Application of Environmental DNA in the Air for Monitoring Biodiversity

Abstract

There is a profound interdependence between biodiversity and the UN Sustainable Development Goals (SDGs). Biodiversity underpins the functioning of global ecosystems and human welfare, and the achievement of numerous SDGs is directly or indirectly linked to protecting and sustainably managing biodiversity. In recent years, environmental DNA (eDNA) technology has exerted a great impact in the field of biodiversity monitoring. Airborne eDNA plays a significant role due to its non-invasive nature and the ability to monitor multiple taxonomic groups simultaneously. This review summarizes the technical principles, sampling methods, data analysis strategies of airborne eDNA and its application in biodiversity monitoring. In addition, it discusses the current technical challenges (e.g., pollution control, degradation mechanisms, and quantitative analysis) in the field, as well as future development directions, including optimizing sampling strategies, developing specific primers, integrating environmental RNA (eRNA), and establishing standardized monitoring systems. This review aims to provide a comprehensive assessment of airborne eDNA technology to promote airborne wide application of eDNA in global biodiversity monitoring.

1. Introduction

Biodiversity is crucial for the ecological balance of the Earth and the survival and development of human beings, and it has been shown to be a core element in maintaining the function and stability of ecosystems [1]. Yet, global biodiversity is currently facing unprecedentedly severe threats [2]. According to the report of the Intergovernmental Science–Policy Platform on Biodiversity and Ecosystem Services, human activities, including habitat destruction, climate change, overexploitation, and the introduction of alien species, have significantly accelerated the rate of species extinction (69% of decline in population since 1970), resulting in severe damage to the structure and function of many ecosystems [3].

Traditional biodiversity monitoring methods, such as visual surveys, camera traps, and acoustic monitoring, have played significant roles in long-term ecological research and conservation practices [4]. Visual surveys rely on professional personnel to identify and record species through direct observation, but this method is highly associated with the experience and professional skills of the observers [5]. Visual surveys, while intuitive, are prone to observer bias, as detectability varies with species behavior, environmental conditions, and observer expertise [5]. For example, cryptic, nocturnal, or rare species are often underreported [5]; bird identification requires long-term professional training for accurate distinguishing among similar species [6]. Although camera traps can obtain high-resolution images and identify individual animals, this method is mainly suitable for medium- and large-sized animals and is not effective for monitoring small organisms [7]. Moreover, the installation and maintenance costs of the equipment are high, and there are also limitations in monitoring range [7]. Acoustic monitoring has certain advantages in the monitoring of songbirds, but environmental noise easily interferes [8].Acoustic monitoring, though effective for vocal taxa (e.g., bats and birds), faces challenges from ambient noise interference, habitat-dependent sound propagation, and difficulties in distinguishing closely related species with overlapping call frequencies [7]. These constraints reduce the scalability and reproducibility of traditional methods, particularly in complex or inaccessible ecosystems [6,8]. In noisy environments like cities, the accuracy of monitoring data will be seriously affected. In addition, these traditional methods generally cannot achieve synchronous monitoring of multiple groups of organisms (e.g., birds, insects, and microorganisms), and it is difficult to comprehensively reflect the status of biodiversity in complex ecosystems [8].

With the continuous development of molecular biology technology, environmental DNA (eDNA) technology has emerged [3,9]. eDNA technology infers the distribution, abundance, and dynamic changes of species by detecting DNA fragments shed in environmental media (e.g., soil, water, and air) [9]. These DNA fragments come from the skin cells, feces, hair, pollen, etc., of organisms and contain genetic information. Currently, eDNA technology has been widely applied in various fields [4]. In fish monitoring, Doi et al. (2017) [10] successfully detected the presence of multiple fish species in different water bodies using eDNA technology, providing important evidence for fish resource investigation and ecological protection. In amphibian research, Vanni et al. (2016) [11] effectively monitored amphibian diversity by analyzing eDNA in water, providing a new means for the protection of endangered amphibian species. In the field of microbiology, Zhang et al. (2020) [12] used eDNA technology to deeply study the structure and function of soil microbial communities, revealing the important role of microorganisms in ecosystems. In terrestrial ecosystems, early eDNA monitoring mainly relies on soil or water samples, but these methods have certain limitations in application scenarios [12]. The discovery of eDNA in the air has opened up a new path for terrestrial biodiversity monitoring [13].While aquatic and soil eDNA methods have revolutionized biodiversity monitoring, their spatial detection range is inherently limited by localized dispersion [10,12,13]. Studies in freshwater ecosystems demonstrate that waterborne eDNA primarily reflects species presence within immediate hydrological connectivity, with detection rapidly declining downstream due to dilution and degradation [10]. Similarly, soil eDNA is constrained by patchy distribution and substrate-specific DNA retention [12,13].

eDNA in air refers to the DNA carried by biological particles (i.e., skin debris, pollen, spores, and microorganisms) suspended in the air [9]. Compared with traditional eDNA sources in water and soil, eDNA in the air has unique advantages [14]. Firstly, this technology is non-invasive, requiring no direct contact with the target organisms and avoiding interference with them [4]. It is particularly suitable for monitoring rare, endangered, or hard-to-reach species. Secondly, it enables the simultaneous detection of multiple groups of organisms including vertebrates, insects, plants, and microorganisms [15]. The eDNA they carry may all be present in the air, greatly enhancing monitoring efficiency and the comprehensiveness of biodiversity information acquisition [16]. Additionally, due to the mobility of air, eDNA can spread over a large area, meaning that eDNA monitoring in the air has a high spatiotemporal resolution, which is suitable for the large-scale and long-term dynamic monitoring of biodiversity [17].Airborne eDNA offers unparalleled spatial coverage, as atmospheric currents can transport genetic material over kilometers, integrating biodiversity signals across landscapes [9,15,17]. This enables ecosystem-wide assessments from single sampling points, which is a critical advantage for monitoring mobile or elusive species [9,15,17]. However, air eDNA’s diffuse nature also poses challenges in pinpointing exact source locations compared to water/soil methods [9,15,17]. Thus, while aquatic and soil eDNA excel in fine-scale studies, airborne eDNA emerges as a transformative tool for macroecological surveys [12,13].However, airborne eDNA also presents challenges, such as uncertainty in source attribution(e.g., distinguishing resident vs. transient species) and the need for robust reference databases. Despite these limitations, its capacity for high-throughput, scalable biodiversity assessment positions airborne eDNA as a powerful complement to traditional methods in large-scale ecological monitoring [17].

This review aims to summarize the technological progress of eDNA monitoring of biodiversity in the air and analyze its practical application, as well as discuss the current technical challenges, limiting factors, and future development direction. This study provides an assessment of eDNA technology in the air to promote the application of this technology in global biodiversity monitoring for biodiversity conservation and environment management.

2. Methods for Detecting eDNA in the Air

2.1. Sources and Release Mechanisms of eDNA

The sources of eDNA in the air are extensive and originate from different groups of organisms [13] (Figure 1). Vertebrates are one of the important sources of eDNA. The shedding of skin cells, replacement of feathers, and excretion of feces all can release DNA into the air environment [1]. Lynggaard et al. (2022) [18] found that, during the activities of birds, behaviors such as flapping wings increase the diffusion of skin cells and feather debris, thereby increasing the content of vertebrate eDNA in the air. Insects also release eDNA into the air through forms such as body surface debris and scales. Pollen and leaf fragments produced by plants are also important sources of eDNA in the air [19]. During the flowering period of plants, a large amount of pollen is spread into the air through wind pollination. Johnson et al. (2019) [20] successfully detected the presence of multiple plant pollens in the air by analyzing plant eDNA, providing a new approach for plant diversity research. In addition, microorganisms including bacteria and fungal spores are widely present in the air environment, and the eDNA they carry is also an important component of eDNA in the air. De Groot et al. (2021) [21] found that microbial eDNA in the air can reflect the composition and changes of microbial communities in different ecological environments.

The release rate of eDNA is influenced by a variety of factors [19]. Biomass is an important factor [19]. Larger organisms release more eDNA into the air. The behavior of organisms is an important factor that affects the release of eDNA. For example, behaviors such as flapping wings among birds and running among animals promote the diffusion of biological particles and accelerate the release of eDNA [13]. Environmental factors play an equally crucial role. The speed of wind directly affects the diffusion range and speed of eDNA, with stronger wind speeds enabling eDNA to spread over a larger area [20]. Humidity also influences the stability of biological particles. Excessively high and low humidity levels can affect the release and preservation of eDNA [3].

2.2. Sampling Methods

2.2.1. Active Sampling

Active sampling involves the use of specific equipment to collect biological particles from the air to obtain eDNA (Table 1). Common active sampling devices include filter samplers, water-based samplers, and electrostatic adsorption devices [14,22]. Filter samplers are currently the most widely used active sampling devices. These samplers typically use fiber filters with pore sizes ranging from 0.22 to 1.2 μm, where a pump forces air through the filter, capturing biological particles in the air [14]. Lynggaard et al. (2022) [18] captured various biological particles in different environments using filter samplers, providing effective samples for subsequent eDNA analysis. Filter samplers have the advantages of relatively simple operation and low cost, but the selection of filter pore size is crucial during sampling [23]. Pore sizes that are too small may increase air resistance and affect sampling efficiency, while those that are too large may fail to effectively capture smaller biological particles [14,22].

Water-based samplers work by allowing air to pass through a liquid (e.g., sterile water), concentrating biological particles in the liquid to enrich eDNA [24]. Clare et al. (2022) [24] conducted sampling experiments in indoor and outdoor environments using water-based samplers. This study demonstrates that water-based samplers can effectively collect eDNA from the air. The advantage of water-based samplers is that they can avoid issues such as filter clogging and are suitable for sampling in environments with high concentrations of particles [24]. However, in the subsequent eDNA extraction process, the impact of the liquid on DNA and approaches for efficiently extracting eDNA from the liquid need to be considered.

Electrostatic adsorption devices enhance the capture efficiency of biological particles by using an electric field [25]. Gusareva et al. (2019) [25] experimentally verified the feasibility of electrostatic adsorption technology in eDNA sampling from the air. This method can capture smaller biological particles and has unique advantages in collecting eDNA from microorganisms that are difficult to capture through traditional filtration methods [25]. However, the equipment cost is relatively high, and professional operation and maintenance are required.

Table 1. The evaluations across sampling equipment performance, including metrics such as capture efficiency, cost, and suitability for different environments [14,22,24,26,27].

Sampling Method	Capture Efficiency	Cost	Best Use Case	Limitations
Filter samplers	High (0.22–1.2 µm)	Low	General biodiversity	Clogging in high-particle areas
Water-based samplers	Moderate	Medium	Pollen/microorganisms	DNA dilution in liquid
Electrostatic adsorption	Very high (small particles)	High	Microorganisms	High maintenance

Table 1. The evaluations across sampling equipment performance, including metrics such as capture efficiency, cost, and suitability for different environments [14,22,24,26,27].

Sampling Method	Capture Efficiency	Cost	Best Use Case	Limitations
Filter samplers	High (0.22–1.2 µm)	Low	General biodiversity	Clogging in high-particle areas
Water-based samplers	Moderate	Medium	Pollen/microorganisms	DNA dilution in liquid
Electrostatic adsorption	Very high (small particles)	High	Microorganisms	High maintenance

2.2.2. Passive Sampling

Passive sampling utilizes natural carriers or artificially placed collection devices to passively collect biological particles in the air (Table 1). Natural carriers such as spider webs and flowers, due to their special structures and properties, can naturally capture biological particles in the air [26]. Newton et al. (2023) [26] found that spider webs can effectively capture insects, pollen, and other biological particles. By analyzing eDNA in spider webs, information on the biodiversity of the surrounding environment can be obtained. Flowers, in the process of attracting pollinators, also capture pollen and other biological particles in the air, making them natural passive sampling carriers [26]. Dust traps are common artificial passive sampling devices that are suitable for long-term monitoring [27]. Johnson et al. (2023) [27] set up dust traps in different environments to collect dust deposited in the air and extract eDNA for analysis, successfully monitoring various plant pollens and fungi. Dust traps have the advantages of low cost, simple operation, and long-term placement [27]. However, during the sampling process, they are easily affected by environmental factors (e.g., wind speed and precipitation). Moreover, dust may contain a large amount of non-biological impurities, which increases the difficulty of subsequent eDNA extraction and analysis [27].

2.3. Molecular Analysis Techniques

2.3.1. DNA Extraction and Contamination Control

Due to the low concentration of eDNA in the air, highly sensitive DNA extraction methods are required [28]. Currently, commonly used DNA extraction kits (e.g., Tissue Kits) can effectively extract DNA from various samples [28]. During the extraction process, it is necessary to follow operating procedures to ensure the quality of the extracted DNA. For air eDNA samples, which are complex in origin and have low DNA content, as well as many impurities, pre-treatment is usually required before extraction, such as grinding filter membrane samples or concentrating liquid samples [29].

Contamination control is a crucial step in the molecular analysis of air eDNA [30]. There is a large amount of exogenous DNA in the laboratory environment, such as skin cells from laboratory personnel and commonly used microorganisms, which may contaminate the samples and lead to false positive results [14]. To effectively control contamination, a series of strict anti-contamination measures need to be taken. Before the experiment, the laboratory space and equipment should be disinfected with ultraviolet light to reduce DNA residues in the environment [15]. During the experiment, operators should wear sterile gloves, masks, and other protective equipment to avoid the self-DNA contamination of the samples. At the same time, negative control samples (e.g., un-sampled blank filter membranes, sterile water) should be set up to determine whether contamination during the experiment exists [18]. Through analyzing the negative control samples, if DNA signals are detected, it indicates that there is contamination during the experiment, and the experiment needs to be redone [29].

2.3.2. Primer Design and Amplification

Primer design plays a crucial role in the molecular analysis of air eDNA [31]. Primers are short nucleotide fragments that are complementary to the target DNA sequence, which are used to guide DNA amplification in PCR (polymerase chain reaction). According to different monitoring purposes, primers can be divided into universal primers and species-specific primers [28]. Universal primers can amplify DNA fragments from multiple species and are suitable for comprehensive monitoring of biodiversity. For example, the 12S rRNA (MiBird—U) primer is often used for amplifying bird eDNA, and the 16S rRNA (165mam) primer can be used for detecting mammal eDNA [12,29]. The advantage of universal primers is that they can simultaneously detect multiple species. However, in practical applications, there may be amplification bias issues [12,29]. A higher amplification efficiency for some species and lower for others may lead to affect the accuracy of biodiversity analysis [19].

The process of designing species-specific primers involves several crucial steps to ensure accuracy, specificity, and efficiency [12,28,29].First, the selection of target gene regions is fundamental. For accurate species identification, conserved gene regions are preferred. For instance, in the case of animals, the COI (Cytochrome c Oxidase Subunit I) gene is commonly used; meanwhile, for microorganisms and vertebrates, the 12S rRNA gene serves as an excellent choice. These regions strike a balance between conservation, which is essential for primer binding, and variability, enabling effective species differentiation. Once the target gene regions are determined, the next step is to collect reference sequences. Databases such as NCBI GenBank, BOLD (Barcode of Life Data Systems), and UNITE (specifically for fungi) are valuable resources. By using relevant keywords, like “Species name COI gene,” and filtering according to taxonomic groups, homologous sequences of the target gene can be retrieved. It is important to download these sequences in FASTA format, including both the target species and its close relatives, as this will be crucial for specificity testing later on. After gathering the sequences, multiple sequence alignment is performed using tools like MAFFT Version 7, which is known for its speed and accuracy, especially when dealing with large datasets, or Clustal Omega Version 1.2.4, which offers a user-friendly web interface. Through this alignment process, conserved and variable regions can be identified. Conserved regions, which are stable across target species, are ideal for primer binding sites. Variable regions, on the other hand, highlight the differences between the target species and its relatives, helping to ensure primer specificity. Subsequently, bioinformatics tools are employed to design the primers. Primer-BLAST, an NCBI tool, allows users to input the aligned conserved region as the target template and set various primer parameters. These parameters include a length of 18–25 bp to balance specificity and annealing efficiency, a melting temperature (Tm) of 58–62 °C to avoid primer–dimer formation, and a GC content of 40–60% to ensure stability. Additionally, specifying “Species-specific primers” and excluding non-target taxa in the “Exclude sequences” field further enhances specificity. Another option is Primer3, which can be used either as a standalone application or via a web interface, where users can upload the aligned region and adjust the settings for optimal specificity.

Before experimental use, it is essential to evaluate primer specificity and efficiency [12,28,29]. In silico testing through Primer-BLAST helps one to confirm that the primers bind only to the target species and not to close relatives, ensuring no off-target hits. Tools like OligoAnalyzer can be used to calculate PCR efficiency by assessing potential issues such as hairpin structures, dimer formation, and self-complementarity. Experimental validation is the next critical step. Candidate primers are synthesized and tested using methods such as gradient PCR to optimize the annealing temperature and minimize non-specific amplification [28,29]. Agarose gel electrophoresis is then used to verify that the product size matches the expected amplicon, typically within the range of 200–500 bp for COI barcoding. Sanger sequencing is also performed to confirm that the amplicon sequence aligns with the target gene in the database. If non-specific bands appear or amplification fails during the experimental validation, the primers need to be refined. This may involve modifying the primer sequences, such as adjusting the 3′ ends to target unique nucleotides in the alignment. After making these modifications, the in silico testing and experimental validation processes are repeated until specific and efficient primers are achieved. Finally, it is important to document and store the primers properly. All relevant information, including primer sequences, target genes, species, and validation results, should be recorded in a database, such as an Excel sheet, for future reference. Validated primers can also be deposited in public repositories, like NCBI Primer-Bank, to facilitate broader use within the scientific community [12,28,29]. This comprehensive process ensures that the designed primers are reliable and can be effectively used for species identification and related research. Amplification bias in molecular biology can distort results, but multiplex PCR and touchdown PCR offer effective solutions [12,28,29]. Multiplex PCR allows simultaneous amplification of multiple targets in a single reaction, reducing variability, reagent use, and time. It ensures proportional amplification, making it ideal for pathogen detection and genetic screening. Touchdown PCR minimizes bias by starting with high annealing temperatures to favor specific primer binding, then gradually lowers them to reduce non-specific amplification [12,28,29]. This improves efficiency for low-abundance targets and balances amplification across sequences. In silico testing enhances these methods by simulating PCR reactions to optimize primer design, predict binding efficiency, and identify potential biases before wet-lab experiments. Computational validation saves resources, increases success rates, and ensures these protocols effectively mitigate amplification bias in practice [12,28,29].

Species-specific primers are designed for the DNA sequences of specific species or groups and have high specificity [32]. They are mainly used for monitoring endangered species and detecting invasive species [33]. For example, in the monitoring of black crakes, researchers designed specific primers for the COI gene of black crakes, which can accurately detect the eDNA of black crakes with low numbers [34]. Foremost, the design of species-specific primers requires an understanding of the gene sequences of the target species [32]. Additionally, in the application process, factors including species gene variation may result in primer failure. During the PCR amplification process, it is also necessary to optimize reaction conditions, such as primer concentration, DNA template quantity, annealing temperature, and reaction time [32]. The optimization condition can ensure the specificity and efficiency of amplification [32]. Meanwhile, to reduce PCR amplification bias, quantitative PCR (qPCR) technology can be adopted. Monitoring the amplification process in real time enables the relative quantitative analysis of eDNA content [32].

2.3.3. High-Throughput Sequencing and Bioinformatics

The development of high-throughput sequencing technology has provided a powerful tool for the analysis of eDNA in the air [35]. Through high-throughput sequencing, a large amount of DNA sequence data can be obtained, thereby allowing one to build a comprehensive understanding of the diversity information of organisms in the sample [14,35]. In the data analysis process, sequences are usually classified based on operational taxonomic units or amplicon sequence variants [14,35]. Operational taxonomic units cluster sequences into different units based on sequence similarity with a threshold of 97% similarity, and each operational taxonomic unit represents a taxonomic unit [14,35]. On the other hand, amplicon sequence variants classify sequences based on precise sequence variations, providing higher resolution and more accurately reflecting the true diversity of species [14,35]. When classifying eDNA sequences in the air, the obtained sequences need to be compared with reference databases [14,35]. However, there are still deficiencies in the bird barcode data in current reference databases, which need to be further expanded and improved to enhance the accuracy of species identification.

Bioinformatics plays a core role in the analysis of eDNA data in the air [36]. Through bioinformatics analysis, quality control of sequencing data can be performed, low-quality sequences and chimeras can be removed, and effective sequences can be clustered, annotated, and classified, thereby allowing us to obtain information on the species and abundances of organisms in a sample [28]. In addition, bioinformatics methods can be used to compare the biodiversity of different samples and study the spatiotemporal variation patterns of biodiversity [36]. With the continuous development of bioinformatics technology, more and more new algorithms and software are supposed to be applied to the analysis of eDNA data in the air, providing stronger support for biodiversity research [28].

The cost of airborne eDNA technology varies depending on the sampling and analysis methods used. Active sampling devices like filter samplers and electrostatic adsorption devices can range from USD 500 to USD 5000, while passive methods like dust traps are cheaper, typically under USD 100. Molecular analysis, including DNA extraction, PCR, and high-throughput sequencing, adds significant costs, ranging from USD 50 to USD 200 per sample [14,24,25]. In contrast, traditional methods such as camera traps cost between USD 100 and USD 1000 per unit, and visual or acoustic surveys require less equipment but more labor, which can be costly over time [14,20]. While airborne eDNA technology may have higher initial costs due to molecular analysis, its ability to monitor multiple species simultaneously and cover larger areas can make it more cost-effective in the long run compared to traditional methods that require extensive labor and multiple devices [28,29].

3. Case Studies

3.1. Synchronous Monitoring of Multiple Groups

Technology for obtaining air eDNA samples has advantages in the synchronous monitoring of multiple groups of organisms (Figure 2). In vertebrate monitoring, Lynggaard et al. (2022) [18] conducted eDNA sampling and analysis in a zoo environment and successfully detected the presence of 13 species of birds and mammals. The study revealed variations in species detection rates across different seasons. For instance, the detection rate reached 85% in spring, while it was 62% in winter. The findings indicate that airborne eDNA can effectively capture terrestrial vertebrate diversity across different ecosystems, highlighting significant differences (p < 0.05) in community composition between habitats. Through the study of eDNA from these vertebrates, this method can be used in understanding the diversity of animals in the zoo and providing references for the health monitoring and management of animals. In the field of insect monitoring, Pumkaeo et al. (2021) [37] used air eDNA technology to identify mosquito species. Researchers collected air samples in different areas and accurately identified multiple mosquito species by analyzing the eDNA in them, providing important preliminary information for the prevention and control of mosquito-borne diseases. Additionally, this technology can also be applied to the monitoring of other insect groups (e.g., agricultural pests), which can aid our understanding of pest dynamics and support effective control measures.

In the monitoring of plants and microorganisms, Johnson et al. (2023) [27] detected 40 types of plant pollen and fungi through the analysis of eDNA in the air. This research achievement provides new ways for plant diversity research, plant pollination ecology, and the study of fungal community structure and function. By analyzing the spatiotemporal changes of plant eDNA in the air, the findings can support our understanding of the flowering period, the distribution range of plants, and the interrelationship between plants and the environment; meanwhile, the study of microbial eDNA helps in revealing the role and ecological functions of microorganisms in the ecosystem (Figure 3).

3.2. Comparison with Traditional Methods

Compared with traditional biodiversity monitoring methods, the eDNA technology in the air has unique advantages and limitations [39] (

Table 2. The advantages and limitations of traditional methods of monitoring diversity.

Method	Advantages	Limitations
Camera traps	High-resolution images, individual identification	Only suitable for medium–large-sized animals, high installation cost
Auditory survey	Applicable to songbird monitoring	Subject to interference from environmental noise
Air eDNA	Simultaneous detection of multiple taxa, non-invasive	Difficult to conduct quantitative analysis, susceptible to contamination influence

). Camera traps [7] can be used to obtain high-resolution images of animals and identify individual animals, which is of great value for studying animal behavior and population dynamics. However, this method is mainly suitable for medium- and large-sized animals, and is not effective for monitoring small animals, insects, or plants [7]. Moreover, the installation, maintenance, and data processing costs of the equipment are relatively high. Auditory surveys have certain advantages in monitoring songbirds, as they can identify species by recording the birds’ calls [39,40]. However, in practical applications, environmental noise (e.g., traffic noise, industrial noise) can interfere with the monitoring results, reducing the accuracy of species identification [39,40] The greatest advantage of the eDNA technology in the air is that it can simultaneously detect multiple groups of organisms without direct contact with the target organisms, which is non-invasive [39,40]. This enables the technology to quickly obtain rich biodiversity information without disturbing the normal activities of organisms [39,40]. However, this technology also faces some challenges at present, such as difficulties in quantitative analysis; moreover, the relationship between eDNA concentration and species abundance is not yet fully clear, making it difficult to accurately assess the quantity and distribution of species [39,40]. In addition, due to the low concentration of eDNA in the air and the complexity of sample sources, laboratory contamination and environmental background DNA easily interfere, affecting the accuracy of the detection results.

3.3. Forest Ecosystem Monitoring

Lynggaard et al. (2024) [41] conducted a forest ecosystem monitoring study with eDNA technology in the air. They set up multiple air sampling points in different forest areas, collected air samples, and analyzed the eDNA in the samples. They detected various vertebrates including deer, squirrels, and birds, as well as discovering the existence of numerous invertebrates such as insects and spiders. Through further in-depth analysis of the eDNA data, they revealed the distribution patterns of different species in the forest. For example, the eDNA of some tree-dwelling birds was more concentrated in the upper air samples of the forest, while the eDNA of ground-dwelling mammals was more abundant in the air samples closer to the ground. At the same time, the study concluded that different species had obvious differentiation in ecological niches, which is of great significance for understanding the structure and function of forest ecosystems [41]. This study proved the effectiveness and reliability of the eDNA technology in the air in monitoring the biodiversity of complex forest ecosystems, which may provide a new scientific basis for forest ecological protection and management.

3.4. Urban and Farmland Bird Diversity

Polling et al. (2024) [39] conducted research on the diversity of birds in urban and farmland areas through eDNA technology in the air to accurately reflect the composition and changes in bird communities. In urban areas, they collected air samples from different parks and green spaces, and detected various birds that had adapted to the urban environment, such as sparrows, pigeons, and magpies, as well as some migratory birds that occasionally appeared in urban areas. In farmland areas, researchers found that the eDNA technology in the air could effectively monitor the diversity of farmland birds, including some beneficial birds that feed on farmland pests. Through long-term monitoring, the study found that the composition of bird communities in urban and farmland areas would change with the seasons, urban construction, and farming methods. For instance, in the early stage of new park construction in the city, the number of bird species was relatively small, but as the vegetation gradually became rich, the diversity of birds significantly increased. During the fallow period of farmland, the number and types of birds would also change [39]. These research results provide important scientific guidance for urban ecological planning and farmland biodiversity conservation, helping us to rationally plan urban green spaces and formulate scientific farmland management measures to protect and enhance bird diversity.

3.5. Dynamic Changes in Biodiversity Across Seasons

Johnson et al. (2023) [27] conducted a comprehensive study on the dynamic changes in biodiversity across seasons by collecting air eDNA samples over a long period. They collected air samples regularly throughout the year according to different seasons and analyzed the composition and changes of eDNA in the samples. The study found that there were significant differences in the composition and structure of biological communities across seasons. In spring, as temperatures increased, plants began to sprout and grow, and the concentration of eDNA from insects and birds that feed on plant pollen and tender leaves gradually increased in the air. During the summer season, biodiversity reached a relatively rich level, and eDNA from various animals could be detected. In autumn, with the withering of plants and the migration of some animals, the types and concentrations of eDNA in the air changed significantly. During the winter period, due to the limitations of environmental conditions, biological activities decreased, and the types and quantities of detectable eDNA also decreased accordingly [27]. This research provides new results to understand seasonal changes in ecosystems, and better grasp the dynamic balance, as well as change patterns of ecosystems in different seasons.

4. Challenges and Limitations

4.1. Production, Transport, and Degradation of eDNA in the Ambient Environment

4.1.1. Factors Affecting eDNA Production

The production of eDNA is influenced by a variety of factors such as biomass, individual size, and behavior [25,36]. From the perspective of biomass, large organisms or populations with high biomass, due to their relatively vigorous metabolism, release more cells and secretions into the environment, and thus release more eDNA. However, the relationship between individual size and eDNA release is not a simple linear one, but is also regulated by the physiological state and behavioral patterns of organisms [25]. Some small organisms in the breeding period, due to their more active physiological activities, may release more eDNA than usual [25,36]. In comparison, for some large organisms in hibernation or dormancy states, the release of eDNA will significantly decrease. The behavioral activities of organisms, including migration, foraging, and reproduction, have a significant impact on the release rate and spatial distribution of eDNA [25,36]. During migration, a large number of organisms move in a concentrated manner, releasing a large amount of eDNA in the areas they pass through areas with frequent foraging activities. Since increased contact between organisms and the environment, migration activities may lead to an increase in eDNA concentration.

4.1.2. Influencing Factors in eDNA Transport

In the air, the transport of eDNA is influenced by various factors, including wind, rainfall, and human activity [14,29,42]. Wind plays a major role in eDNA dispersal, as strong winds can carry eDNA over long distances, potentially skewing monitoring results. During periods of high wind, eDNA from distant organisms may be blown into urban areas, leading to the detection of genetic signals from species not actually present in the local environment [14,29,42]. This can distort assessments of regional biodiversity. Rainfall also affects eDNA distribution by causing airborne particles to settle with raindrops, reducing their concentration in the air [14,29,42]. After heavy rain, a significant portion of eDNA is washed out of the atmosphere, leading to lower detection levels in subsequent air sampling. This fluctuation can impact the reliability of eDNA-based monitoring. Additionally, human activities such as vehicle traffic and construction disrupt airflow patterns, further complicating eDNA transport. In urban areas, air turbulence from dense traffic, as well as dust and altered wind currents from construction sites, can affect how eDNA moves through the environment [14,29,42]. These disturbances introduce additional variability, making it more challenging to obtain precise biodiversity measurements.

Aerosols are carried over long distances in the air and may not always indicate the biodiversity of a particular territory [14,29,42]. To address the issue of long-distance aerosol transport, researchers can implement several strategies including localized sampling, meteorological data integration, reference databases, temporal sampling, and multi-method validation [14,29,42]. Localized sampling should focus on areas with minimal wind influence, such as sheltered or enclosed environments, to reduce the impact of distant aerosols. Meteorological data integration involves using wind direction and speed data to account for potential external eDNA sources, allowing researchers to adjust their interpretations accordingly. Reference databases can be applied to compare detected eDNA with known local species lists to distinguish between resident and transient DNA [14,29,42].Temporal sampling can be conducted, repeating sampling over time to identify consistent patterns and filter out sporadic detections caused by wind-blown DNA [14,29,42].Multi-method validation integrates airborne eDNA, with traditional surveys confirming the presence of species detected through eDNA [14,29,42].

4.1.3. Factors Affecting the Degradation of eDNA

The degradation process of eDNA in the air is influenced by multiple factors, including microorganisms, temperature, ultraviolet (UV) radiation, and pH [14,28]. Microorganisms secrete various enzymes that are capable of breaking down eDNA, thereby accelerating its degradation. In environments with high microbial activity, the degradation rate of eDNA increases significantly. Elevated temperatures and UV radiation can damage the molecular structure of eDNA, hastening its breakdown [14,28]. High temperatures may cause the DNA double helix to unwind, destabilizing its structure and making it more susceptible to fragmentation and degradation [14,28]. Meanwhile, UV radiation can induce the formation of pyrimidine dimers in DNA, disrupting its normal structure and function, which further accelerates eDNA degradation [14,28]. Variations in environmental pH also impact the stability of eDNA. Both highly acidic and highly alkaline conditions can compromise the structural integrity of eDNA, promoting its breakdown [14,28]. These factors make it challenging to accurately predict the concentration and integrity of airborne eDNA, introducing greater uncertainty into monitoring results and presenting significant obstacles for biodiversity assessments.

4.2. Factors Affecting the Accuracy of eDNA Detection

4.2.1. Contamination Sources

Airborne eDNA technology carries a relatively high risk of contamination, particularly within laboratory environments [1,17]. Given the inherently low concentration of eDNA in air samples, even minimal exogenous DNA contamination can substantially distort experimental outcomes. Multiple stages of laboratory processing present potential contamination sources [1,17]. Reagents may become contaminated with foreign DNA during their manufacture, storage, or usage. Similarly, laboratory equipment surfaces can retain residual DNA from prior experiments, which may subsequently be released during later procedures, leading to cross-contamination [1,17]. Furthermore, the clothing and gloves of personnel may harbor external environmental DNA, inadvertently introducing contaminants during sample handling. Compounding these challenges, the long-range atmospheric transport of eDNA can create signal ambiguity. For instance, air samples collected from distinct geographical locations might contain identical eDNA signatures [1,17]. However, due to atmospheric dispersal mechanisms, it becomes problematic to ascertain whether such detections represent indigenous local species, migratory organisms from distant regions, or secondary contamination via airborne transmission from other areas [1,17]. This uncertainty complicates the precise attribution of detected eDNA signals, presenting significant interpretation challenges for biodiversity monitoring.

4.2.2. Measures for Control Contamination Sources

To effectively minimize contamination, it is critical to implement rigorous negative controls throughout the experimental process, including sampling blanks, extraction blanks, and PCR blanks. Sampling blanks involve executing the full sampling procedure without actually collecting a sample to identify potential contamination during field collection [43]. Extraction blanks process no sample material through the DNA extraction protocol to check for contamination in reagents or extraction procedures. PCR blanks run amplification reactions without template DNA to verify the purity of PCR reagents and detect any amplification contaminants [43]. These controls allow for the timely detection and elimination of potential contamination sources. Simultaneously, establishing stringent cleaning protocols is essential. All laboratory equipment, reagents, and workspaces require regular decontamination and sterilization. Equipment must be thoroughly cleaned and disinfected before and after each use to prevent cross-contamination [43]. Reagents should be stored and handled in sterile conditions to avoid external DNA introduction [43]. The laboratory environment itself should undergo frequent cleaning and disinfection to maintain aseptic conditions, ensuring sterile experimental procedures and improving the accuracy and reliability of the results.

4.3. Detection Sensitivity and False Negatives

4.3.1. Risk of Missing Low-Abundance Species

Airborne eDNA technology presents a considerable risk of false negatives when detecting low-abundance species [14,44]. Owing to their limited population sizes, these species release comparatively smaller quantities of eDNA into the atmosphere. During sampling, constraints on sample volume coupled with the heterogeneous distribution of airborne eDNA may result in insufficient capture of genetic material from scarce organisms [14,44]. In the analytical phase, the eDNA signals from low-abundance species may become obscured by the dominant signals of more prevalent species, potentially causing them to be overlooked during detection and interpretation [14,44]. This is particularly problematic for rare and endangered species, which often exist in low densities within ecosystems [14,44]. Their eDNA signatures may prove exceptionally challenging to identify, thereby compromising monitoring efforts and conservation initiatives for these vulnerable organisms.

4.3.2. Impact of Primer Bias

Primer bias is yet another crucial cause of false negatives [29,45]. Different primers exhibit diverse amplification efficiencies when it comes to eDNA (environmental DNA) from various species. In the process of PCR (polymerase chain reaction) amplification, specific primers might possess a higher affinity and amplification efficiency for the eDNA of certain species [29,45]. Conversely, they could have a subpar amplification effect or even fail to amplify the eDNA of other species altogether. This situation can lead to the eDNA of particular species being present within the sample, but not being effectively detected due to primer bias, thereby giving rise to false negative results [29,45]. In addition, the existing reference databases have a limited scope of coverage. The DNA sequences of some species have not been incorporated into these databases. When attempting to annotate the species within the eDNA of a sample, species lacking corresponding sequences in the database cannot be accurately identified [29,45]. This further boosts the likelihood of false negatives and restricts the comprehensiveness and accuracy of species detection utilizing air eDNA technology.

4.4. Limitations of Quantitative Analysis

Inferring species abundance from eDNA data represents a significant challenge for air eDNA technology [10,35,46]. The concentration of eDNA does not exhibit a straightforward linear relationship with species abundance and is instead influenced by a multitude of environmental and biological factors [10,35,46]. Environmental physical and chemical conditions, including temperature, humidity, light, and soil pH, impact the release, diffusion, and degradation of eDNA [10,35,46]. For instance, high temperatures and humidity may hasten the degradation of eDNA, decreasing its concentration and consequently preventing it from accurately reflecting the actual species abundance [10,35,46]. Varying soil pH levels also affect the release of eDNA by organisms and its stability within the environment, disrupting the correlation between eDNA concentration and species abundance [10,35,46]. The behavioral activities and physiological states of organisms likewise result in the uneven release of eDNA [10,35,46]. The eDNA released by the same species can differ considerably during different physiological states, such as the breeding and non-breeding periods [10,35,46]. The activity range and patterns of organisms also influence the distribution and concentration of eDNA in the environment [10,35,46]. Additionally, eDNA release efficiency varies among different species; some species release more eDNA, while others release less [10,35,46]. These factors render quantitative analysis based on eDNA concentration ineffective in accurately representing the actual species abundance. The question of how we can account for the impact of environmental factors and establish precise quantitative analysis models is a pressing issue that requires immediate attention.

5. Future Directions

5.1. Technical Optimization

In the future, it will be necessary to further optimize the sampling strategies and filter membrane materials for air eDNA [47]. Regarding sampling strategies, the design of sampling equipment needs to be improved to boost sampling efficiency and the eDNA capture rate [22]. For example, developing new kinds of air samplers and optimizing the control of sampling flow rate and time can allow for the more efficient collection of eDNA in the air [22]. Designing a more rational layout of sampling points and considering the different ecological environments, topographies, and distribution characteristics of organisms ensures that the collected air samples can comprehensively and accurately reflect the biodiversity in a region.

When it comes to the research and development of filter membrane materials, efforts should focus on enhancing the retention capacity for aerosol particles of various sizes and properties while reducing background interference [22]. Developing filter membranes with special surface structures or chemical properties that can specifically adsorb eDNA can increase the eDNA capture efficiency [22]. By improving the material and manufacturing process of the filter membranes, the interference of the filter membranes themselves on the experimental results can be minimized, and the detection accuracy can be enhanced.

Additionally, for specific biological groups like birds, more specific primers should be designed [28]. Using bioinformatics technology, the characteristics of bird gene sequences should be analyzed in-depth to create primers that can accurately amplify bird eDNA and explore the method of using multiple markers in combination [28]. By using multiple primer pairs simultaneously, the accuracy and coverage of species detection can be improved.

5.2. Standardization and Database Construction

The standardization of airborne eDNA technology represents the cornerstone for its widespread adoption [14]. Notably, airborne eDNA technology currently lacks globally unified protocols, and this methodological gap compromises the comparability of results across studies while impeding broader adoption in ecological monitoring. Presently, the absence of unified protocols has resulted in poor comparability and reproducibility between studies, significantly constraining technological advancement and practical implementation. Consequently, the establishment of comprehensive standard operating procedures is imperative, encompassing all aspects from sampling to bioinformatics analysis.

Key areas requiring standardization include the following:

• Sampling protocols

(a)

Clear specifications regarding sampler selection, including operational parameters and performance criteria

(b)

Defined standard operating parameters (e.g., sampling duration, flow rate, and frequency)

• Molecular processing

(a)

Optimized DNA extraction methodologies with prescribed reagents and protocols

(b)

Standardized primer design principles with validation requirements

• Bioinformatics pipeline

(a)

Implementation of uniform data processing and analytical frameworks

(b)

Quality control metrics to enable cross-study data integration

Simultaneously, expansion of DNA barcode reference libraries for avian and other taxa is essential. This should involve the following:

(a)

Coordinated large-scale specimen collection and sequencing initiatives

(b)

Enhanced quality assurance systems for reference sequences

(c)

Rigorous vetting procedures for newly submitted sequences to ensure database integrity

Such measures will significantly improve taxonomic resolution and detection reliability, while providing robust foundational support for airborne eDNA applications in ecological monitoring [28]. The implementation of these standards will facilitate proper validation studies and inter-laboratory comparisons, ultimately determining the technique’s operational limits and optimal deployment strategies.

5.3. Integration of Multiple Technologies

The integration of multiple technologies represents the future development trend of biodiversity monitoring. Combining airborne eDNA technology with traditional monitoring methods can capitalize on their respective advantages and enable complementary monitoring. For instance, when eDNA technology is combined with camera traps and visual surveys, eDNA technology’s high sensitivity allows it to detect the presence of rare species [39]. Meanwhile, camera traps and visual surveys can capture behavioral and ecological information about organisms, including animals’ activity patterns, reproductive behaviors, and interspecies relationships. By integrating these approaches, we can not only gain a more comprehensive understanding of biodiversity, we can also conduct in-depth research on the ecological habits of organisms. Moreover, the combination of eDNA technology with remote sensing technology can furnish extensive spatial information, such as terrain, vegetation coverage, and land use [14,48]. Performing a correlation analysis on the biodiversity data obtained through eDNA technology and the spatial information provided by remote sensing technology can help us gain a deep understanding of the relationship between species distribution and environmental factors. This provides a more scientific foundation for ecological protection and resource management. At the same time, applying artificial intelligence technology to analyze large volumes of eDNA data holds great potential. Artificial intelligence algorithms can rapidly process and analyze massive eDNA datasets, enhancing the efficiency and accuracy of data analysis. They can also uncover potential information, such as biodiversity change trends and potential species associations, offering stronger support for biodiversity monitoring and ecological protection decision making.

5.4. Emerging Application Scenarios

With the continuous development of technology, technology for gathering air eDNA will be applied in more emerging fields [39]. In the monitoring of migratory bird routes, through setting up air sampling points in different locations, regularly collecting eDNA samples in the air, and analyzing the DNA information of birds, we can track the migratory routes and stopover sites of birds [39]. This is of great significance for understanding the migration patterns of birds, protecting their habitats, and formulating reasonable ecological protection policies. Accordingly, by monitoring, the key stopover sites that birds rely on during migration can be discovered, thereby strengthening the protection of these areas to ensure the survival and reproduction of birds during migration. For the protection assessment of endangered species, these technologies can monitor the distribution and population changes of endangered species in real time. By promptly identifying changes in the habitats of endangered species, fluctuations in population numbers, and potential threat factors such as human disturbance and habitat destruction, it can provide a scientific basis for formulating targeted protection measures and improve the protection of endangered species.

6. Conclusions

Airborne environmental DNA (eDNA) technologies provide a revolutionary non-invasive means for biodiversity monitoring that is capable of simultaneously detecting multiple groups including vertebrates, insects, plants, and microorganisms, making up for the shortcomings of traditional methods (e.g., camera traps and acoustic monitoring) in coverage and efficiency. Its technical core includes active (filter-based, water-based samplers) and passive sampling methods (e.g., spider webs, dust traps), combined with high-throughput sequencing and bioinformatics analysis, which have been successfully applied in the monitoring of forest, urban, and agricultural ecosystems, revealing species distribution and seasonal dynamics. However, these technologies still face challenges such as the complexity of the eDNA production–transport–degradation process, contamination risks, primer bias, and difficulties in quantitative analysis. Future development directions should focus on optimizing sampling strategies, developing specific primers, integrating eRNA data, and establishing standardized procedures and improving DNA barcode databases. The integration of multiple technologies (such as remote sensing and AI) and emerging applications (such as migratory bird tracking and endangered species assessment) will further expand its potential, promoting the precision and efficiency of global biodiversity conservation.