Mosquitoes and Mosquito-Associated RNA Viruses of Finland

Mosquitoes (Diptera, Culicidae) are amongst the most socioeconomically important animals in the world, with many species being vectors of disease-causing pathogens, including bacteria, helminths, protozoa and viruses. While not subjected to the same mosquito-borne disease burden as the tropics, Finland nonetheless has three known mosquito-borne viruses which cause disease in humans. Sindbis virus (Togaviridae: Alphavirus), which causes Pogosta disease, as well as Inkoo Virus and Möhkö strains of Chatanga virus (Peribunyaviridae: Orthobunyavirus). Three insect-specific flaviviruses have also been isolated from mosquitoes in Finland, Hanko, Ilomantsi and Lammi viruses (Flaviviridae: Flavivirus) as well as one Negevirus, Mekrijärvi negevirus. Knowledge of the mosquitoes, however, had become outdated, with no significant records of mosquitoes published since 1979 when distribution maps for each of the 38 recorded species were created using historical and contemporary collections. Additionally, the viruses which have been isolated from mosquitoes in Finland have been found in pools of unidentified specimens, which means that no vector or invertebrate host species have been confirmed for five of the seven aforementioned viruses. The aims were therefore to increase the knowledge of the composition and distribution of the Finnish mosquito fauna and the viruses associated with them. Adult and immature mosquito collections were made around the country in all 19 regions (corresponding to the European NUTS-3 divisions) between 2012–2018. The main collection efforts were made in 2014–2017 along transects in Lapland, southern Finland and the mainland of the Åland Archipelago (Ahvennanmaa). All specimens were morphologically identified, where possible, and stored in one of a variety of ways suitable for either virus cell culture experiments, virus PCR experiments, DNA studies or morphological studies. Study I was the first to focus on the mosquitoes of the Åland Archipelago, which is situated between Finland and Sweden in the Baltic Sea. From the collections made on mainland (Fasta) Åland in late 2015 and across 2016, 12 new species records were observed. This included the first country record for Aedes geminus Peus and reconfirmation of Anopheles maculipennis s.s. Meigen and Ochlerotatus sticticus (Meigen) following their recent removal from the fauna of Finland. It also recorded what later became the only confirmed record of Dahliana geniculata (Olivier) in Finland. The Finnish mosquito fauna increased from 38 to 41 species. Study II used DNA sequences extracted from specimens of the Anopheles maculipennis complex to identify which species are present in Finland. This resulted in the discovery of a new country record for Anopheles daciae Linton, Nicolescu & Harbach, a sibling species of An. messeae Falleroni. The Finnish mosquito fauna increased from 41 to 42 species. Study III utilised records for all 52,466 specimens from 1,031 collections to create distribution maps for 40 of the 43 recorded species that were collected for this thesis. Specimens of Aedes rossicus Dolbeškin, Gorickaja & Mitrofanova, Culiseta subochrea (Edwards) and Ochlerotatus cyprius (Ludlow) were not collected, nor mapped, but have been recorded previously. These new collection data significantly extended the recorded distributions for several species, while other common species were still found across the country. Questions were raised about the presence of Ochlerotatus riparius, since only adult specimens were identified, which are easily confused with closely related species. This study provides a solid foundation for future studies to build upon. Study IV explored the RNA viromes of nine man-biting Ochlerotatus species collected in Finland using next generation sequencing. In total, 514 viral polymerases were sequenced, which grouped into 159 species belonging to 25 families or equivalent taxonomic groups as follows: Aliusviridae (1), Aspiviridae (1), Botybirnavirus (8), Chrysoviridae (5), Chuviridae (14), Endornaviridae (2), Flaviviridae (9), Iflaviridae (17), Negevirus (41), Partitiviridae (55), Permutotetraviridae (6), Phasmaviridae (13), Phenuiviridae (58), Picornaviridae (5), Qinviridae (7), Quenyavirus (2), Rhabdoviridae (21), Sedoreoviridae (10), Solemoviridae (15), Spinareoviridae (1), Togaviridae (1), Totiviridae (205), Virgaviridae (7), Xinmoviridae (9) and Yueviridae (1). Twelve of these species have previously been described, while 147 were novel viruses. The host-vector associations of these viruses are yet to be established. Overall, studies I–IV contribute a wealth of contemporary knowledge about the mosquitoes of Finland. More research is required to complete our understanding of mosquito distributions in the country and mosquito-virus associations and interactions, but this thesis provides a solid foundation upon which future research can now be built.Hyttyset (Diptera, Culicidae) ovat sosioekonomisesti maailman tärkeimpiä eläimiä, ja monet lajit ovat sairauksia aiheuttavien patogeenien - bakteerien, loismatojen, alkueläinten ja virusten - levittäjiä. Vaikka Suomessa ei ole samanlaista hyttysten levittämää tautitaakkaa kuin tropiikissa, Suomessa tunnetaan kuitenkin kolme hyttysten levittämää virusta, jotka ovat ihmisen taudinaiheuttajia: Sindbis-virus (Togaviridae: Alphavirus), joka aiheuttaa pogostantautia, Inkoo-virus ja Chatanga-virus (Peribunyaviridae: Orthobunyavirus). Suomessa on hyttysistä eristetty myös kolme hyönteisspesifistä flavivirusta, Hanko-, Ilomantsi- ja Lammi-virukset (Flaviviridae: Flavivirus) sekä yksi Negevirus, Mekrijärvi-negevirus. Tieto hyttysistä on kuitenkin vanhentunut, eikä merkittävää tietoa Suomen hyttyslajeista ole julkaistu sitten vuoden 1979, jolloin kunkin 38 rekisteröidyn lajin levinneisyyskartat luotiin käyttämällä historiallisia ja silloin tehtyjä keräyksiä. Lisäksi virukset on aiemmin eristetty lajitasolle tunnistamattomista hyttyspooleista, joten viidelle edellä mainitusta seitsemästä viruksesta ei ole varmistettu yhtään levittäjähyttyslajia tai selkärangatonta isäntälajia. Tämän väitöskirjan tavoitteena oli lisätä tietoa Suomen hyttyslajien kirjosta ja levinneisyydestä sekä niihin liittyvistä viruksista. Aikuisten ja kehittymättömien hyttysten keräyksiä tehtiin ympäri maata kaikissa 19 maakunnassa (vastaten Euroopan NUTS-3-jakoa) vuosina 2012–2018. Tärkeimmät keräystyöt tehtiin vuosina 2014–2017 poikkileikkaustutkimuksilla Lapissa, Etelä-Suomessa ja Ahvenanmaan saaristossa (Åland). Kaikki näytteet tunnistettiin morfologisesti, mikäli mahdollista, ja säilytettiin lisäksi virusviljelyä, virologisia PCR_tutkimuksia, sekä DNA- ja morfologisia tutkimuksia varten. Osatyö I keskittyi Ahvenanmaan saariston hyttysiin. Manner-Ahvenanmaalla (Fasta) vuoden 2015 lopulla ja vuoden 2016 aikana tehdyistä keräyksistä havaittiin 12 uutta lajimerkintää. Tämän osatyön myötä Aedes geminus Peusille havaittiin ensimmäistä kertaa Suomessa ja Anopheles maculipennis s.s. Meigen ja Ochlerotatus sticticus (Meigen), jotka oli äskettäin poistettu Suomen hyttyslajistosta, lisättiin siihen uudestaan. Tässä tutkimuksessa tehtiin myös ainoa vahvistettu havainto Dahliana geniculata (Olivier) -lajista Suomessa. Löytöjen myötä Suomen hyttyslajien hyttyslajien määrä kasvoi 38 lajista 41 lajiin. Osatyössä II käytettiin Anopheles maculipennis -kompleksin näytteiden DNA-sekvenssejä Suomessa esiintyvien lajien selvittämiseen. Tämän seurauksena löydettiin Suomessa uusi laji Anopheles daciae Linton, Nicolescu & Harbach, joka on An. messeae Falleronin sisaruslaji ja tämän myötä Suomen hyttyslajisto kasvoi 41 lajista 42 lajiin. Osatyössä III käytettiin tietueita kaikista 52 466 yksilöstä, jotka kuuluivat yhteensä 1 031 erilliseen kokoelmaan levinneisyyskarttojen luomiseksi tätä väitöskirjatyötä varten kerätyille 40:lle 43:sta Suomessa kirjatusta lajista. Aedes rossicus Dolbeškin, Gorickaja & Mitrofanova, Culiseta subochrea (Edwards) ja Ochlerotatus cyprius (Ludlow) yksilöitä ei kerätty eikä kartoitettu, mutta ne on tallennettu aiemmin. Nämä uudet keräystiedot laajensivat merkittävästi useiden lajien kirjattuja levinneisyysalueita, kun taas muita yleisiä lajeja löydettiin edelleen eri puolilla maata. Ochlerotatus riparius -lajin esiintyminen herätti kysymyksiä, koska siitä tunnistettiin vain aikuisia yksilöitä, jotka on mahdollista sekoittaa läheisiin lajeihin. Tämä osatyö antaa hyvän pohjan tuleville tutkimuksille. Osatyössä IV tutkittiin RNA-viromeja kaikkiaan yhdeksästä Suomessa ihmisiä pistävästä Ochlerotatus-lajinsta uuden sukupolven sekvensointimenetelmillä. Yhteensä sekvensoitiin 514 viruksen polymeraasigeeniä, jotka ryhmiteltiin kuuluviksi 159 lajiin, ja edelleen 25 heimoon tai vastaavaan taksonomiseen ryhmään seuraavasti: Aliusviridae (1), Aspiviridae (1), Botybirnavirus (8), Chrysoviridae (5), Chuviridae (14), Endornaviridae (2), Flaviviridae (9), Iflaviridae (17), Negevirus (41), Partitiviridae (55), Permutotetraviridae (6), Phasmaviridae (13), Phenuiviridae (58), Picornaviridae (5), (7), Quenyavirus (2), Rhabdoviridae (21), Sedoreoviridae (10), Solemoviridae (15), Spinareoviridae (1), Togaviridae (1), Totiviridae (205), Virgaviridae (7), Xinmoviridae (9) ja Yueviridae (1). Vain 12 näistä ehdotetuista lajeista on kuvattu aiemmin, ja 147 on uusia viruksia. Vielä ei täysin ymmärretä, toimivatko nämä yhdeksän lajia näiden virusten isäntinä vai vektoreina. Kaiken kaikkiaan tämän väitöskirjatyön tutkimukset tuovat runsaasti uutta tietoa Suomen hyttysistä. Lisää tutkimusta tarvitaan täydentämään ymmärrystämme hyttysten leviämisestä maassa sekä hyttysten ja virusten yhteyksistä ja vuorovaikutuksista, mutta tämä väitöskirjatyö tarjoaa vankan perustan, jolle tulevaa tutkimusta voidaan nyt rakentaa

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.Peer reviewe

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 sampled more densely. 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 (‘non-homogeneous’) 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, whilst 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

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

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

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

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