Insektmobilen: A large-scale survey of flying insects assessed with citizen science and DNA metabarcoding

Although insects are the most species-rich and abundant group of terrestrial animals on Earth, many species have not yet been discovered and described. Many species or groups are, e.g. essential pollinators of crops and wild plants, significant decomposers of organic matter and thereby contribute to the nutrient turnover in the soil, disease transmitters, and a sizable food source for higher trophic levels. Despite the limited knowledge and data on insects, an increasing number of studies show a long-term decline in insects both locally and regionally in several places in the world, though primarily in Europe and North America. The increased focus on the global biodiversity crisis and the decline of insects has been covered by international and Danish news media and increased awareness of the increased human-driven pressure on nature among the general population.There is a limited amount of long-term time series data on a large spatial scale on insects in Denmark. Therefore, it is unknown whether insects are declining nationally or which factors could play a role in a possible decline. With a large number of samples collected simultaneously, i.e. within the same period and over a large spatial scale, one can examine how different land uses are associated with insects and develop a qualified guess on how human activity affects insect populations. However, long-term trends cannot be accounted for.This PhD thesis aims to investigate the distribution of flying insects in Denmark and investigate which factors influence the distribution of insect biomass, species richness, and species composition. It is a relevant topic to examine for the following three reasons: 1) the overall landscape-based distribution of flying insects has not previously been studied simultaneously, 2) additional data on insects are needed to examine trends in growth or declines in insect populations, and finally 3) nature management measures for the conservation of insect populations depend on the anchoring in knowledge about the distribution of insects.The dissertation consists of four chapters examining flying insects collected by citizen scientists throughout Denmark, primarily in June 2018. The samples were collected with a net rooftop net, along predetermined 5 km routes primarily located in urban areas, farmland, open nature, wetlands, and forests. DNA was extracted from the mixed insect samples, and the individuals in the samples were identified by their unique DNA sequences. This method is called DNA metabarcoding and is a useful tool for quickly obtaining diversity and species composition data from complex, multi-taxon samples. The extracted DNA was captured and copied in PCR reactions using three different universal insect or arthropod primers and was sequenced on a HiSeq platform.Chapter I presents the car net collection method for collecting flying insects on a large spatial scale and DNA metabarcoding with one of the CO1 primer pairs (fwhF2 & fwhR2n) used in the laboratory. A comparison is made between the distribution of insects at order level in the carnet samples with the order distribution of what is already known to Denmark, by comparing the data to the National Species Database (www.allearter.dk) and species occurrences in the Global Biodiversity Information Facility. In addition, the distribution of insects at order level in the carnet samples is compared with the distribution of insect orders from the national Swedish Malaise trap project, where insects are identified by morphological methods, as it is expected that the species composition of insects in Sweden is in broad strokes similar to the Danish fauna. The results show that car nets and DNA metabarcoding generally can be used for insect monitoring. However, car nets may be more likely to capture specific insect orders, just as other insect traps also have an inherent bias. The results also show that new species for the country can be captured using car nets and DNA metabarcoding and that the distribution of insect orders agrees well with the distribution of insect orders recently estimated nationally in Sweden. Furthermore, we find that Diptera is the most species-rich group of flying insects in both countries.Chapter II evaluates various methodological steps in the laboratory. Two non-destructive DNA extraction methods are compared; ethanol precipitation and an extraction buffer developed for DNA extraction of museum specimens. In addition, a manual and an automatic purification of DNA are compared. The comparison between DNA extraction methods shows that the extraction buffer is better at capturing different insect species compared to ethanol precipitation, but that the DNA purification method does not have an overall significance for which species are captured.Chapter III examines the effect of five land covers: urban, farmland, grassland, wetland and forest on flying insect biomass in Denmark and parts of Germany. Furthermore, the effect of land use intensity in urban and agricultural in Denmark are investigated. The study shows a strong negative effect of urban areas on insect biomass at a large spatial scale (1000 m buffer around the routes) in both countries. The results also show a positive effect of grasslands, forests, wetlands and agriculture on insect biomass in Denmark, with some variation where the spatial effect is greatest.Chapter IV examines the effect of the same five land covers used in Chapter III, on flying insect diversity and species composition in Denmark. The results show a strong negative effect of urban areas on insect diversity and an impact on the species composition in urban areas compared to the other land uses. Besides, there is a positive effect of farmland, grasslands and forests on species richness, with a more pronounced positive grassland effect. The majority of the 20 most frequently occurring insect species in the car net samples are positively associated with farmland, which indicates that the widespread agricultural area in Denmark affects the species composition on a landscape scale.The results of this dissertation confirm the possibilities of studying insects with the help of volunteers on a large spatial scale and the current applicability of DNA metabarcoding to monitor terrestrial insects. This dissertation also contributes much-needed information about the distribution of insects in Denmark and can serve as a baseline for future insect monitoring activities. Furthermore, the dataset is one of the few large-scale studies of insects and can provide a clearer picture of how insects are distributed and affected by human activity. This dissertation presents the following implications for future conservation and monitoring of insects in Denmark: 1) urbanisation in its current form has a pronounced negative effect on insect populations, 2) widespread agricultural areas have an impact on the species composition of insects on a large spatial scale, 3) focus on, and measures for, expansion of semi-natural areas are essential for the conservation of insects.