Effects of Hibernation Site, Temperature, and Humidity on the Abundance and Survival of Overwintering Culex pipiens pipiens and Anopheles messeae (Diptera: Culicidae).

Knowledge of the hibernation site preferences and the factors which influence winter survival in these hibernation sites may enhance understanding of mosquito population dynamics after winter and how arboviruses persist in temperate regions. Our study quantified the number of adult overwintering mosquitoes in cellars and aboveground constructions and analyzed survival rates in relation to the environmental conditions in these sites. During the winters 2016/2017 and 2018/2019, 149 different constructions in Northwest Germany were sampled for mosquitoes. Mosquitoes were detected in 44% of the cellars and in 33% of the aboveground constructions. Culex p. pipiens Linnaeus was the most abundant species in cellars, whereas high numbers of Anopheles messeae Falleroni were collected from a single barn. Subsequently, an enclosure study was conducted during 2019/2020. Overwintering field-collected Cx. p. pipiens and An. messeae were divided into groups with or without fructose availability, and placed in cages with different man-made hibernations sites, where temperature and relative humidity were recorded hourly. For both species, increasing mean temperatures (5-16°C) but not mean relative humidity (58-94%) were correlated with winter mortality rates of the mosquitoes. The lipid measurements were greater and mortality rates were lower when both species were provided fructose. Larger specimens (determined by wing length) stored more lipids, and in Cx. p pipiens, but not in An. messeae, survival probability of large specimens was significantly greater than for small females. Mosquitoes showed a distinct pattern in the selection of overwintering sites, while temperature was an important driver for survival.

Habitat and microclimate affect the resting site selection of mosquitoes.

Mosquitoes (Diptera: Culicidae) use certain resting sites during their inactive phase. The microclimatic conditions of these resting sites might affect their physiology and vectorial capacity. In this study, we combined a field and a laboratory study to investigate the natural resting site and temperature preferences of mosquitoes. The field study was conducted at a forest close to Oldenburg (Lower Saxony, Germany) from May to October 2018. Mosquitoes were collected in five different natural habitats with seven replicates each. Temperature was recorded hourly at each site. Significantly more mosquitoes were collected in deadwood (predominantly Culiseta morsitans/fumipennis) and shaded herb layer (predominantly Aedes species) compared to unshaded herb layer or broadleaf and coniferous trees. GLMMs revealed resting site habitats as the best predictor to explain the observed preference patterns, but microclimatic conditions are also involved in mosquito resting site selection. Most mosquitoes were collected at resting sites with relatively colder and more stable temperatures. In concert, laboratory choice experiments with a thermal gradient ring demonstrated that Cs. morsitans/fumipennis avoid temperatures over 30 °C. Understanding the small-scaled resting site preferences and the related microclimatic conditions can improve mosquito collection techniques and refine the prediction of mosquito-borne pathogen transmission.

Geometric morphometric wing analysis represents a robust tool to identify female mosquitoes (Diptera: Culicidae) in Germany.

Accurate species identification is the prerequisite to assess the relevance of mosquito specimens, but is often hindered by missing or damaged morphological features. The present study analyses the applicability of wing geometric morphometrics as a low-cost and practical alternative to identify native mosquitoes in Germany. Wing pictures were collected for 502 female mosquitoes of five genera and 19 species from 80 sampling sites. The reliable species identification based on interspecific wing geometry of 18 landmarks per specimen was tested. Leave-one-out cross validation revealed an overall accuracy of 99% for the genus and 90% for the species identification. Misidentifications were mainly due to three pairings of Aedes species: Aedes annulipes vs. Aedes cantans, Aedes cinereus vs. Aedes rossicus and Aedes communis vs. Aedes punctor. Cytochrome oxidase subunit I (COI) gene region was sequenced to validate the morphological and morphometric identification. Similar to the results of the morphometric analysis, the same problematic three Aedes-pairs clustered, but most other species could be well separated. Overall, our study underpins that morphometric wing analysis is a robust tool for reliable mosquito identification, which reach the accuracy of COI barcoding.

Comparison of emergence traps of different shape and translucency in the trapping of Culicoides (Diptera: Ceratopogonidae).

Various types of emergence traps are available for investigations of the breeding habitats of Culicoides (Diptera: Ceratopogonidae). In order to assess the potential impact of the trap design on the trapping success, we compared the efficiency of opaque and white (more translucent) emergence traps and two trap shapes (cone-shaped and quadratic), to sample Culicoides emerging from cowpats. Significantly higher numbers of Culicoides chiopterus and Culicoides dewulfi were trapped with opaque traps, while there was no obvious effect of the trap shape. There were no distinct differences in the microclimate among different trap types.

Impact of experimental flooding on larvae and pupae of dung-breeding Culicoides.

Culicoides biting midges (Diptera: Ceratopogonidae) spend the greatest part of their life in the larval stage. However, knowledge on the immature stages and the impact of abiotic factors on their development is still poor. Therefore, we investigated the effect of flooding on the larvae and pupae of Culicoides chiopterus (Meigen, 1830) and C. dewulfi Goetghebuer, 1936. In water, the larvae of both species showed head-to-tail flexions and sinuous flexions, at slow rates, but were not able to swim. Flooding of larvae for 24 h did not affect the number of emerging adults; flooding of pupae significantly reduced the emergence rate of C. chiopterus, compared to the control group, while C. dewulfi was not affected. Pupae were not able to float and no pupae survived flooding for 10 days. After flooding of larvae for 10 days, 50 % of C. chiopterus and 4 % of C. dewulfi completed the pre-adult development. During this treatment, 84 % of C. chiopterus and 48 % of C. dewulfi larvae pupated in water.

Distribution of individual members of the mosquito Anopheles maculipennis complex in Germany identified by newly developed real-time PCR assays.

Owing to their role as vectors of malaria parasites, species of the Anopheles maculipennis complex (Diptera: Culicidae) Meigen were intensively studied in the past, but with the disappearance of malaria in Germany in the middle of the last century, the interest in this field of research declined. A comprehensive ecological analysis of the current species distribution for Germany is lacking. Between 2010 and 2013, a total of 1445 mosquitoes of the An. maculipennis complex were collected at 72 different sites in Germany. The samples comprise 722 single individuals as well as 723 individuals in 90 pools of up to 25 mosquitoes. All samples were analysed with newly developed species-specific qPCR assays for the identification of the four German species using nucleotide differences within the internal transcribed spacer 2 (ITS2) ribosomal DNA. All gathered data were used for species distribution modelling. The overall prevalence of An. messeae s.l. was highest with 98.89% of all pools; An. daciae with 6.93% of all individuals and An. messeae s.s. with 69.53%. The prevalence of the other two species was relatively low: An. maculipennis s.s. with 13.30% of all individuals (6.67% of all pools) and An. atroparvus with 1.80% of all individuals (1.11% of all pools).

Emergence of Culicoides obsoletus group species from farm-associated habitats in Germany.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) may transmit several arboviruses to ruminant livestock. The species of the Obsoletus group are considered to be among the most important vectors of bluetongue virus (BTV) in northern Europe. As agricultural environments offer suitable habitats for the development of their immature stages, the emergence of adult Culicoides from potential breeding sites was investigated at 20 cattle farms throughout Germany in 2012 and 2013. In analyses of species-specific habitat preferences and relationships between Culicoides abundance in breeding substrates and their physicochemical characteristics, dungheaps emerged as the most important substrate for the development of Culicoides obsoletus sensu stricto (s.s.) (Meigen), whereas Culicoides chiopterus (Meigen) and Culicoides dewulfi Goetghebuer were generally restricted to cowpats. A decreasing pH value was associated with a higher abundance or a higher probability of observing these three species. Furthermore, the abundance of C. obsoletus s.s. was positively related to increasing moisture. Dungheaps were very productive breeding sites for this species and are therefore suggested as a target for potential control measures.

Impact of freezing on the emergence of Culicoides chiopterus and Culicoides dewulfi from bovine dung.

The emergence of Culicoides chiopterus (Meigen), 1830 and C. dewulfi Goetghebuer, 1936 (Diptera: Ceratopogonidae) from cowpats in northwestern Germany was investigated. In order to investigate the survival of both species at low temperatures, cowpat subsamples were frozen for 48h at -18 and -21°C. Emergence from frozen and non-frozen samples was compared. The number of emerging adults of C. chiopterus from samples frozen at -18°C was greatly reduced and no emergence was observed from samples frozen at -21°C. No adult C. dewulfi emerged from frozen samples, suggesting this species is less resistant to these temperatures, compared to C. chiopterus.

Assessment of the abundance of Culicoides chiopterus and Culicoides dewulfi in bovine dung: a comparison of larvae extraction techniques and emergence traps.

We compared two larvae extraction methods involving sugar-flotation and an adapted Berlese funnel-extraction with emergence traps. This was done in order to analyse the colonisation of cowpats by Culicoides chiopterus (Meigen 1830) and Culicoides dewulfi (Goetghebuer 1936) (Diptera: Ceratopogonidae) and to gain information on the comparability and efficacy of these three methods. With all three methods, a considerable number of individuals was obtained. Significantly more individuals were obtained via sugar-flotation and Berlese funnel-extraction compared to the emergence traps. These differences, likely due to natural mortality and sample processing are discussed. We recommend Berlese as an efficacious method for extracting Culicoides larvae from bovine dung. It produces data rapidly and extracted larvae are viable. In comparison with Berlese, slightly more larvae were obtained by sugar-flotation, but this method was very labour intensive.

Mosquitoes (Diptera: Culicidae) of metropolitan Hamburg, Germany.

In Europe, mosquito-related public health concerns are growing due to the increasing spread of invasive mosquito species and the recent emergence of mosquito-borne arboviruses. A vital backbone in the assessment of these issues is detailed knowledge of the mosquito fauna, i.e. regional mosquito inventories. It was therefore decided to intensify nationwide investigations on the occurrence and distribution of mosquitoes in Germany in order to update old records and to detect possible faunal changes. This paper is focussing on a densely populated metropolitan region, the federal state of Hamburg and its adjacent environs, taking two historical baseline inventories into consideration, spanning almost 100 years of mosquito research in Hamburg. In the period between 2010 and 2014, more than 10,000 juvenile, neonate and adult mosquito specimens were sampled and trapped at 105 sites in Hamburg and its environs, of which about 60% have been identified to species level, resulting in a total of 33 recorded species. Of these, Anopheles algeriensis, Culex modestus, Ochlerotatus caspius, Ochlerotatus nigrinus and Ochlerotatus sticticus are new to the area. The most common species in Hamburg are Culex pipiens/torrentium and Ochlerotatus annulipes/cantans. In contrast, two previously common species, Anopheles atroparvus and Ochlerotatus excrucians, were not detected. Despite substantial environmental changes due to reconstruction, urbanisation and renaturation in the Hamburg metropolitan region in recent decades, there has been remarkably little change within the mosquito fauna during the last century.