Genetic diversity and population structure of Culex modestus across Europe: does recent appearance in the United Kingdom reveal a tendency for geographical spread?

In mainland Europe, the mosquito species Culex modestus Ficalbi (1890) is a bridge vector for West Nile virus (WNV) from its natural bird-mosquito cycle to mammals. The present study assessed the genetic diversity of Cx. modestus, as well as related Culex species, using the mitochondrial COI DNA barcoding region and compared this with the population structure across Europe. A haplotype network was mapped to determine genealogical relationships among specimens. The intraspecific genetic diversity within individual Culex species was below 2%, whereas the interspecific genetic divergence varied from 2.99% to 13.74%. In total, 76 haplotypes were identified among 198 sequences. A median-joining network determined from 198 COI sequences identified two major lineages that were separated by at least four mutation steps. A high level of intraspecific genetic diversity was not detected in Cx. modestus in samples submitted from different European populations, which indicates that morphologically identified specimens represent a single species and not a species complex. Therefore, it is deduced that different populations of Cx. modestus will show a similar potential to transmit WNV, lending support to concerns that the population present in southeast England represents a risk of transmission to humans.

Detection of Borrelia and Babesia species in Haemaphysalis punctata ticks sampled in Southern England.

The distribution and population size of the red sheep tick (Haemaphysalis punctata) are increasing in Northern Europe, and in the United Kingdom reports of human biting by this species have increased in recent years. To assess the risk of tick-borne disease (TBD) transmission to humans and livestock by H. punctata, ticks sampled from sites in Southern England were screened using PCR for either Borrelia species or piroplasms over a three year period, 2018-2020. A total of 302 H. punctata were collected from eight locations. From these, two Babesia species associated with TBD infections in livestock, Babesia major and Babesia motasi, and the human pathogen Borrelia miyamotoi were detected, predominantly from a single location in Sussex. Consequently, the range expansion of this tick across Southern England may impact public and livestock health.

First report of human exposure to Hyalomma marginatum in England: Further evidence of a Hyalomma moulting event in north-western Europe?

Hyalomma marginatum is widely distributed across the Mediterranean, Northern Africa and the Middle East. Current climate conditions in Northern Europe are thought to limit the species' ability to moult to the adult stage. It is a vector of several pathogens of human and veterinary concern, including Crimean-Congo haemorrhagic fever virus, for which it is the primary vector in Europe. Here, we report the first human exposure to a locally acquired adult H. marginatum in England, and the second detection in England of Rickettsia aeschlimannii associated with imported Hyalomma.

First Record of Culex (Microculex) daumastocampa (Diptera: Culicidae) in Mexico, with Notes on Cx. rejector and Cx. imitator.

Culex (Microculex) daumastocampa Dyar & Knab was originally described by Dyar and Knab (Proc US Nat Mus 35:53-70, 1908) from larvae collected at axils of bromeliads in Port San Felipe, Panama. Culex daumastocampa is found in Colombia, Venezuela, Panama, Suriname, Costa Rica, Nicaragua, Guatemala, and Mexico, although its presence had not been reported suggesting its northernmost distribution. In Mexico, the subgenus Microculex had included Cx. rejector Dyar and Knab, and Cx. imitator Theobald. However, after that collection specimens were re-examined along with other specimens collected during 2016 in Chiapas (all specimens are available in the Culicidae Collection of the Universidad Autonoma Agraria Antonio Narro Unidad Laguna [UAAAN-UL], Mexico). Culex daumastocampa is now reported for the first time in Mexico, Cx. rejector for Chiapas, and Cx. imitator removed from the checklist of previous reports as to be present in Mexico.

Blood-feeding patterns of native mosquitoes and insights into their potential role as pathogen vectors in the Thames estuary region of the United Kingdom.

BACKGROUND: The range of vertebrate hosts on which species of mosquito blood-feed is an important parameter for identifying potential vectors and in assessing the risk of incursion and establishment of vector-borne pathogens. In the United Kingdom, studies of mosquito host range have collected relatively few specimens and used techniques that could only broadly identify host species. This study conducted intensive collection and analysis of mosquitoes from a grazing marsh environment in southeast England. This site provides extensive wetland habitat for resident and migratory birds and has abundant human nuisance biting mosquitoes. The aim was to identify the blood-feeding patterns of mosquito species present at the site which could contribute to the transmission of pathogens. METHODS: Twice-weekly collections of mosquitoes were made from Elmley Nature Reserve, Kent, between June and October 2014. Mosquitoes were collected using resting boxes, by aspiration from man-made structures and using a Mosquito Magnet Pro baited with 1-octen-3-ol. Blood-fed specimens were classified according to the degree of blood meal digestion using the Sella scale and vertebrate origin determined using sequencing of a fragment of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes that were morphologically cryptic were identified to species level using multiplex PCR and sequencing methods. RESULTS: A total of 20,666 mosquitoes of 11 species were collected, and 2,159 (10.4%) were blood-fed (Sella scale II-VI); of these 1,341 blood-fed specimens were selected for blood meal analysis. Vertebrate origin was successfully identified in 964 specimens (72%). Collections of blood-fed individuals were dominated by Anopheles maculipennis complex (73.5%), Culiseta annulata (21.2%) and Culex pipiens form pipiens (10.4%). Nineteen vertebrate hosts comprising five mammals and 14 birds were identified as hosts for mosquitoes, including two migratory bird species. Feeding on birds by Culex modestus and Anopheles atroparvus populations in England was demonstrated. CONCLUSIONS: This study expands the vertebrate host range of mosquitoes in the Thames estuary region of the UK. Feeding on both resident and migratory bird species by potential arbovirus vectors including Cx. pipiens f. pipiens and Cx. modestus indicates the potential for enzootic transmission of an introduced arbovirus between migratory and local bird species by native mosquito species.

Detection of Theileria orientalis in mosquito blood meals in the United Kingdom.

Theileria spp. are tick-borne protozoan parasites that infect a wide range of wild and domestic animals. In this study, the utility of xenosurveillance of blood-fed specimens of Culiseta annulata for detecting the presence of piroplasms in livestock was investigated. Blood-fed mosquitoes were collected at Elmley National Nature Reserve, Kent, United Kingdom. All specimens were morphologically identified, and DNA barcoding was used to confirm the morphological identification. Both the vertebrate host species and Theileria genome was detected within the bloodmeal by real-time PCR. Sequencing was used to confirm the identity of all amplicons. In total, 105 blood-fed mosquitoes morphologically identified as Cs. annulata were collected. DNA barcoding revealed that 102 specimens were Cs. annulata (99%), while a single specimen was identified as Anopheles messeae. Two specimens could not be identified molecularly due to PCR amplification failure. Blood meal analysis revealed that Cs. annulata fed almost exclusively on cattle at the collection site (n=100). The application of a pan-piroplasm PCR detected 16 positive samples (15.2%) and sequence analysis of the amplicons demonstrated that the piroplasms present in the blood meal belonged to the Theileria orientalis group. This study demonstrates how xenosurveillance can be applied to detecting pathogens in livestock and confirms the presence of Theileria species in livestock from the United Kingdom.

Recovery of DNA barcodes from blackfly museum specimens (Diptera: Simuliidae) using primer sets that target a variety of sequence lengths.

In this study, we evaluated the efficacy of various primers for the purpose of DNA barcoding old, pinned museum specimens of blackflies (Diptera: Simuliidae). We analysed 271 pinned specimens representing two genera and at least 36 species. Due to the age of our material, we targeted overlapping DNA fragments ranging in size from 94 to 407 bp. We were able to recover valid sequences from 215 specimens, of which 18% had 500- to 658-bp barcodes, 36% had 201- to 499-bp barcodes and 46% had 65- to 200-bp barcodes. Our study demonstrates the importance of choosing suitable primers when dealing with older specimens and shows that even very short sequences can be diagnostically informative provided that an appropriate gene region is used. Our study also highlights the lack of knowledge surrounding blackfly taxonomy, and we briefly discuss the need for further phylogenetic studies in this socioeconomically important family of insects.