ABSTRACT Understanding the status and global trends of soil invertebrate diversity requires accurate and comparable data across geographical regions. However, soil animal extraction approaches still vary among laboratories, and no commonly accepted, openly available and well‐documented protocols exist across taxa. Here, we present harmonized methodologies, assembled by an international group of experts, for extracting soil‐ and litter‐inhabiting nematodes, enchytraeids, microarthropods, and larger invertebrates. Illustrated with images and videos, the protocols include advice for overcoming the most frequently encountered issues (’expert tips’) for maximizing extraction efficiency and reproducibility. In addition, we provide results from two pilot experiments that test certain steps in nematode and large invertebrate extractions. We show that using two layers of milk filters instead of one in wet extraction of nematodes yields very similar extraction efficiency and has little effect on the sample cleanliness. Further, we demonstrate that on average 31.3% of large soil animals (body length 3 mm and longer) are overlooked during hand sorting but were captured by heat extraction of the sorted samples across three geographically distinct laboratories. Observed differences among the laboratories call for standardized tests of extraction efficiency of large soil animals across regions and research groups. Overall, we provide openly available expert protocols for assessing soil animal taxa in ecological studies worldwide, ultimately facilitating comparisons for a better understanding of the distribution and dynamics of soil biodiversity.

Understanding the resource utilization of soil invertebrates is essential for elucidating nutrient cycling and energy flow in terrestrial ecosystems. The soil environment offers a wide range of resources to its consumers, including microbes, soil organic matter (SOM), and plant litter. Soil invertebrates are small and cryptic, and typically rely on diverse arrays of basal resources, making the determination of its specific contributions to the diets of distinct animal groups challenging. This study investigated the carbon and nitrogen flows from different organic resources to soil meso-and macrofauna using stable isotope dual-labelling (13C and 15N) in an experimental temperate forest mesocosm over a one-month period. Studied resources included bacterial and fungal biomass, leaf litter and artificial mineral-associated organic matter 'a-MaOM' made of microbial necromass and vermiculite-a newly developed substrate produced in-house to experimentally mimic stabilized SOM. Our findings indicate that mesofauna incorporated the isotopic label from bacteria and fungi within three days after label introduction, demonstrating their role as primary microbial consumers in soil food webs and highlighting the importance of microbial biomass as nutrient sources for soil mesofauna. In contrast, macrofauna showed no detectable label uptake during the entire experimental period, suggesting either dietary preferences for other, unidentified resources or reflecting physiological factors such as lower metabolic turnover and slower assimilation of labelled materials. No detectable label was found in the studied invertebrate groups in the a-MaOM and litter treatments during the short experimental period, indicating that its consumption by soil fauna was negligible in comparison to microbial biomass. Beyond feeding ecology, our study introduces a methodological innovation by producing stable 13C-and 15N-labelled a-MaOM, offering a new tool for experimentally tracking stabilized SOM pathways in soil food webs while its bioavailability to soil organisms remains to be studied. Overall, our results reveal distinct feeding strategies among soil invertebrates, emphasizing the importance of mesofauna-microorganism interactions in soil nutrient cycling and the differentiated feeding modes of meso-and macrofauna in temperate forest ecosystems.

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Viruses of soil-dwelling invertebrates remain poorly studied. Viruses of eusocial insects are of special interest as they can serve as a model for studying the spread of viruses via social interactions. In this review, we aim to compile and actualize the available information on the diversity of viruses associated with soil-dwelling social insects, termites and ants. Both groups are among the most functionally important soil invertebrates and include numerous pests and invasive species. We analyzed 93 articles dedicated to viral findings in these groups. Viruses were found in 54 species of ants and 28 species of termites. In sum, 270 viruses and viral genetic variants from over 16 viral orders were found to date in soil-dwelling social insects. Complete information on viruses detected in termites and ants, including insect species, viral name, species, replication status, and GenBank accession number is provided. For most of the novel viruses, replication in the insect was not yet confirmed. We encourage more studies of the virome of ants and termites, which should pay more attention to viral replication and infection symptoms.

The extraradical mycelium of mycorrhizal fungi is among the major carbon pools in soil that is hard to quantitatively assess in-situ. Established method of in-growth mesh bags in temperate ecosystems is difficult to apply in the tropics, where mesh bags are often damaged by termites. Here we introduce a modification of the ingrowth mesh bag technique, in which mesh bags are enforced by stainless steel mesh. Its performance was tested in the Dong Nai (Cat Tien) National Park in Vietnam across two monsoon tropical forests, dominated by tree species associated with either ectomycorrhizal (ECM) or arbuscular mycorrhizal (AM) fungi. Armored ingrowth mesh bags remained intact, while about 60 % of non-armored mesh bags were damaged by termites after 180 days of exposure. The biomass of extraradical mycelium of ectomycorrhizal fungi estimated by PLFA analysis was similar in the armored and non-armored mesh bags and did not differ between studied forests. However, fungal community composition slightly differed between armored and non-armored mesh bags in the ECM-but not in the AM-dominated forest. Fungal mycelium gathered in the AM-dominated forest was depleted in N-15 compared to that collected in the ECM-dominated forest. Overall, our results argue for using armored mesh bags as a robust tool for harvesting the biomass of extraradical mycelium of mycorrhizal fungi in tropical ecosystems.

The role of earthworms in the direct transfer of dissolved soil organic carbon to plants is poorly understood. We quantified this effect by examining the root uptake of carbon from 13C-labeled rice straw in a greenhouse experiment with three non-flooded Philippine soils that greatly differed in texture (from light sandy to loam). Measurements of carbon stable isotope signatures of aerobic rice and maize over a two-month period revealed that earthworms positively affected 13C transfer from rice straw to rice plants but not to maize plants. However, this effect was significant only in loamy and not in sandy soils. Although the direct uptake of dissolved carbon from soil had no impact on rice production, it indicated a widely ignored option to mitigate carbon loss from crop residue decomposition and associated climate risks. Its enhancement by earthworms confirms the important role of this taxon as soil engineers.

Mycorrhizal fungi represent a potentially abundant carbon resource for soil animals, but their role in soil food webs remains poorly understood. To detect taxa that are trophically linked to the extraradical mycelium of mycorrhizal fungi, we used stable isotope (13C) labelling of whole trees in combination with the in-growth mesh bag technique in two coniferous forests. This allowed us to detect the flux of carbon in the mycelium of mycorrhizal fungi, and consequently in the tissues of soil invertebrates. The mycorrhizal fungal genera constituted 93.5% of reads in mycelium samples from the in-growth mesh bags. All mycelium from in-growth mesh bags and about 32% of the invertebrates sampled (in total 11 taxa) received the 13C label after 45 days of exposure. The extent of feeding of soil invertebrates on the mycelium of mycorrhizal fungi depended on the taxonomic affinity of the animals. The strongest trophic link to the mycorrhiza-derived carbon was detected in Isotomidae (Collembola) and Oppiidae (Oribatida). The label was also observed in the generalist predators, indicating the propagation of mycorrhiza-derived carbon into the higher trophic levels of the soil food web. Higher 13C labelling in the tissues of euedaphic Collembola and Oribatida compared to atmobiotic and hemiedaphic families indicates the importance of mycorrhizal fungi as a food resource for invertebrates in deeper soil horizons.

New data on the distribution of 2 species of the genus Gymnometriocnemus Edwards, 1932: Gymnometriocnemus (Rhaphidocladius) brumalis (Edwards, 1929) and Gymnometriocnemus (Rhaphidocladius) kamimegavirgus Sasa et Hirabayashi, 1993 in Russia are presented.The species were caught using an emergent trap installed on the territory of Moscow.The distribution of the species on the territory of the European part of Russia was proved using morphological and molecular methods.РЕЗЮМЕ.Представлены новые данные о распространении 2 видов рода Gymnometriocnemus Edwards, 1932: Gymnometriocnemus (Rhaphidocladius) brumalis (Edwards, 1929) и Gymnometriocnemus (Rhaphidocladius) kamimegavirgus Sasa et Hirabayashi, 1993 на территории России.Виды были пойманы при помощи эмергенц-ловушки, установленной на территории Москвы.Распространение видов на территории европейской части России было доказано с использованием морфологического и молекулярного методов.

Fruiting bodies of ascomycete fungi that appear in temperate forests after the melting of snow cover but before the active plant vegetation, were probed as an ephemeral food resource for soil and terrestrial invertebrates for the first time. Studied apothecia of Sarcoscypha austriaca were inhabited only by Nematoda, while the diversity of invertebrates inhabiting the hollow ascocarps of Gyromitra esculenta was considerably higher (at least 31 families). Stable isotope analysis confirmed the trophic status of G. esculenta as a humus saprotroph and showed very limited trophic links of invertebrates to ascocarps, which suggests a more pronounced function of the mature fruiting bodies of G. esculenta as a hideout, rather than a food substrate.

Marine macroalgae are often used as biofertilizers in the coastal regions across the world. However, the effects of soil fauna in macroalgae decomposition are not fully understood. We conducted a microcosm experiment to assess the possibility of using a mixture of native Enchytraeidae and the model earthworm species Eisenia fetida to accelerate decomposition of dominant algae taxa from White Sea (Fucus spp.) and Black Sea (Cystoseira spp.) after their amendment to soil with simultaneous mitigation of the associated carbon dioxide (CO2) emissions. During 45 days of the microcosm experiment, we measured CO2 emission from the soil and evaluated changes in macroalgal biomass, 813C and 815N isotopic signatures of soil, macroalgae, earthworm faeces and enchytraeid tissues as well as bulk carbon and nitrogen content in soil. The mass of Cystoseira spp. residues, unlike that of Fucus spp., significantly decreased during the experiment (by approximately 73 % of the initial level). This effect was amplified by the co-action of E. fetida and Enchytraeidae (by 65 % of the initial). The addition of Enchytraeidae into microcosms led to the more than two-fold reduction of CO2 emission in all treatments. Differential enrichment of soil and earthworm casts with macroalgae-derived carbon and nitrogen, as suggested by stable isotope analysis, indicated that macroalgae-originating carbon pathway is rather driven by a synergistic activity of enchytraeids and earthworms (813C enrichment up to 4.8 parts per thousand). Nitrogen transfer was dependent on the microbial activity and could be modulated and channelized by enchytraeids, whose 815N values strictly corresponded to algal taxa. This highlights the importance of annelids in increasing the value of macroalgae as biofertilizers.

The features of the distribution of the stable isotope composition of carbon and nitrogen for saprotrophic and mycorrhizal macromycetes growing on an oligotrophic peatland were characterized for the first time. The molecular identification of the free-growing mycelium of mycorrhizal fungi detected the presence of both ecto- and arbuscular mycorrhizal fungi. Stable isotope composition of fruiting bodies and mycelium of mycorrhizal fungi of peatland differed fractionally. The fruiting bodies of saprotrophic fungi growing on the surface of the moss cover were enriched in 13 С, but not in 15 N, compared to their main substrate, sphagnum. Differences in the isotopic composition of functionally different parts of the fruiting bodies of macromycetes (hymenophores and stipes) were minor. The mycelium of mycorrhizal fungi of the peatland is enriched in 15 N by 1.4 to 9.5 ‰ compared to the tissues of host plants. These values noticeably exceed similar values that were previously shown for forest ecosystems.

Ectomycorrhizal fungi are a prominent component of the soil biota of boreal forests, but the role of mycorrhizal mycelium as a food source for soil Collembola remains controversial. We addressed this question in a trenching experiment in young (70 years old) and old (180 years old) stands of Scotch pine, combined with stable isotope analysis. Trenching halved the biomass of ectomycorrhizal mycelium, estimated using in-growth mesh bags. In the young forest, the abundance of two euedaphic Collembola species, Mesaphorura yosiii and Willemia anophthalma, decreased after trenching by 99 and 97% respectively, while in the old forest the abundance of Collembola was not affected. In both forests, trenching reduced δ15N values of the dominant euedaphic species Isotomiella minor and W. anophthalma, indicating a shift in trophic niches. Thus, we obtained convincing evidence of species-specific trophic links of euedaphic Collembola species to the mycelium of mycorrhizal fungi.

Soil animals perform a range of essential ecosystem functions and can modify the effects of global change on terrestrial ecosystems. We evaluated responses of six major groups of soil animals (Acari (all groups), Oribatida, Collembola, Insecta, Nematoda, and Oligochaeta) to controlled changes in air temperature, precipitation level or carbon dioxide concentration by using random-effects modelling and mixed-effects meta-regression modelling. Along with the three global change factors, sixteen local climatic characteristics (such as mean annual temperature, Ko & BULL;ppen climate classification, vegetation type) were tested. Overall, 86 studies comprising 236 observations with mean duration of 51 months were selected as relevant for the analysis. Quantitative links between global change factors, local climate characteristics and changes in abundance of four taxonomic groups of soil animals were revealed. Warming and precipitation level were associated most strongly with population dynamics of soil invertebrates compared to elevated atmospheric CO2. Each 1 & DEG;C increase in air temperature was correlated with a mean of 12.5% (95% CI: 2.5%-22.6%) increase in Acari abundance, while populations of Collembola were declined by 9.6% (95% CI: -17.8% to -1.4%). Meanwhile, each 10% increase in precipitation level was correlated with the increase in the abundance of Nematoda by 1.4% (95% CI: -7.6% to 10.4%) and Oligochaeta by 34.7% (95% CI: 8.1%-61.2%). Considering IPCC estimates (SSP3-7.0 Scenario) of an average climate warming by 3.6 & DEG;C and a substantial variation in local precipitation levels (up to & PLUSMN;20%) by the end of the 21st century, strong local changes in the structure of detrital food webs are predicted by meta-regression models. In regions with decreased precipitation, the formation of soil food webs promoting carbon mineralization may be expected, while in regions with increased precipitation, the changes in detrital food web structure can contribute to the accumulation of carbon in the soil.

The genetic diversity of gamasid mite, Hoploseius oblongus Masan & Halliday, 2016, found in France, Denmark, Ukraine, and widely spread in Russia (from the Caucasus to the Arctic), is characterized. This species, known so far from the six finds in Slovakia and Poland, lives only in the hymenophore of living fruiting bodies of the bracket fungus Fomitopsis pinicola . A total of 502 living F. pinicola fruiting bodies from 62 localities were studied, and DNA barcoding of H. oblongus individuals from 28 localities in Europe, the Caucasus, and Western and Central Siberia was performed. At least 90% of the fruiting bodies of F. pinicola in the studied regions were inhabited by the H. oblongus mite. The association of F. pinicola with the H. oblongus mite was observed throughout the Western Palearctic, up to the Yenisei River valley in the east. Throughout the territory of the extensive range examined, H. oblongus is characterized by the low sequence variation of the mitochondrial COI gene BOLD fragment and is monomorphic in the nucleotide sequence of nuclear ribosomal repeat fragment. Low genetic diversity points to the relatively recent origin of the population of H. oblongus , possibly associated with the restoration of the forest belt of Northern Eurasia in the Holocene. The efficiency of H. oblongus dispersal is ensured by the phoresy of adult mites on ovipositing females of dark-winged fungus gnats (Sciaridae) in late summer and early autumn. More rarely, mites disperse on adult gall midges (Cecidomyiidae) from the tribe Brachineurini or on adults of some other small insect species from the order Diptera.

The development of methods for exclusion or reduction of the abundance of the target group in a field experiment is one of the important methodological tasks in studying the interactions of soil fungi and invertebrates. The possibility of using cypermethrin in order to reduce the abundance of invertebrates during the research of interaction with mycorrhizal fungi was studied in two model coniferous forests (in the reserves “Kivach” and the Central Forest State Reserve). A short-term (30 days) experiment showed a significant decrease in the abundance of soil invertebrates and no differences in the mycelium biomass of mycorrhizal fungi. In a longer experiment (90 days), the effect of soil defaunation was similar, but the production of mycelium of mycorrhizal fungi decreased compared to the control. The method of soil defaunation with cypermethrin is effective, but can be recommended for use only in short-term field experiments.

Gastropods from the family Eulimidae have different life strategies in inhabiting their host echinoderms, but it is currently assumed that they derive most of their nutrition from the host’s tissues. This paper presents the results of stable carbon and nitrogen isotope analyses of different tissues of echinoderm hosts (crinoids Comaster nobilis and holothurians Holothuria atra) and their ectosymbionts (Annulobalcis wareni and Peasistilifer nitidula). Our data show that the stable nitrogen isotope enrichment level in symbionts is practically indistinguishable from that of their hosts. The symbionts are depleted in 13C compared to their hosts. We suggest that these molluscs may not feed on host tissues but instead obtain food particles from the environment, possibly stealing detritus particles, which, in turn, are food for these species of echinoderms. We revealed the proximity of the location and slight overlap of the crinoids’ visceral tissue and their symbiont tissues. This suggests a greater food variety for the symbionts. The detected differences in δ13C values and the almost complete absence of overlap of the holothurians’ body walls and symbionts allow us to conclude that in this symbiotic association, the hosts and symbionts rely on different food sources. In addition, the two studied echinoderm species differed significantly in δ13C signatures, which is confirmed by their different lifestyles. Thus, stable isotope analysis suggests that kleptoparasitism is at least an additional and possibly exclusive feeding mode for eulimids A. wareni and P. nitidula. However, this hypothesis will need to be followed up with morphological, biochemical, and experimental data.

Marine organic matter can potentially enter terrestrial ecosystems via abiotic or biotic vectors. The former is facilitated by e.g. waves, tides, winds, etc.; the latter - by the activity of organisms, e.g. seabird migration. However, differences in its assimilation rate in detrital food webs at different distance from water remain generally unexplored. To fill this gap, we compared the consumption of marine organics by terrestrial in-vertebrates within coastal ecosystems of the Black sea using stable isotope analyses of carbon and nitrogen. We focused on relatively inland (100 m from the coast) forested areas with the recently established overwintering colonies of the great cormorant (Phalacrocorax carbo) and control ecosystems isolated from any marine organic matter input. Guano and cormorant food leftover input led to the significant increase of delta N-15 values of soil, litter and all trophic groups of soil invertebrates. However, delta C-13 values changed to a smaller extent. The importance of marine resources (in a form of guano) as a carbon source for soil arthropods was relatively low: approximately 15% of their diet. In contrast, coastal soil invertebrates were significantly enriched with C-13 compared to control and cormorant-influenced ecosystems as they were greatly dependent on the seaweed and other marine resources (on average 45% of coastal invertebrate diet). We conclude that marine resources delivered by an abiotic vector may form the basis of the soil food web diets in a 15 m zone from the water edge. Alternatively, the biotic transfer further inland (forest on a cliff 100 m away from the water edge) with e.g. nitrogen-rich seabird guano or food remains may geographically extend proliferation of marine nutrients into terrestrial ecosystems. As far as we know, our results are among the first to demonstrate the spatial zoning of mechanisms securing bottom-up subsidy of biogenic elements to soil ecosystems having originated from the sea.

The relative importance of belowground and aboveground energy inputs for the decomposer communities in soil remains largely unknown. In particular, no research has been done on the significance of root-derived resources for nematode communities in boreal forests. In two spruce stands in the taiga zone, we set up a field experiment in which girdling of spruce trees and clipping of dwarf shrubs was performed. Root-derived resources were hypothesized to be highly important; accordingly, we expected to observe a suppression of the nematode community after experimental manipulations. To obtain information on the nature of changes in the soil food web, nematode community structure indices were applied. In partial confirmation of our hypothesis, spruce girdling decreased mycorrhizal hyphae biomass as assessed via in-growth mesh bags, as well as the abundance of fungivorous nematodes, mostly of the Aphelenchoides and Filenchus genera. The enrichment index (EI) value decreased, indicating reduction of organic matter inputs into the soil food web, whereas nematode channel ratio (NCR) index value increased, indicating a shift towards domination of the bacterial energy channel. Total nematode abundance, genera richness, and abundance of herbivores, omnivores, and predators did not change in response to spruce girdling. Clipping of dwarf shrubs decreased fungal and bacterial PLFA biomarkers, but did not affect nematode communities. Thus, the resources channeled in soil by the roots of canopy trees are of different relative importance for nematodes having different trophic habits. Fungivorous nematodes are at least partly dependent on root-derived resources, suggesting feeding on ectomycorrhizal mycelium. Rhizodeposits of understory vegetation are likely of low importance for nematodes.

Size-structured food webs form integrated trophic systems where energy is channeled from small to large consumers. Empirical evidence suggests that size structure prevails in aquatic ecosystems, whereas in terrestrial food webs trophic position is largely independent of body size. Compartmentalization of energy channeling according to size classes of consumers was suggested as a mechanism that underpins functioning and stability of terrestrial food webs including those belowground, but their structure has not been empirically assessed across the whole size spectrum. Here we used stable isotope analysis and metabolic regressions to describe size structure and energy use in eight belowground communities with consumers spanning 12 orders of magnitude in living body mass, from protists to earthworms. We showed a negative correlation between trophic position and body mass in invertebrate communities and a remarkable nonlinearity in community metabolism and trophic positions across all size classes. Specifically, we found that the correlation between body mass and trophic level is positive in the small-sized (protists, nematodes, arthropods below 1 μg in body mass), neutral in the medium-sized (arthropods of 1 μg to 1 mg), and negative in the large-sized consumers (large arthropods, earthworms), suggesting that these groups form compartments with different trophic organization. Based on this pattern, we propose a concept of belowground food webs being composed of (1) size-structured micro-food web driving fast energy channeling and nutrient release, for example in microbial loop; (2) arthropod macro-food web with no clear correlation between body size and trophic level, hosting soil arthropod diversity and subsidizing aboveground predators; and (3) "trophic whales," sequestering energy in their large bodies and restricting its propagation to higher trophic levels in belowground food webs. The three size compartments are based on a similar set of basal resources, but contribute to different ecosystem-level functions and respond differently to variations in climate, soil characteristics and land use. We suggest that the widely used vision of resource-based energy channeling in belowground food webs can be complemented with size-based energy channeling, where ecosystem multifunctionality, biodiversity, and stability are supported by a balance across individual size compartments.