes (Diptera: Culicidae) in the Åland archipelago, southwestern Finland listed 19 species (Utrio, 1979). To determine the current species diversity, one collection trip was made to mainland Åland in 2015 and three in 2016. Mosquitoes (n = 3286) were collected as both adult and immature life stages from 88 collections within 29 1-km2 areas. Fifteen of the 19 previously reported species were obtained, leaving the current status of four species uncertain. At least 11 species previously not reported from Åland, but confirmed on the Finnish mainland, were collected. Aedes geminus Peus was identified based on examination of the gonostylus, and represents a new species distribution for Finland. Anopheles maculipennis s.s. Meigen was confirmed from cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) sequence data and is reinstated on the list of Finnish species, along with Ochlerotatus sticticus (Meigen). Dahliana geniculata (Olivier) was found in two locations, in 2 months, indicating that there is an established population in Åland. The present data confirm that at least 27 species inhabit mainland Åland, rising to 31 when historical data are included. The Finnish mosquito fauna is increased from 38 to 41 species.

Aedes nigrinus (Eckstein, 1918) (Diptera, Culicidae), a new country record for England, contrasted with Aedes sticticus (Meigen, 1838).

We report the discovery of Aedes nigrinus (Eckstein, 1918) in the New Forest of southern England, bringing to 36 the number of mosquito species recorded in Britain. Because it seems that this species has been misidentified previously in Britain as the morphologically similar Aedes sticticus (Meigen, 1838), the two species are contrasted and distinguished based on distinctive differences exhibited in the adult and larval stages. The pupa of Ae. nigrinus is unknown, but the pupa of Ae. sticticus is distinguished from the pupae of other species of Aedes by modification of the most recent key to British mosquitoes. The history of the mosquito fauna recorded in the UK is summarized and bionomical information is provided for the two species.

Family-Level Sampling of Mitochondrial Genomes in Coleoptera: Compositional Heterogeneity and Phylogenetics.

Mitochondrial genomes are readily sequenced with recent technology and thus evolutionary lineages can be densely sampled. This permits better phylogenetic estimates and assessment of potential biases resulting from heterogeneity in nucleotide composition and rate of change. We gathered 245 mitochondrial sequences for the Coleoptera representing all 4 suborders, 15 superfamilies of Polyphaga, and altogether 97 families, including 159 newly sequenced full or partial mitogenomes. Compositional heterogeneity greatly affected 3rd codon positions, and to a lesser extent the 1st and 2nd positions, even after RY coding. Heterogeneity also affected the encoded protein sequence, in particular in the nad2, nad4, nad5, and nad6 genes. Credible tree topologies were obtained with the nhPhyML ("nonhomogeneous") algorithm implementing a model for branch-specific equilibrium frequencies. Likelihood searches using RAxML were improved by data partitioning by gene and codon position. Finally, the PhyloBayes software, which allows different substitution processes for amino acid replacement at various sites, produced a tree that best matched known higher level taxa and defined basal relationships in Coleoptera. After rooting with Neuropterida outgroups, suborder relationships were resolved as (Polyphaga (Myxophaga (Archostemata + Adephaga))). The infraorder relationships in Polyphaga were (Scirtiformia (Elateriformia ((Staphyliniformia + Scarabaeiformia) (Bostrichiformia (Cucujiformia))))). Polyphagan superfamilies were recovered as monophyla except Staphylinoidea (paraphyletic for Scarabaeiformia) and Cucujoidea, which can no longer be considered a valid taxon. The study shows that, although compositional heterogeneity is not universal, it cannot be eliminated for some mitochondrial genes, but dense taxon sampling and the use of appropriate Bayesian analyses can still produce robust phylogenetic trees.

A new subgenus and species of Topomyia (Diptera: Culicidae: Sabethini) based on a remarkable male mosquito from Sabah, Malaysia.

Miyagiella Harbach, subgen. nov., is introduced as a new subgenus of Topomyia Leicester for a remarkable male mosquito, Topomyia discors Harbach, sp. nov., from Sabah, Malaysia. A diagnosis of the subgenus is provided that features unique anatomical characters of the genitalia of the holotype male. Miyagiella is very distinct from the two previously recognised subgenera of Topomyia, but is perhaps more closely related to the nominotypical subgenus than to subgenus Suaymyia Thurman. Salient differences that distinguish the three subgenera are contrasted; the holotype male of To. discors is described and its unique genitalia are illustrated.

Novel virus discovery and genome reconstruction from field RNA samples reveals highly divergent viruses in dipteran hosts.

We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected.

DNA barcoding of Schistosoma haematobium on Zanzibar reveals substantial genetic diversity and two major phylogenetic groups.

To shed light on the genetic diversity of Schistosoma haematobium on Zanzibar a DNA barcoding study was performed on parasite material isolated from different time-points 4 years apart. Substantive sequence variation was found within the mitochondrial cytochrome oxidase subunit I (cox1) and the NADH-dehydrogenase subunit 1 (nad1) with 27 and 22 unique haplotypes identified respectively and 38 when both gene regions were considered. Upon phylogenetic analysis and comparison with other S. haematobium isolates, haplotypes or barcode types partitioned into two discrete major groups, designated Group 1 and Group 2. Whilst Group 1 isolates were recovered from both Zanzibar and the African mainland, Group 2 isolates were exclusive to Zanzibar. A mixture of Group 1 and 2 parasites were recovered from individual children with no child shedding parasites of a single group haplotype alone. Whilst changes in general levels of genetic diversity between the two parasite isolation time-points were observed, no obvious change in genetic diversity was detected, despite large-scale drug distribution of praziquantel during the intervening period and there was no biased of Group 1 or 2 parasites persisting at the different time-points. To assist in future genetic screening of schistosome larval stages e.g. eggs, miracidia or cercariae, two new DNA-typing assays based on group-specific PCR primers and SNaPshot™ probes have been developed to distinguish Group 1 and 2 haplotypes.

Nyx pholeocola, a new genus and cavernicolous species of tribe Aedini (Diptera: Culicidae) from southern Thailand based on morphological and molecular data.

Nyx Harbach & Linton, gen. nov., is introduced as a new mosquito genus of tribe Aedini for a previously unknown cave-dwelling species, Nyx pholeocola Linton & Harbach, sp. nov., from southern Thailand. A diagnosis of the genus is provided that features unique anatomical characters of the adult, pupal and larval stages of the type species. The affinities of Nyx are discussed in terms of its position in the phylogeny of Aedini. Nyx is more closely related to Borichinda and Isoaedes than to other genera of tribe Aedini. Salient differences that distinguish these three genera are contrasted. The male and female genitalia, pupa and fourth-instar larva of the new species are illustrated. DNA sequence for the second nuclear internal spacer region (ITS2) and the 658-bp barcode fragment of the mitochondrial cytochrome c oxidase I (COI) gene reveal very low similarity with published sequences, supporting the unique status of the new species.

Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics.

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags ('barcodes'). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three 'bait' sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species 'barcodes' that currently use the cox1 gene only.

Lack of influence of test meal fatty acid composition on the contribution of intestinally-derived lipoproteins to postprandial lipaemia.

The extent and duration of postprandial lipaemia have been linked to risk of CHD but the influence of dietary variables on, and the relative contributions of, exogenous (chylomicron) and endogenous (VLDL) triacylglycerols to the total lipaemic response have not been comprehensively evaluated. In the present study the triacylglycerol, apolipoprotein (apo) B-48 and retinyl ester (RE) responses to three test meals of varying monounsaturated (MUFA) and saturated fatty acid (SFA) content were measured in the triacylglycerol-rich lipoprotein (TRL) fraction of plasma (rho = 1.006 g/ml) for 9 h after meal consumption. Fifteen healthy normolipidaemic young men consumed, on separate occasions, three test meals which were identical apart from their MUFA and SFA contents. Expressed as a percentage of total energy the MUFA/SFA contents of the meals were: (1) 12%/17%; (2) 17%/12% and (3) 24%/5%. The contribution of the intestinally-derived lipoproteins (chylomicrons) to the lipaemic response was investigated by determining the time to reach peak concentration and the total and incremental areas under the time response curves (AUC and incremental AUC) for RE, apoB-48 and triacylglycerol in the TRL fraction. No significant differences in these measurements were observed for the three meals. However, visual comparison of the postprandial responses to the three meals suggested that as meal MUFA content increased there was a tendency for the triacylglycerol, apoB-48 and RE responses to become biphasic as opposed to the typical monophasic response seen with the 12% MUFA/17% SFA meal. Comparison of the apoB-48 and RE responses for the three test meals confirmed other workers' findings of delayed entry of RE relative to apoB-48 in TRL. The value of the two markers in investigating dietary fat absorption and metabolism is discussed.