Large-scale performance assessment of the BG-Counter 2 used with two different mosquito traps.

BACKGROUND: Mosquitoes are important vectors of pathogens. They are usually collected with CO2-baited traps and subsequently identified by morphology. This procedure is very time-consuming. Automatic counting traps could facilitate timely evaluation of the local risk for mosquito-borne pathogen transmission or decision-making on vector control measures, but the counting accuracy of such devices has rarely been validated in the field. METHODS: The Biogents (BG)-Counter 2 automatically counts mosquitoes by discriminating the size of captured objects directly in the field and transmits the data to a cloud server. To assess the accuracy of this counting device, 27 traps were placed at 19 sampling sites across Germany and used in daily, weekly or bimonthly intervals from April until October 2021. The BG-Counter 2 was attached to a CO2-trap (BG-Pro trap = CO2-Pro) and the same trap was converted to also attract gravid mosquitoes (upside-down BG-Pro trap with a water container beneath = CO2-Pro-gravid). All captured mosquitoes were identified by morphology. The number of females (unfed and gravid), mosquito diversity and the number of identified specimens in relation to the counting data of the BG-Counter were compared between the two trapping devices to evaluate sampling success and counting accuracy. RESULTS: In total 26,714 mosquitoes were collected during 854 trap days. The CO2-Pro-gravid trap captured significantly more mosquitoes per trap day for all specimens, gravid females and non-gravid females, while there was no difference in the mosquito diversity. The linear model with the captured mosquitoes as a response and the counted specimens as a predictor explained only a small degree of the variation within the data (R2 = 0.16), but per individual trap the value could reach up to 0.62 (mean R2 = 0.23). The counting accuracy for the daily samples had a significant positive correlation with sample size, resulting in higher accuracy for the CO2-Pro-gravid trap and higher accuracy for sites and sampling months with high mosquito abundance. CONCLUSIONS: While the accuracy of the BG-Counter 2 is quite low, the device is able to depict mosquito phenology and provide information about local population dynamics.

The first record of the invasive mosquito species Aedes albopictus in Chisinӑu, Republic of Moldova, 2020.

BACKGROUND: In Europe, Aedes albopictus is an important vector of chikungunya virus and Dirofilaria nematodes and has been involved in local autochthonous circulation of dengue and Zika viruses. Due to the ongoing spread, targeted field surveillance at potential points of entry of invasive Aedes mosquitoes was initiated by the Republic of Moldova in 2020 as part of the transboundary "Invasive Aedes Mosquitoes COST-Action project." METHODS: In 2020, ovitraps were positioned at each of three locations: the border crossing to Romania in Leuseni (Hancesti region), Chisinӑu International Airport and Chisinӑu Botanical Garden. RESULTS: A total of 188 Aedes spp. eggs were collected at the Chisinӑu International Airport between August and September 2020. Twenty-three adults reared in the laboratory were identified morphologically as Ae. albopictus (Skuse, 1895), and 12 selected specimens were confirmed by molecular barcoding of the cytochrome oxidase subunit I gene region. In addition, one adult Ae. albopictus female at the same site was caught with a manual aspirator. CONCLUSIONS: This is the first documented report of Ae. albopictus in the Republic of Moldova. The presence of immature and adult stages indicates the local reproduction of the species in the country. Therefore, it is crucial to extend and strengthen surveillance of the invasive Aedes mosquitoes to prevent Ae. albopictus and other exotic mosquito species from becoming established in the Republic of Moldova.

Phlebotomine sand fly survey in the Republic of Moldova: species composition, distribution and host preferences.

BACKGROUND: Phlebotomine sand flies (Diptera: Psychodiae) in the Republic of Moldova have been understudied for decades. Our study provides a first update on their occurrence, species composition and bloodmeal sources after 50 years. METHODS: During 5 seasons (2013-2017), 58 localities from 20 regions were surveyed for presence of sand flies using CDC light traps and manual aspirators. Species identification was done by a combination of morphological and molecular approaches (DNA barcoding, MALDI-TOF MS protein profiling). In engorged females, host blood was identified by three molecular techniques (RFLP, cytb sequencing and MALDI-TOF peptide mass mapping). Population structure of most abundant species was studied by cox1 haplotyping; phylogenetic analyses of ITS2 and cox1 genetic markers were used to resolve relationships of other detected species. RESULTS: In total, 793 sand flies were collected at 30 (51.7%) localities from 12 regions of Moldova. Three species were identified by an integrative morphological and molecular approach: Phlebotomus papatasi, P. perfiliewi and Phlebotomus sp. (Adlerius), the first being the most abundant and widespread, markedly anthropophilic based on bloodmeal analyses, occurring also indoors and showing low population structure with only five haplotypes of cox1 detected. Distinct morphological and molecular characters of Phlebotomus sp. (Adlerius) specimens suggest the presence of a yet undescribed species. CONCLUSIONS: Our study revealed the presence of stable sand fly populations of three species in Moldova that represent a biting nuisance as well as a potential threat of pathogen transmission and shall be further studied.

Circulation of Dirofilaria repens and Dirofilaria immitis in Moldova.

BACKGROUND: Over the last two decades, a significant spread of dirofilariasis has been observed in eastern and central Europe. However, data on the circulation of Dirofilaria spp. in Moldova were absent although direct neighbor states reported high incidence rates of human dirofilariasis. METHODS: Daily mean temperature data were used to calculate Dirofilaria spp. development units, which were used to estimate the potential for complete extrinsic development in the mosquitoes (= sum of potential Dirofilaria spp. transmission days). In addition, 4,481 adult female mosquitoes were collected from 25 trapping sites. From 2010 to 2015, sampling was conducted with Centers for Disease Control miniature light traps, indoor resting mosquito collections as well as human landing catches in urban, rural and natural areas. Mosquitoes were analyzed for the presence of D. repens and D. immitis DNA using a duplex real-time PCR assay targeting nucleotide differences within the cytochrome c oxidase subunit 1 (D. repens) and 16S rRNA gene fragment (D. immitis). RESULTS: The average of the yearly sum of potential Dirofilaria spp. transmission days between 2010 and 2015 ranged from 90 to 140 days with an increasing gradient from the North to the South of Moldova. Positive mosquito pools for D. repens were found countrywide at 13 of the 25 trapping sites and in 17 of the 22 screened mosquito taxa (26.51% of all 347 tested pools), while D. immitis was detected only at 4 of the trapping sites (Center and South) in 4 different mosquito species (8.65% of all 347 tested pools). Highest infection rates (EIR) per 100 specimens for both Dirofilaria species were found in An. maculipennis (s.l.) (D. repens: EIR = 4.91; D. immitis: EIR = 2.01), whereas the most frequent mosquito taxon Cx. pipiens (s.l.)/torrentium had significantly lower infections rates (D. repens: EIR = 0.88; D. immitis: EIR = 0.47). CONCLUSIONS: The temperature conditions in Moldova are suitable for transmission of Dirofilaria spp. within the entire country, which is supported by a wide distribution of Dirofilaria spp.-positive mosquitoes with high infection rates. The low number of reported human cases most likely does not reflect the current epidemiological situation of dirofilariasis in Moldova.

Detection of Dirofilaria repens and Dirofilaria immitis DNA in mosquitoes from Belarus.

During the last two decades, Belarus faces an increase of human cases of Dirofilaria (Nematoda, Spirurida, Onchocercidae) infections. However, comprehensive analyses explaining this development and the identification of mosquito vector species are missing. Here, we present results using temperature data from Belarus and show that the annual number of human Dirofilaria cases is significantly correlated with the yearly average temperatures (Spearman's rho = 0.49, p < 0.05) and the average sum of potential Dirofilaria transmission days (Spearman's rho = 0.46, p < 0.05), suggesting that autochthonous transmission is at least in part responsible for the increasing number of clinical Dirofilaria cases in the country. In addition, 467 female mosquitoes were collected from different sampling sites in the regions of Brest and Minsk, which were analyzed by molecular methods for the presence of Dirofilaria repens and Dirofilaria immitis DNA, respectively. Two pools (5.56 %) were tested positive for Dirofilaria (estimated infection rate per 100 specimens = 0.44, 95 % confidence interval = 0.08-1.43), comprising one Anopheles claviger s.l. pool that was positive for D. repens and one Culex pipiens s.l./Culex torrentium pool positive for D. immitis DNA. This, to our knowledge, is the first molecular evidence for the presence of Dirofilaria in mosquitoes from Belarus, suggesting a high probability of autochthonous Dirofilaria transmission in the country.