Synthesis of Novel 1-Oxo-2,3,4-trisubstituted Tetrahydroisoquinoline Derivatives, Bearing Other Heterocyclic Moieties and Comparative Preliminary Study of Anti-Coronavirus Activity of Selected Compounds.

A series of novel 1-oxo-2,3,4-trisubstituted tetrahydroisoquinoline (THIQ) derivatives bearing other heterocyclic moieties in their structure were synthesized based on the reaction between homophthalic anhydride and imines. Initial studies were carried out to establish the anti-coronavirus activity of some of the newly obtained THIQ-derivatives against two strains of human coronavirus-229E and OC-43. Their antiviral activity was compared with that of their close analogues, piperidinones and thiomorpholinones, previously synthesized in our group, with aim to expand the range of the tested representative sample and to obtain valuable preliminary information about biological properties of a wider variety of compounds.

In Vitro Antineoplastic and Antiviral Activity and In Vivo Toxicity of Geum urbanum L. Extracts.

This study evaluated the in vitro antineoplastic and antiviral potential and in vivo toxicity of twelve extracts with different polarity obtained from the herbaceous perennial plant Geum urbanum L. (Rosaceae). In vitro cytotoxicity was determined by ISO 10993-5/2009 on bladder cancer, (T-24 and BC-3C), liver carcinoma (HEP-G2) and normal embryonic kidney (HEK-293) cell lines. The antineoplastic activity was elucidated through assays of cell clonogenicity, apoptosis induction, nuclear factor kappa B p65 (NFκB p65) activation and total glutathione levels. Neutral red uptake study was applied for antiviral activity. The most promising G. urbanum extract was analyzed by UHPLC-HRMS. The acute in vivo toxicity analysis was carried out following OEDC 423. The ethyl acetate extract of aerial parts (EtOAc-AP) exhibited the strongest antineoplastic activity on bladder cancer cell lines (IC50 = 21.33-25.28 µg/mL) by inducing apoptosis and inhibiting NFκB p65 and cell clonogenicity. EtOAc and n-butanol extracts showed moderate antiviral activity against human adenovirus type 5 and human simplex virus type I. Seventy four secondary metabolites (gallic and ellagic acid derivatives, phenolic acids, flavonoids, etc.) were identified in EtOAc-AP by UHPLC-HRMS. This extract induced no signs of acute toxicity in liver and kidney specimens of H-albino mice in doses up to 210 mg/kg. In conclusion, our study contributes substantially to the detailed pharmacological characterization of G. urbanum, thus helping the development of health-promoting phytopreparations.

Anti-enteroviral activity of new MDL-860 analogues: Synthesis, in vitro/in vivo studies and QSAR analysis.

A series of 60 nitrobenzonitrile analogues of the anti-viral agent MDL-860 were synthesized (50 of which are new) and evaluated for their activity against three types of enteroviruses (coxsackievirus B1, coxsackievirus B3 and poliovirus 1). Among them, six diaryl ethers (20e, 27e, 28e, 29e, 33e and 35e) demonstrated high in vitro activity (SI > 50) towards at least one of the tested viruses and very low cytotoxicity against human cells. Compound 27e possesses the broadest spectrum of activity towards all tested viruses in the same way as MDL-860 does. The most active derivatives (27e, 29e and 35e) against coxsackievirus B1 were tested in vivo in newborn mice experimentally infected with 20 MLD50 of coxsackievirus B1. Compound 29e showed promising in vivo activity (protection index 26% and 4 days lengthening of mean survival time). QSAR analysis of the substituent effects on the in vitro cytotoxicity (CC50) and anti-viral activity of the nitrobenzonitrile derivatives was carried out and adequate QSAR models for the anti-viral activity of the compounds against poliovirus 1 and coxsackievirus B1 were constructed.

Synthesis and anti-enterovirus activity of new analogues of MDL-860.

A series of twelve novel compounds, analogues of antiviral agent MDL-860 were synthesized and their antiviral activity was evaluated in vitro against enteroviruses poliovirus 1 (PV1), Coxsackieviruses B1 (CVB1) and Coxsackieviruses B3 (CVB3). Compounds 14, 24 and 25 manifested strong antiviral effects against CVB1 and PV1 (SI values of 405 and 118 for CVB1 and PV1 respectively). In contrast to the wide anti-enteroviral activity of MDL-860, these three compounds were inactive against CVB3. Compounds 14, 24 and 25 along with MDL-860 were tested in vivo in mice infected with CVB1. Marked protective effects of compounds 14 and 24 were established, PI values of 50% and 33.3%, respectively. In addition, almost all of the tested compounds manifested very low toxicity.

Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.

Selection and characterization of naturally occurring high acidification rate Streptococcus thermophilus strains.

Among Streptococcus thermophilus cultures, the principle component of yoghurt and cheese starters, a minority of strains forms the group of 'H'-strains which show an unusually high acidification rate, grow faster and coagulate milk 3-5 hours earlier than the typical S. thermophilus cultures. A large-scale screening study was performed to select 'H'-strains of S. thermophilus from more than 100 samples of home-made yoghurt, industrial yoghurt starters and single cultures, maintained in the LBB culture collection. Only four strains - LBB.TN1, LBB.M23, LBB.M34 and LBB.M60 - were isolated/selected due to their ability to form large yellowish colonies on milk agar, supplemented with beta-glycerophosphate and bromocresol purple. While in general S. thermophilus is described as a species with limited proteolytic capacity and in contrast to all other tested S. thermophilus cultures, the four selected strains invariably gave positive amplification product with the polymerase chain reaction when primers, specific for the membrane proteinase-coding gene prtS were used. The macrorestriction profiles of the genomic DNA of the four strains confirmed that they are non-isogenic and not related to each other. When grown in milk and compared to the control industrial strain LBB.A, the four strains showed a dramatically faster acidification, coagulating milk within four hours. The application of strain TN1 or M23 as adjunct culture to industrial yoghurt starter LBB.BY5-12 resulted in shortening the fermentation time with more than 30 min.

Spiders (Araneae) of Churchill, Manitoba: DNA barcodes and morphology reveal high species diversity and new Canadian records.

BACKGROUND: Arctic ecosystems, especially those near transition zones, are expected to be strongly impacted by climate change. Because it is positioned on the ecotone between tundra and boreal forest, the Churchill area is a strategic locality for the analysis of shifts in faunal composition. This fact has motivated the effort to develop a comprehensive biodiversity inventory for the Churchill region by coupling DNA barcoding with morphological studies. The present study represents one element of this effort; it focuses on analysis of the spider fauna at Churchill. RESULTS: 198 species were detected among 2704 spiders analyzed, tripling the count for the Churchill region. Estimates of overall diversity suggest that another 10-20 species await detection. Most species displayed little intraspecific sequence variation (maximum <1%) in the barcode region of the cytochrome c oxidase subunit I (COI) gene, but four species showed considerably higher values (maximum = 4.1-6.2%), suggesting cryptic species. All recognized species possessed a distinct haplotype array at COI with nearest-neighbour interspecific distances averaging 8.57%. Three species new to Canada were detected: Robertus lyrifer (Theridiidae), Baryphyma trifrons (Linyphiidae), and Satilatlas monticola (Linyphiidae). The first two species may represent human-mediated introductions linked to the port in Churchill, but the other species represents a range extension from the USA. The first description of the female of S. monticola was also presented. As well, one probable new species of Alopecosa (Lycosidae) was recognized. CONCLUSIONS: This study provides the first comprehensive DNA barcode reference library for the spider fauna of any region. Few cryptic species of spiders were detected, a result contrasting with the prevalence of undescribed species in several other terrestrial arthropod groups at Churchill. Because most (97.5%) sequence clusters at COI corresponded with a named taxon, DNA barcoding reliably identifies spiders in the Churchill fauna. The capacity of DNA barcoding to enable the identification of otherwise taxonomically ambiguous specimens (juveniles, females) also represents a major advance for future monitoring efforts on this group.

Antiviral Potential of Specially Selected Bulgarian Propolis Extracts: In Vitro Activity against Structurally Different Viruses.

Propolis is a natural mixture of resins, wax, and pollen from plant buds and flowers, enriched with enzymes and bee saliva. It also contains various essential oils, vitamins, mineral salts, trace elements, hormones, and ferments. It has been found that propolis possesses antimicrobial, antiviral, and anti-inflammatory properties. We have studied the antiviral activity of six extracts of Bulgarian propolis collected from six districts of Bulgaria. The study was conducted against structurally different viruses: human coronavirus strain OC-43 (HCoV OC-43) and human respiratory syncytial virus type 2 (HRSV-2) (enveloped RNA viruses), human herpes simplex virus type 1 (HSV-1) (enveloped DNA virus), human rhinovirus type 14 (HRV-14) (non-enveloped RNA virus) and human adenovirus type 5 (HadV-5) (non-enveloped DNA virus). The influence of the extracts on the internal replicative cycle of viruses was determined using the cytopathic effect (CPE) inhibition test. The virucidal activity, its impact on the stage of viral adsorption to the host cell, and its protective effect on healthy cells were evaluated using the final dilution method, making them the focal points of interest. The change in viral infectivity under the action of propolis extracts was compared with untreated controls, and Δlgs were determined. Most propolis samples administered during the viral replicative cycle demonstrated the strongest activity against HCoV OC-43 replication. The influence of propolis extracts on the viability of extracellular virions was expressed to a different degree in the various viruses studied, and the effect was significantly stronger in those with an envelope. Almost all extracts significantly inhibited the adsorption step of the herpes virus and, to a less extent, of the coronavirus to the host cell, and some of them applied before viral infection demonstrated a protective effect on healthy cells. Our results enlarge the knowledge about the action of propolis and could open new perspectives for its application in viral infection treatment.

Enhancing DNA barcode reference libraries by harvesting terrestrial arthropods at the Smithsonian's National Museum of Natural History.

The use of DNA barcoding has revolutionised biodiversity science, but its application depends on the existence of comprehensive and reliable reference libraries. For many poorly known taxa, such reference sequences are missing even at higher-level taxonomic scales. We harvested the collections of the Smithsonian's National Museum of Natural History (USNM) to generate DNA barcoding sequences for genera of terrestrial arthropods previously not recorded in one or more major public sequence databases. Our workflow used a mix of Sanger and Next-Generation Sequencing (NGS) approaches to maximise sequence recovery while ensuring affordable cost. In total, COI sequences were obtained for 5,686 specimens belonging to 3,737 determined species in 3,886 genera and 205 families distributed in 137 countries. Success rates varied widely according to collection data and focal taxon. NGS helped recover sequences of specimens that failed a previous run of Sanger sequencing. Success rates and the optimal balance between Sanger and NGS are the most important drivers to maximise output and minimise cost in future projects. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, the Global Genome Biodiversity Network Data Portal and the NMNH data portal.

Phagocytosis and killing of Salmonella by 7-hydroxycoumarin activated macrophages.

Coumarin and its derivatives have potent immunomodulatory activities. Here we describe the parameters of the protective effect of 7-hydroxycoumarin (7-OHC) in experimental Salmonella enterica Serovar Typhimurium infection in mice. The protective effect depended on the duration of treatment reaching its maximum after 10 days of pretreatment and lasted for at least 15 days after its end. Electron microscopy studies revealed that 7-OHC induced ultrastructural changes in macrophages consistent with their activation as well as faster destruction of ingested salmonellae. Superoxide and hydrogen peroxide secretion by macrophages was decreased in both healthy and Salmonella-infected 7-OHC treated animals, which is in line with the current view that some coumarins possess antioxidant and radical scavenging activity. Thus, 7-OHC pretreatment also appears beneficial to the host by limiting the harmful tissue damaging and immunosuppressive effects of the oxidative stress during a Salmonella infection but still activates the microbicidal capacity of exposed phagocytes.

Bulgarian Medicinal Extracts as Natural Inhibitors with Antiviral and Antibacterial Activity.

BACKGROUND: Bulgaria is a country with a wide range of medicinal plants, with uses in traditional medicine dating back for centuries. METHODS: Disc diffusion assay was used to evaluate the antimicrobial activity of the plant extracts. A cytopathic effect inhibition test was used for the assessment of the antiviral activity of the extracts. The virucidal activity of the extracts, their influence on the stage of viral adsorption, and their protective effect on uninfected cells were reported using the end-point dilution method, and Δlgs was determined as compared to the untreated controls. RESULTS: The results of the study reveal that the antibacterial potential of G. glabra and H. perforatum extracts in Gram-positive bacteria is more effective than in Gram-negative bacteria. When applied during the replication of HSV-1 and HCov-OC-43, only some of the extracts showed weak activity, with SI between 2 to 8.5. Almost all tested extracts inhibited the extracellular virions of the studied enveloped viruses (HSV-1 and HCov-OC-43) to a greater extent than of the non-enveloped viruses (PV-1 and HAdV-5). They inhibited the stage of viral adsorption (HSV-1) in the host cell (MDBK) to varying degrees and showed a protective effect on healthy cells (MDBK) before they were subjected to viral invasion (HSV-1). CONCLUSION: The antipathogenic potential of extracts of H. perforatum and G. glabra suggests their effectiveness as antimicrobial agents. All 13 extracts of the Bulgarian medicinal plants studied can be used to reduce viral yield in a wide range of viral infections.

Integrated Approaches for the Identification of Mosquitoes (Diptera: Culicidae) from the Volcanoes of Central America Physiographic Subprovince of the State of Chiapas, Mexico.

Nowadays, there is a lack of information on the mosquito's fauna and DNA barcoding sequence reference library from many areas in Mexico, including the Volcanoes of Central America physiographic subprovince in the state of Chiapas. Consequently, a survey was undertaken to delineate the mosquito (Diptera: Culicidae) fauna in this region across different seasons using different collecting techniques. All species were identified by morphology and DNA barcoding, and their ecological features were also defined. In total, 62 taxa were morphologically examined, 60 of these were successfully identified based on morphological characteristics, but two were unable to be identified at the species level. The genera Aedes, Anopheles, Culex, and Wyeomyia are the most diverse among mosquito genera collected and include several species of medical and veterinary importance. Ecological characteristics of the immature habitats indicated that they were grouped into four categories namely, (1) large water bodies at ground level, (2) small and shady phytotelmata (e.g., tree holes and bamboo internodes), (3) large phytotelmata (e.g., plant leaves and axis bromeliad), and (4) artificial containers. The cytochrome c oxidase subunit I (COI) DNA barcoding sequences successfully separated the majority of these species, although specific species showed >2% intraspecific genetic divergences.

DNA Barcoding of Mosquitoes from the Pantanos de Centla Biosphere Reserve, Southeastern Mexico.

Accurate identification of mosquito species is essential to support programs that involve the study of distribution and mosquito control. Numerous mosquito species are difficult to identify based only on morphological characteristics, due to the morphological similarities in different life stages and large numbers of some species that are members of morphologically similar species complexes. In the present study, the mosquitoes collected in the Pantanos de Centla Biosphere Reserve, southeastern Mexico, were evaluated using a combination of morphological and molecular approaches (mitochondrial cytochrome c oxidase subunit I [COI] DNA barcode). A total of 1,576 specimens of 10 genera and 35 species, mostly adult stages, were collected. A total of 225 COI DNA barcode sequences were analyzed; most species formed well-supported groups in the neighbor joining, maximum likelihood, and Bayesian inference trees. The intraspecific Kimura 2-parameter (K2P) genetic distance averaged 1.52%. An intraspecific K2P distance of 6.20% was observed in Anopheles crucians s.l., while a deep split was identified in Culex erraticus and Cx. conspirator. This study showed that COI DNA barcodes offer a reliable approach to support mosquito species identification in Mexico.

Identification of mosquitoes (Diptera: Culicidae) from Mexico State, Mexico using morphology and COI DNA barcoding.

Mosquitoes are commonly identified to species level using morphological traits, but complementary methods for identification are often necessary when specimens are collected as immature stages, stored inadequately, or when delineation of species complexes is problematic. DNA-barcoding using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene is one such tool used for the morphological identification of species. A comprehensive entomological survey of mosquito species in Mexico State identified by COI DNA barcoding and morphology is documented in this paper. Specimens were collected from all the physiographic provinces in Mexico State between 2017 and 2019. Overall, 2,218 specimens were collected from 157 localities representing both subfamilies Anophelinae and Culicinae. A species checklist that consists of 6 tribes, 10 genera, 20 subgenera, and 51 species, 35 of which are new records for Mexico State, is provided. Three hundred and forty-two COI sequences of 46 species were analysed. Mean intraspecific and interspecific distances ranged between 0% to 3.9% and from 1.2% to 25.3%, respectively. All species groups were supported by high bootstraps values in a Neighbour-Joining analysis, and new COI sequences were generated for eight species: Aedes chionotum Zavortink, Ae. vargasi Schick, Ae. gabriel Schick, Ae. guerrero Berlin, Ae. ramirezi Vargas and Downs, Haemagogus mesodentatus Komp and Kumm, Culex restrictor Dyar and Knab, and Uranotaenia geometrica Theobald. This study provides a detailed inventory of the Culicidae from Mexico State and discusses the utility of DNA barcoding as a complementary tool for accurate mosquito species identification in Mexico.

Effect of yeast superoxide dismutase treatment on some mediators of inflammation during adjuvant-induced arthritis in mice.

* Author for correspondence and reprint requests Z. Naturforsch. 65c, 141-147 (2010); received June 17/July 21, 2009 The superoxide radical (O2-), hydrogen peroxide (H2O2) and nitric oxide (NO) are pleiotropic inflammatory mediators which play an important role in inflammatory joint diseases. They are overproduced during rheumatoid arthritis and its experimental model - adjuvant-induced arthritis in rodents--and may be detected both systemically and intra-articularly. Their secretion is up-regulated by proinflammatory cytokines such as IFN-gamma, IL-12, IL-6 and TNF-alpha, and they are responsible for the destruction of joint tissue. In this work, the effect of superoxide dismutase (SOD) from a thermotolerant yeast strain, Kluyveromyces marxianus, on the production of proinflammatory cytokines, reactive oxygen and nitrogen species was studied. Mice received three intraperitoneal injections of yeast SOD at a dose of 10 mg/ kg body weight (30,000 U/kg) on consecutive days starting on the day after arthritic induction. On days 3, 8 and 14 post induction peritoneal macrophages were isolated and both spontaneous and stimulated production of reactive oxygen and nitrogen metabolites were measured. Early in arthritic development yeast SOD treatment did not influence the O2- production, but on day 14 both spontaneous and PMA-induced secretion were dramatically reduced. Spontaneous H2O2 release was inhibited on day 14, while PMA-stimulated production was decreased from the beginning of the arthritic development. Yeast SOD treatment effectively suppressed the spontaneous and recombinant mouse IFN-gamma + LPS induced release of NO as well. Serum levels of proinflammatory cytokines, IL-12, IFN-gamma, IL-6 and TNF-alpha, were also significantly reduced. The obtained results show some of the mechanisms of action of SOD in reducing the severity of arthritic inflammation. Besides direct inhibition of joint tissue destruction exogenous SOD substantially limits the existing positive feedback between secretion of reactive oxygen species and inflammatory cytokine production.

Integrated Approaches in Support of Taxonomic Identification of Mosquitoes (Diptera: Culicidae) in Vector Surveillance in Spain.

The pandemic of Zika virus in 2016 and other arboviruses prompted La Rioja Government in Spain to implement an entomological surveillance program of mosquitoes (Diptera; Culicidae) in the region of La Rioja. The morphological identification was supported by genetic analysis using the COI (cytochrome c oxidase subunit I) and the ITS2 (internal transcribed spacer 2) genes. In total, we identified 24 species arranged in 6 genera: Aedes (7 species), Anopheles (4 species), Coquillettidia (1 species), Culex (7 species), Culiseta (4 species), and Uranotaenia (1 species). Aedes sticticus and Aedes geniculatus are newly reported for La Rioja region. In total, 465 COI sequences were analyzed for Culicinae and Anophelinae and 54 ITS2 sequences for Anophelinae; all individuals identified as the same species clustered together in the Neighbor Joining trees. The levels of sequence divergence based on COI ranged between 0% and 2.62%, while the interspecific genetic divergence ranged from 3.05% to 20.07%. Within the genus Culiseta, certain specimens of Culiseta annulata, Culiseta litorea, and Culiseta subochrea were morphologically misidentified due to variation in the main diagnostic characters. The interspecific genetic divergence based on the ITS2 ranged from 0% to 2.98%. An accurate identification of mosquito vectors is the first step to establish a vector surveillance program for preventing pathogen transmission.

Antiviral, Cytotoxic and Antioxidant Effects of Tanacetum Vulgare L. Crude Extract In Vitro.

INTRODUCTION: Due to the high prevalence of viral infections having no specific treatment and the constant emergence of resistant viral strains, searching for effective antiviral compounds is crucial. The present study explores in vitro the antiviral activity of ethanolic extract from aerial parts of. AIM: The aim of the current study was to evaluate antiviral activity of ethanolic extract from herbaceous plant. MATERIALS AND METHODS: The crude aqueous ethanolic extract from aerial parts of. RESULTS: The results show that the extract has the lowest toxicity on the MDBK cell line and similar cytotoxicity in Hep-2, whereas in the MDCK cells it has more than twice the highest toxicity. Testing the antiviral activity of. CONCLUSION: The crude extract from aerial parts of the medicinal plant.

Ticks on the Channel Islands and implications for public health.

The Channel Islands are British Crown dependencies located in the English Channel to the west of the Normandy coast in northern France. Whilst there have been studies investigating tick occurrence and distribution in different habitats on the mainland of the UK and in France, the Channel Islands have been relatively understudied. As such, little is known about whether the sheep tick, Ixodes ricinus, is present, and whether there is a potential risk of Lyme borreliosis on the Channel Islands. To ascertain the presence of I. ricinus on the three largest islands in the archipelago: Jersey, Guernsey and Alderney, surveys of ticks questing in the vegetation and ticks feeding on hosts were undertaken during April and May 2016. Across all three islands, the highest numbers of ticks were found in woodland habitats. Ixodes ricinus was the predominant questing tick species found on Jersey, and Ixodes ventalloi the most common questing tick species on Alderney and Guernsey, with little or no evidence of questing I. ricinus on either island. During field studies on small mammals, I. ricinus was the predominant tick species feeding on Jersey bank voles (Myodes glareolus caesarius), with Ixodes hexagonus the most common species infesting hedgehogs on Guernsey. We propose that the greater diversity of small mammals on Jersey may be important in supporting immature stages of I. ricinus, in contrast to Guernsey and Alderney. Morphological identification of tick species was confirmed by PCR sequencing based on amplification of the cytochrome c oxidase subunit one (cox1) gene (COI DNA barcoding). To date, there have been few records of human tick bites in the Channel Islands, suggesting that the current risk from tick-borne disease may be low, but continued reporting of any human tick bites, along with reporting of cases of Lyme borreliosis will be important for continued assessment of the impact of tick-borne diseases in the Channel Islands.

An Integrated Molecular Approach to Untangling Host-Vector-Pathogen Interactions in Mosquitoes (Diptera: Culicidae) From Sylvan Communities in Mexico.

There are ~240 species of Culicidae in Mexico, of which some are vectors of arthropod-borne viruses such as Zika virus, dengue virus, chikungunya virus, and West Nile virus. Thus, the identification of mosquito feeding preferences is paramount to understanding of vector-host-pathogen interactions that, in turn, can aid the control of disease outbreaks. Typically, DNA and RNA are extracted separately for animal (insects and blood meal hosts) and viral identification, but this study demonstrates that multiple organisms can be analyzed from a single RNA extract. For the first time, residual DNA present in standard RNA extracts was analyzed by DNA barcoding in concert with Sanger and next-generation sequencing (NGS) to identify both the mosquito species and the source of their meals in blood-fed females caught in seven sylvan communities in Chiapas State, Mexico. While mosquito molecular identification involved standard barcoding methods, the sensitivity of blood meal identification was maximized by employing short primers with NGS. In total, we collected 1,634 specimens belonging to 14 genera, 25 subgenera, and 61 morphospecies of mosquitoes. Of these, four species were new records for Mexico (Aedes guatemala, Ae. insolitus, Limatus asulleptus, Trichoprosopon pallidiventer), and nine were new records for Chiapas State. DNA barcode sequences for >300 bp of the COI gene were obtained from 291 specimens, whereas 130 bp sequences were recovered from another 179 specimens. High intraspecific divergence values (>2%) suggesting cryptic species complexes were observed in nine taxa: Anopheles eiseni (5.39%), An. pseudopunctipennis (2.79%), Ae. podographicus (4.05%), Culex eastor (4.88%), Cx. erraticus (2.28%), Toxorhynchites haemorrhoidalis (4.30%), Tr. pallidiventer (4.95%), Wyeomyia adelpha/Wy. guatemala (7.30%), and Wy. pseudopecten (4.04%). The study increased the number of mosquito species known from 128 species to 138 species for Chiapas State, and 239 for Mexico as a whole. Blood meal analysis showed that Aedes angustivittatus fed on ducks and chicken, whereas Psorophora albipes fed on humans. Culex quinquefasciatus fed on diverse hosts including chicken, human, turkey, and Mexican grackle. No arbovirus RNA was detected by reverse transcriptase-polymerase chain reaction in the surveyed specimens. This study demonstrated, for the first time, that residual DNA present in RNA blood meal extracts can be used to identify host vectors, highlighting the important role of molecular approaches in both vector identification and revealing host-vector-pathogen interactions.

DNA barcoding of British mosquitoes (Diptera, Culicidae) to support species identification, discovery of cryptic genetic diversity and monitoring invasive species.

Correct mosquito species identification is essential for mosquito and disease control programs. However, this is complicated by the difficulties in morphologically identifying some mosquito species. In this study, variation of a partial sequence of the cytochrome c oxidase unit I (COI) gene was used for the molecular identification of British mosquito species and to facilitate the discovery of cryptic diversity, and monitoring invasive species. Three DNA extraction methods were compared to obtain DNA barcodes from adult specimens. In total, we analyzed 42 species belonging to the genera Aedes Meigen, 1818 (21 species), Anopheles Meigen, 1818 (7 species), Coquillettidia Theobald, 1904 (1 species), Culex Linnaeus, 1758 (6 species), Culiseta Felt, 1904 (7 species), and Orthopodomyia Theobald, 1904 (1 species). Intraspecific genetic divergence ranged from 0% to 5.4%, while higher interspecific divergences were identified between Aedesgeminus Peus, 1971/Culisetalitorea (Shute, 1928) (24.6%) and Ae.geminus/An.plumbeus Stephens, 1828 (22.5%). Taxonomic discrepancy was shown between An.daciae Linton, Nicolescu & Harbach, 2004 and An.messeae Falleroni, 1828 indicating the poor resolution of the COI DNA barcoding region in separating these taxa. Other species such as Ae.cantans (Meigen, 1818)/Ae.annulipes (Meigen, 1830) showed similar discrepancies indicating some limitation of this genetic marker to identify certain mosquito species. The combination of morphology and DNA barcoding is an effective approach for the identification of British mosquitoes, for invasive mosquitoes posing a threat to the UK, and for the detection of hidden diversity within species groups.