Quantitative assessment of omega-3 and omega-6 polyunsaturated fatty acid fluxes between aquatic and terrestrial ecosystems of a small shaded river

doi:10.21203/rs.3.rs-5150104/v1

Boreal forests are rich in small rivers, whose primary productivity is limited by shading. The diet of benthivorous fish in such rivers is based on autochthonous and allochthonous organic matter. Aquatic algae and invertebrates produce and accumulate polyunsaturated fatty acids of n-3 family (n-3 PUFA). Terrestrial organisms are poor in these substances but are rich in n-6 PUFA. We aimed to assess fluxes of biomass and n-3 and n-6 PUFA between aquatic and terrestrial ecosystems of the shaded Krutaya Kacha River. The production of zoobenthos in the river was 11.7 mg of dry weight (DW)·m^− 2·day^− 1, while the export of biomass of amphibious insects was 4.3 mg (DW)·m^− 2·day^− 1. The import of invertebrate biomass into the river was 56.2 mg (DW)·m^− 2·day^− 1, which was one order of magnitude higher than the export of amphibious insects and 5 times higher than the production of zoobenthos. The import of n-3 PUFA, and n-6 PUFA into the river via invertebrates was 0.55 and 0.909 mg (DW)·m^− 2·day^− 1, respectively, while the export of these substances from the river with emergent insects was lower by factors of 6.6 and 20.7, respectively. Thus, in such rivers, benthivorous fish feeding on aquatic and terrestrial resources receive food of biochemically different quality: the amounts of food being equal, fish consuming terrestrial invertebrates receive less n-3 PUFA but more n-6 PUFA than fish consuming aquatic invertebrates. The predominance of allochthonous food in the diet of fish can be the reason for the decrease in the nutritional value of fish inhabiting small shaded rivers.

zoobenthos

emergence

terrestrial subsidies

arthropods

eicosapentaenoic acid

food webs

Aquatic and terrestrial ecosystems are closely linked by fluxes of matter and energy that occur in both directions: from land to water and from water to land (Baxter et al. 2005; Marcarelli et al. 2011; Vander Zanden and Gratton 2011; Richardson and Sato 2015). The quantity and the quality of these fluxes vary. One of the important indicators of the quality of transferred substances is the content of polyunsaturated fatty acids (PUFA) (Hixson et al. 2015).

In aquatic ecosystems, primary producers synthesize significant amounts of long-chain (LC) PUFA of the n-3 family, namely eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), which have high physiological value for most animals (Lands 2009; Gladyshev et al. 2013; Gladyshev and Sushchik 2019), and alpha-linolenic (ALA, 18:3n-3) and linoleic (LA, 18:2n-6) acids (Makhutova et al. 2022). In terrestrial ecosystems, primary producers synthesize significant amounts of ALA and LA, but not EPA and DHA (Hixson et al. 2015; Twining et al. 2017; Guo et al. 2021; Bogatov et al. 2021). There are exceptions, though, in terrestrial ecosystems, for example, some terrestrial mosses produce significant amounts of LC-PUFA (Filippova et al. 2023).

In terrestrial invertebrates, the contents of PUFA in general and EPA and DHA in particular are significantly lower, and LA is higher than in aquatic and semi-aquatic animals (Twining et al. 2019; Parmar et al. 2022). A dichotomy is evident between the FA profiles of aquatic and terrestrial invertebrates, with the division being driven primarily by the higher n-3 PUFA content in aquatic invertebrates and the higher n-6 PUFA content in terrestrial invertebrates (Fontaneto et al. 2011; Hixson et al. 2015; Gladyshev and Sushchik 2019; Parmar et al. 2022). Invertebrates inhabiting water surface such as some species of beetles (whirligig beetles), bugs (water striders, creeping water bugs), spiders (pisaurid spiders), springtails, etc. are of considerable interest (Hendawy et al. 2005). These organisms are able to feed on both aquatic and terrestrial resources, which is reflected in their fatty acid composition. They can also serve as food for aquatic and terrestrial inhabitants. Thus, aquatic ecosystems play a unique role in the biosphere as a major source of n-3 LC-PUFA for animals, including those living in terrestrial ecosystems (Gladyshev et al. 2013; Yan et al. 2024). At the boundary of the aquatic and terrestrial environments, an exchange of substances differing in biochemical quality constantly occurs.

Amphibious insects make a significant contribution to the transfer of aquatic resources from the water, where they spend their larval period, to land, where the adults serve as food for a variety of coastal inhabitants, including bats, birds, lizards, beetles, spiders, etc. (Nakano and Muralami 2001; Gladyshev et al. 2013; Richardson and Sato 2015; Twining et al. 2016; Popova et al. 2017; Kowarik et al. 2021; Bashinskiy et al. 2023). Calculations suggest that amphibious insect biomass export from lotic ecosystems varies widely: 0.4–174 g of dry weight (DW)·m^− 2·yr^− 1 (Jackson and Fisher 1986; Gladyshev et al. 2009; Popova et al. 2017; Moyo et al. 2017; Raitif et al. 2018; Kowarik et al. 2023). At the same time, according to the few data available, the amount of exported LC-PUFA varies from 0.1 mg (DW)·m^− 2·yr^− 1 to 2.5 g (DW)·m^− 2·yr^− 1 (Gladyshev et al. 2009; Moyo et al. 2017). The export of amphibious insect biomass from lentic ecosystems is 0.1–3.7 g (DW)·m^− 2·yr^− 1, which is significantly lower than from lotic ecosystems (Gratton and Vander Zanden 2009; Gladyshev et al. 2011; Dreyer et al. 2015; Borisova et al. 2016; Martin-Creuzburg et al. 2017; Gladyshev et al. 2019). The amount of exported PUFA varies within a range of 1.9–80.5 mg (DW)·m^− 2·yr^− 1 (Gladyshev et al. 2011; Borisova et al. 2016; Popova et al. 2017; Martin-Creuzburg et al. 2017), of which EPA or EPA + DHA comprise 0.03–32.8 mg (DW)·m^− 2·yr^− 1 (Borisova et al. 2016; Martin-Creuzburg et al. 2017; Gladyshev et al. 2019). Thus, the export of biomass and LC-PUFA from rivers is several orders of magnitude higher than from lakes.

Aquatic food webs are subsidized by energy and nutrients from terrestrial ecosystems (Twining et al. 2017). In aquatic ecosystems, terrestrial invertebrates fall from trees and tall grass or are carried by air currents. Arthropods that consume resources of both aquatic and terrestrial origin accumulate on the water surface or near the shoreline and may serve as food for aquatic consumers (Marcarelli et al. 2011; Zakeyuddin et al. 2017). Additionally, according to limited data, 1–3.1% of emerging amphibious insects return to aquatic ecosystems (Gray 1989; Ballinger and Lake 2006; Dreyer et al. 2015), and their amount reaches 0.7 g (DW)·m^− 2·yr^− 1 (Jackson and Fisher 1986). The terrestrial invertebrates that are imported into rivers and streams in the temperate zone are dominated by the representatives of Hymenoptera, Diptera, Coleoptera, Lepidoptera, Homoptera, Orthoptera, Hemiptera, Arachnida, and Collembola (Baxter et al. 2005; Erős et al. 2012). The amount of terrestrial invertebrates imported into water bodies depends on the type of vegetation of the coastal or riparian zone. Studies have shown higher import into streams crossing forests, with deciduous forests having higher infall than coniferous forests. Additionally, the highest import was found in areas with greater canopy density of deciduous trees (Webster and Hartman 2005; Kraus et al. 2016; Milardi et al. 2016). Accurate quantitative assessment of export-import is methodologically difficult, and, therefore, data are scarce. It was shown that rivers and streams receive 8.5–679 mg (DW)·m^− 2·day^− 1 of terrestrial invertebrates from the riparian zones and trees, with which 0.3–3 mg·m^− 2·day^− 1 of LC-PUFA are imported into the water (Erős et al. 2012; Moyo et al. 2017).

We showed previously that in the small shaded Krutaya Kacha River, the diet of fish (Thymallus baicalensis) was based not only on autochthonous invertebrates, but also on allochthonous ones, which resulted in a decrease in the content of EPA and DHA in the muscle tissue of grayling, and, therefore, in a decrease in their nutritional value for consumers (Makhutova et al. 2024). The aim of the present study was to measure fluxes of invertebrate biomass and n-3 and n-6 PUFA between aquatic and terrestrial ecosystems in the Krutaya Kacha River, flowing under a mixed coniferous and deciduous forest canopy, over the ice-free season.

Study area

The study was conducted in the upper reaches of the Krutaya Kacha River, Siberia, from May 1st (after the river was cleared of ice) to September 26th (the beginning of frost), in 2022 and, occasionally, in June and July in 2023, for verification. The 2022 field season lasted 148 days. The river is a left second-order tributary in the middle reaches of the Yenisei River; it is a small river, flowing in the boreal forest. Its length is 17 km, and its width in the sampling area was 1–2 m; the temperature varied from 2.9°C in May to 12.8°C in August. The degree of shading of the river was assessed by measuring photosynthetically active radiation (PAR) intensity at the surface of the river and in the unshaded area close to the river. In situ PAR was measured using terrestrial sensor LI-190SA (Li-Cor Ltd., Lincoln, Nebraska, U.S.A.). On average, over the study period, PAR was 250 ± 27 µmol·m^− 2·s^− 1 in the riverbed and 1088 ± 163 µmol·m^− 2·s^− 1 in the open area (n = 11). In clear weather, PAR in the riverbed and in the field was 314 ± 51 vs. 1581 ± 10 µmol·m^− 2·s^− 1 in May; 412 ± 96 vs. 1820 ± 12 µmol·m^− 2·s^− 1 in June; 345 ± 82 vs. 1949 ± 16 µmol·m^− 2·s^− 1 in July; 250 ± 45 vs. 1379 ± 33 µmol·m^− 2·s^− 1 in August; 301 ± 60 vs. 1091 ± 29 µmol·m^− 2·s^− 1 in September. Mean canopy openness in July was 48.9%.

Sampling

On a 100-meter stretch of the river, we placed 6 floating traps for collecting emergent amphibious insects (Export); each trap had an area of 0.237 m² and was equipped with a removable cap. The heterogeneity of zoobenthos and insect behavior and life histories (Malison et al. 2010) were taken into account when setting the traps. In the same area, 6 floating transparent traps with an area of 0.1085 m² (350·310·85 mm) each were placed to collect terrestrial and aquatic invertebrate infall to the surface of the water (Import) (Fig. 1). Each trap was filled 2 cm deep with water, and a drop of odorless surfactant was added to ensure that the invertebrates would sink to the bottom of the traps and would not be able to escape from the traps by climbing up the walls – in order to avoid underestimating invertebrate import.

Invertebrates were collected from the traps every 1–3 days, depending on weather conditions. All traps were then washed, repaired, and placed again. Flooded, drained or damaged traps were not taken into account in the calculation. Samples were placed in refrigerated bags and transported to the laboratory within an hour. In the laboratory, emerging amphibious insects were placed at a temperature of − 20°C for no more than 30 minutes to immobilize animals. The insects from all traps were then combined into one sample, weighed, separated into taxonomic groups, and weighed again by groups on an analytical balance (± 0.1 mg, Mettler Toledo ME204). Then, insects were dried to constant weight at 60°C to estimate moisture content of animals. Additionally, amphibious insect adults from four dominant groups (Ephemeroptera, Plecoptera, Trichoptera, and Diptera) were collected for fatty acid analysis.

All traps with invertebrates on the water surface were combined into one sample. The invertebrates were divided into two size groups. For this purpose, water with invertebrates, transported to the laboratory in 5-liter canisters, was filtered through 1-mm mesh and then through 130-µm mesh. The organisms larger than 1 mm (macroarthropods) remaining on the 1-mm mesh, together with the debris, were placed in containers with distilled water and all invertebrates were collected with tweezers and placed onto filter paper to remove water from their surface, after which invertebrates were weighed. The organisms smaller than 1 mm (microarthropods) remaining on the 130-µm mesh were also dried with filter paper and subjected to a temperature of − 20°C for 30 minutes to immobilize animals, after which they were weighed. Some samples of macroarthropods and microarthropods were dried to constant weight at 60°C to estimate moisture content of animals. Then, these invertebrates were counted and identified under a stereo microscope. Other samples of macroarthropods and microarthropods were placed in a chloroform: methanol mixture (2: 1, v/v) and kept at − 20°C until further analysis. Samplings for moisture content and FA analysis were alternated.

Macrozoobenthos samples were collected monthly in three replicates to determine taxonomic composition, abundance, and biomass and to calculate their daily production. Zoobenthos samples were collected using a hydrobiological scraper (cutting edge length 19 cm, mesh size 213 µm). In the laboratory, macrobenthic animals were removed from the sample and preserved in 70% ethanol. The organisms were identified under a stereo microscope and sorted into species or genera. The organisms of various taxonomic groups were weighed. A detailed description of the calculation of zoobenthos daily production (P_D, g·m^− 2·day^− 1) can be found in Gladyshev et al. (2016). In addition, representatives of different taxa dominant in the zoobenthos were collected using a hydrobiological scraper, for FA analysis and moisture content.

Fatty acid analysis

Lipid extraction, transesterification (methylation) of the lipids, and purification of methyl esters were done using the methods described in detail by Christie and Han (2010). Before the extraction, a definite volume of an internal standard solution (a solution of 19:0 methyl ester in chloroform, 0.5 mg/mL; Sigma-Aldrich, St. Louis, MO, U.S.A.) was added to the samples. Analysis of fatty acid methyl esters (FAMEs) was conducted using a gas chromatograph with a mass spectrometric detector (Model 7000 QQQ, Agilent Technologies, U.S.A.), which was equipped with a 30-m capillary HP-FFAP column with the internal diameter of 0.25 mm. Detailed descriptions of the chromatographic and mass-spectrometric conditions are given elsewhere (Petrov et al. 2020). FAME identification and quantification are given in Makhutova and Stoyanov (2021). In this study, the content of EPA, sum of C18-C22 PUFA of n-3 family, and sum of C18-C22 PUFA of n-6 family were used.

Statistical analysis

Mean values, standard errors, and non-parametric Kruskal–Wallis test were calculated using STATISTICA software, version 10.0 (StatSoft, Inc.). Editing of figures was carried out in CorelDRAW.

Macrozoobenthos

In 2022, macrozoobenthos production varied from 3.8 ± 1.2 mg (DW)·m^− 2·day^− 1 in June to 25.7 ± 6.2 mg (DW)·m^− 2·day^− 1 in August and averaged 11.7 ± 2.6 mg (DW)·m^− 2·day^− 1 for the season (Fig. 2, Table 1, Table S1). The biomass-dominant taxa were Trichoptera, Amphipoda, and Ephemeroptera (Fig. 2). Trichoptera were represented by 17 species, with Rhyacophila sibirica, and Chaetopteryx spp. dominating; Amphipoda were represented by one species Gammarus lacustris; Ephemeroptera were represented by 19 species, with Ephemerella spp., Epeorus pellucidus, and Rhithrogena grandifolia dominating. The complete taxonomic composition is shown in Table S2. The density-dominant taxa were Diptera (the majority of which were representatives of Chironomidae), Ephemeroptera, and Trichoptera (Fig. 2). Amphibious insects accounted for 64.6% of the biomass and 76.7% of the density of the total macrozoobenthos. The production of amphibious insects varied from 2.3 ± 0.2 mg (DW)·m^− 2·day^− 1 in May to 16.5 ± 3.2 mg (DW)·m^− 2·day^− 1 in August and averaged 7.9 ± 1.7 mg (DW)·m^− 2·day^− 1 for the season. The values of macrozoobenthos production in 2023 (June and July) were within the range of 2022 values (Fig. 2, Table S1).

Table 1

Daily secondary production (B), daily production and the contents of EPA, n-3 and n-6 PUFA (M ± SE) of zoobenthos, emergent insects, imported macroarthropods and microarthropods in the Krutaya Kacha River, May-September 2022 (n – number of replicates). Means labelled with the same letters are not significantly different at P < 0.05 according to the non-parametric Kruskal–Wallis H-test
Groups	Zoobenthos	n	Export (Emergent Insects)	n	Import (Macroarthropods)	n	Import (Microarthropods)	n	H
mg·m^− 2·day^− 1
B, dry weight	11.66 ± 2.63^B	15	4.28 ± 0.52^B	33	45.2 ± 5.5^A	34	10.97 ± 1.31^B	10	64.35
EPA	0.109 ± 0.023^A	15	0.039 ± 0.005^B	30	0.122 ± 0.018^A	34	0.006 ± 0.0007^C	10	38.42
n-3 PUFA	0.246 ± 0.052^A	15	0.082 ± 0.011^B	30	0.532 ± 0.079^A	34	0.018 ± 0.0022^B	10	52.62
n-6 PUFA	0.141 ± 0.029^B	15	0.044 ± 0.007^B	30	0.877 ± 0.091^A	34	0.032 ± 0.0039^B	10	65.85
mg·g^− 1 of dry weight
EPA	8.84 ± 0.44^A	15	9.87 ± 0.57^A	29	3.28 ± 0.58^B	17	0.99 ± 0.28^B	9	45.52
n-3 PUFA	19.89 ± 0.82^A	15	20.44 ± 1.19^A	29	12.05 ± 1.64^B	17	2.95 ± 0.31^B	9	33.04
n-6 PUFA	12.17 ± 0.38^AB	15	11.64 ± 0.74^B	29	20.05 ± 2.52^A	17	5.31 ± 1.21^C	9	27.24

Table S1

Zoobenthos production (mg (DW)·m^− 2·day^− 1), and production of EPA, n-3 PUFA, and n-6 PUFA (mg·m^− 2·day^− 1) by zoobenthos in the Krutaya Kacha River in 2022 and 2023
Month	Zoobenthos	EPA	n-3 PUFA	n-6 PUFA
2022
May	5.725	0.045	0.110	0.080
May	2.575	0.034	0.071	0.027
May	6.686	0.063	0.136	0.091
June	1.947	0.019	0.043	0.025
June	3.229	0.025	0.070	0.042
June	6.153	0.073	0.149	0.069
July	25.416	0.273	0.595	0.291
July	10.830	0.091	0.216	0.145
July	14.918	0.180	0.390	0.174
August	25.435	0.203	0.498	0.352
August	15.041	0.172	0.384	0.173
August	36.549	0.267	0.645	0.371
September	3.143	0.030	0.072	0.037
September	8.740	0.075	0.167	0.117
September	8.469	0.085	0.140	0.119
2023
June	7.907	0.118	0.239	0.083
June	4.520	0.064	0.129	0.046
June	10.877	0.167	0.327	0.110
July	6.889	0.070	0.134	0.074
July	19.566	0.133	0.293	0.163
July	9.191	0.100	0.209	0.110

Table S2

The taxonomic composition of zoobenthos in the Krutaya Kacha River in 2022 and 2023
Data Taxonomic affiliation	2022					2023
Data Taxonomic affiliation	M	Jn	Jl	A	S	Jn	Jl
Phylum: Arthropoda Class: Insecta Order: Diptera
Family: Chironomidae
Constempellina brevicosta (Edwards, 1937)			+				+
Cricotopus gr.tremulus		+		+		+	+
Cricotopus gr.bicinctus						+
Cladotanytarsus gr. mancus						+	+
Eukiefferiella gr.claripennis			+
Eukiefferiella gr.devonica			+	+		+
Eukiefferiella gr.gracei		+		+
Heterotrissocladius gr.marcidus				+	+		+
Hydrobaenus sp.	+						+
Larsia curticalcar (Kieffer, 1918)		+			+		+
Limnophyes sp.				+			+
Micrapsectra gr.praecox		+	+				+
Microtendipes gr.pedellus					+		+
Nanocladius sp.				+
Natarsia punctata (Meigen, 1804)		+		+
Orthocladius gr.saxicola		+		+			+
Orthocladius thienemanni (Kieffer & Thienemann, 1906)		+				+
Orthocladius sp.						+
Pagastia lanciolata (Tokunaga, 1936)				+
Pagastia orientalis (Chernovsky, 1949)			+	+		+	+
Paracladopelma camptolabis (Kieffer, 1913)			+	+	+		+
Paracricotopus niger (Kieffer, 1913)				+
Parafiefferiella triquetra (Pankratova, 1970)				+			+
Paratendipes albimanus (Meigen, 1804)					+
Paratrichocladius inaequalis (Kieffer, 1926)							+
Polypedilum scalaenum (Schrank, 1803)			+	+	+
Potthastia gaedii (Meigen, 1838)						+
Procladius ferrugineus (Kieffer, 1918)				+	+		+
Prodiamesa olivacea (Meigen, 1818)					+
Psectrocladius bisetus (Goetghebuer, 1942)				+
Rheotanytarsus sp.		+	+			+	+
Stempellinella minor (Edwards, 1929 )		+		+	+
Stempellina bausei (Kieffer, 1911)							+
Stictochironomus crassiferceps (Kieffer, 1922)		+		+	+
Orthocladius (Symposiocladius) lignicola (Kieffer, 1914)			+
Synorthocladius semivirens (Kieffer, 1909)						+	+
Thienemanniella gr.clavicornis		+				+
Thienemannimiya sp.	+	+	+	+		+	+
Tvetenia gr.bavarica	+	+	+	+		+
Zavrelia pentatoma (Kieffer, 1913)		+
Family: Limoniidae
Dicranota bimaculata (Schummel, 1829)	+	+	+	+	+		+
Limoniidae gen.sp.							+
Hexatoma bicolor (Meigen, 1818)			+		+
Family: Simuliidae		+	+	+	+	+
Family: Ceratopogonidae		+	+	+	+	+
Family: Dixydae
Dixa nebulosa (Meigen, 1830)				+
Family: Empididae
Chelifera sp.		+		+
Family: Psychodidae	+
Family: Blephariceridae
Blephariceridae gen.sp.						+
Order: Trichoptera
Agapetus jakutorum (Martynov, 1934)		+	+	+
Agapetus sp.						+
Apatania crymophila (McLachlan, 1880)			+				+
Apatania zonella (Zetterstedt, 1840)					+
Chaetopteryx sahlbergi (McLachlan, 1876)			+	+			+
Chaetopteryx villosa (Fabricius, 1798)				+			+
Ecclisomyia digitata (Martynov, 1929)	+	+	+	+	+		+
Glossosoma sp.			+	+	+		+
Goera sajanensis (Martynov 1924)		+		+	+		+
Halesus tesselatus (Rambur, 1842)	+
Lepidostoma hirtum (Fabricius, 1775)			+
Micrasema sp.			+	+			+
Molannodes tinctus (Zetterstedt, 1840)					+		+
Rhyacophila angulata (Martynov, 1910)				+
Rhyacophila sibirica (McLachlan, 1879)	+	+	+	+	+	+	+
Semblis phalaenoides (Linnaeus, 1758)				+
Tinodes waeneri (Linnaeus, 1758)		+		+
Order: Ephemeroptera
Ameletus sp.						+
Baetis fuscatus (Linnaeus, 1761)	+		+	+	+
Baetis sp.				+	+	+
Baetis ussuricus (Kluge, 1983)					+
Baetis pseudothermicus (Kluge, 1983)						+
Caenis rivulorum (Eaton, 1884)			+	+	+
Cloeon (Centroptilum) luteolum (Muller 1776)							+
Ecdyonurus (Afronurus) abracadabrus (Kluge, 1983)			+
Ecdyonurus aspersus (Kluge, 1980)				+			+
Epeorus pellucidus (Brodsky, 1930)			+	+	+		+
Ephemerella aurivillii (Bengtsson, 1909)	+		+	+		+
Ephemerella ignita (Poda, 1761)		+	+				+
Ephemerella setigera (Bajkova, 1965)				+	+
Ephemerella (Drunella) triacantha (Tschernova, 1949)		+	+	+		+
Ephemerella sp.							+
Leptophlebia (Neoleptophlebia) chocolata (Imanishi, 1937)	+	+	+	+	+	+	+
Rhithrogena hirasana (Imanishi, 1935)	+			+
Rhithrogena grandifolia (Tshernova, 1952)						+
Rhithrogena sp.		+		+	+
Order: Plecoptera
Alloperla sp.	+		+		+		+
Amphinemura borealis (Morton, 1894)	+	+	+	+	+	+	+
Capnia sp.					+
Isoperla sp.						+	+
Leuctra sp.		+	+	+	+
Leuctra fusca (Linnaeus, 1758)						+	+
Nemoura sp.	+
Nemoura sahlbergi (Morton, 1896)						+
Skwala pusilla (Klapálek, 1912)			+	+	+
Order: Coleoptera
Oulimnius sp.	+	+	+	+	+	+	+
Agabus sp.				+
Dityscidae gen.sp.1						+
Dityscidae gen.sp.2						+
Order: Lepidoptera
Lepidoptera fam.gen.sp.			+
Order: Megaloptera
Sialis sordida (Klingstedt 1932)			+				+
Class: Malacostraca Order: Amphipoda
Gammarus lacustris (G.O.Sars, 1863)	+		+	+	+	+	+
Class: Arachnida Subclass: Acari Order: Trombidiformes
Hydracarina gen.sp.	+	+	+	+	+	+
Phylum: Annelida Class: Clitellata (Oligochaeta)
Family: Lumbricidae
Eiseniella tetraedra (Savigny, 1826)			+			+
Family: Enchytraeidae	+
Family: Naididae
Peloscolex ferox (Eisen, 1879)			+		+
Family: Tubificidae
Linmodrilus sp.							+
Family: Lumbriculidae
Stilodrilus heringianus (Claparède, 1862)	+	+	+	+	+	+	+
Subclass: Hirudinae
Erpobdella octoculata (Linnaeus, 1758)							+
Piscicola geometra (Linnaeus, 1761)							+
Phylum: Mollusca Class: Bivalvia
Family: Pisidiidae							+

Table S3

The export of amphibious adult insects (mg (DW)·m^− 2·day^− 1), and production of EPA, n-3 PUFA, and n-6 PUFA (mg·m^− 2·day^− 1) by amphibious adult insects from the Krutaya Kacha River in 2022 and 2023
Month	Export	EPA	n-3 PUFA	n-6 PUFA
2022
May	0	0	0	0
May	4.305	0.024	0.069	0.095
May	8.469	0.048	0.135	0.187
May	0.795	0.006	0.009	0.010
May	1.310	0.010	0.014	0.016
June	8.382	0.106	0.227	0.061
June	5.194	0.062	0.099	0.076
June	4.667	0.057	0.104	0.049
June	4.685	0.036	0.062	0.056
June	2.386	0.018	0.026	0.028
July	3.790	0.025	0.056	0.033
July	4.071	0.029	0.063	0.037
July	3.041	0.025	0.051	0.029
July	2.948	0.031	0.060	0.028
July	5.194	0.047	0.101	0.068
July	6.551	0.092	0.192	0.051
July	4.562	0.064	0.124	0.048
August	8.071	0.096	0.190	0.074
August	5.025	0.065	0.134	0.042
August	4.909	-	-	-
August	4.239	0.038	0.083	0.055
August	4.492	0.033	0.073	0.070
August	1.628	0.008	0.019	0.019
August	0.526	0.002	0.004	0.006
August	1.397	-	-	-
August	1.918	0.020	0.040	0.018
September	5.580	0.087	0.182	0.040
September	3.884	0.031	0.074	0.027
September	15.113	0.069	0.153	0.051
September	8.198	-	-	-
September	2.779	0.025	0.061	0.020
September	2.315	0.014	0.034	0.018
September	0.559	0.010	0.021	0.005
September	0.338	0.003	0.004	0.004
2023
June	2.421	0.014	0.036	0.048
June	8.949	0.056	0.146	0.199
June	0.351	0.002	0.004	0.005
July	27.215	0.185	0.505	0.197
July	4.387	0.029	0.063	0.063

Table S4

The import of macroarthropods (mg (DW)·m^− 2·day^− 1), and production of EPA, n-3 PUFA, and n-6 PUFA (mg·m^− 2·day^− 1) by macroarthropods in the Krutaya Kacha River in 2022 and 2023
Month	Import	EPA	n-3 PUFA	n-6 PUFA
2022
May	30.876	0.157	0.423	0.659
May	9.217	0.047	0.126	0.197
May	32.258	0.164	0.441	0.689
May	66.135	0.336	0.905	1.412
May	95.440	0.485	1.306	2.037
June	18.433	0.023	0.237	0.278
June	87.250	0.111	1.122	1.317
June	171.190	0.217	2.202	2.585
June	74.194	0.094	0.954	1.120
June	36.206	0.046	0.466	0.547
June	88.940	0.113	1.144	1.343
July	41.868	0.150	0.604	0.910
July	82.642	0.296	1.192	1.796
July	50.384	0.180	0.727	1.095
July	30.932	0.111	0.446	0.672
July	27.266	0.098	0.393	0.593
July	39.993	0.143	0.577	0.869
July	40.553	0.145	0.585	0.881
July	54.102	0.194	0.780	1.176
August	36.098	0.032	0.146	0.931
August	25.346	0.023	0.103	0.654
August	46.770	0.042	0.190	1.207
August	41.782	0.037	0.169	1.078
August	25.907	0.023	0.105	0.668
August	33.180	0.030	0.135	0.856
August	21.270	0.019	0.086	0.549
August	21.198	0.019	0.086	0.547
September	61.956	0.246	0.730	0.949
September	33.410	0.133	0.394	0.512
September	17.127	0.068	0.202	0.262
September	36.150	0.144	0.426	0.554
September	7.450	0.030	0.088	0.114
September	16.882	0.067	0.199	0.259
September	33.333	0.132	0.393	0.510
2023
June	46.851	0.059	0.603	0.707
June	89.631	0.114	1.153	1.353
June	36.060	0.046	0.464	0.544
July	23.848	0.030	0.344	0.360
July	85.138	0.305	1.095	1.851

Table S5

The taxonomic composition of the imported invertebrates in the Krutaya Kacha River in 2022 and 2023
Data Taxonomic affiliation	2022					2023
Data Taxonomic affiliation	M	Jn	Jl	A	S	Jn	Jl
Phylum: Arthropoda Class: Insecta Order: Diptera
Family: Agromyzidae		+	+	+
Family: Anthomyiidae	+
Family: Blephariceridae*		+				+	+
Family: Ceratopogonidae*	+	+	+	+	+		+
Family: Chamaemyiidae			+	+	+
Family: Chironomidae*
Subfamily: Orthocladiinae	+	+		+	+	+	+
Subfamily: Chironominae		+	+	+	+	+
Rheotanytarsus photophilus (Goetghebuer, 1921)			+	+	+
Sphaeromias sp.		+
Subfamily: Tanypodinae Ablabesmyia monilis (Linnaeus, 1758)			+	+	+
Family: Chloropidae						+
Family: Coelopidae							+
Family: Culicidae *		+	+	+
Family: Cylindrotomidae		+	+	+	+	+	+
Family: Dolichopodidae*		+	+	+	+		+
Family: Empididae	+	+	+	+	+		+
Family: Empididae*					+		+
Family: Ephydridae*					+		+
Family: Lonchopteridae		+		+	+
Family: Macroceridae					+
Family: Muscidae		+	+	+	+	+	+
Family: Mycetophilidae	+	+		+	+	+
Family: Pediciidae*	+			+	+		+
Family: Phoridae	+	+	+	+		+
Family: Pipunculidae		+	+	+	+
Family: Psychodidae*			+	+			+
Family: Scathophagidae			+	+
Spaziphora hydromyzina (Fallén, 1819) *			+
Family: Scatopsidae	+				+		+
Family: Sciaridae		+	+	+	+	+	+
Family: Sciomyzidae	+
Family: Simuliidae*				+
Family: Syrphidae	+	+	+	+		+
Family: Tachinidae					+
Family: Tephritidae		+	+
Family: Tipulidae*						+
Family: Hybotidae				+
Order: Trichoptera*
Agapetus jakutorum (Martynov, 1934)				+			+
Apatania crymophila (McLachlan, 1880)	+
Ceratopsyche nevae (Kolenati, 1858)			+
Chaetopteryx villosa (Fabricius, 1798)					+
Lepidostoma hirtum (Fabricius, 1775)				+
Lype phaeopa (Stephens, 1836)			+				+
Micrasema gelidum (McLachlan, 1876)							+
Molannodes tinctus (Zetterstedt, 1840)			+
Rhyacophila sp.					+
Order: Ephemeroptera*
Baetis sp.		+		+
Caenis horaria (Linnaeus, 1758)		+
Ecdyonurus dispar (Curtis, 1834)	+	+
Epeorus pellucidus (Brodsky, 1930)				+
Ephemerella aurivillii (Bengtsson, 1909)		+	+
Ephemerella ignita (Poda, 1761)			+	+	+
Order: Plecoptera*
Alloperla sp.					+
Amphinemura borealis (Morton, 1894)							+
Capnia lepnevae (Zapekina-Dulkeit, 1957)						+
Capnopsis schilleri (Rostock, 1892)		+
Diura majuscula (Klapálek, 1912)						+
Leuctra fusca (Linnaeus, 1758)		+			+
Nemoura cinerea (Retzius, 1783)	+		+	+	+		+
Skwala pusilla (Klapálek, 1912)						+
Order: Hemiptera
Family: Anthocoridae		+		+	+
Family: Lygaeidae			+	+	+
Family: Miridae	+	+		+	+		+
Family: Nabidae Nabis sp.					+
Family: Saldidae	+
Family: Pentatomidae			+	+			+
Suborder: Heteroptera
Family: Gerridae**
Gerris lacustris (Linnaeus, 1758)	+	+
Limnoporus rufoscutellatus (Latreille, 1807)						+
Family: Corixidae** Hesperocorixa linnaei (Fieber, 1848)				+	+
Order: Homoptera
Family: Aphididae	+	+	+	+	+	+	+
Family: Aphrophoridae Aphrophora alni (Fallén, 1805)				+	+	+
Family: Delphacidae	+	+	+
Family: Jassidae		+	+	+	+	+	+
Family: Psyllidae	+	+	+		+	+	+
Order: Hymenoptera
Family: Braconidae		+	+	+
Family: Chalcidoidea	+			+	+
Family: Formicidae	+	+	+	+	+	+	+
Family: Ichneumonidae	+	+	+	+	+	+	+
Family: Proctotrupidae		+			+
Family: Sphecidae						+
Family: Tenthredinidae	+		+
Family: Xyelidae						+
Order: Coleoptera
Family: Brentidae Betulapion simile (W. Kirby, 1811)				+
Family: Carabidae		+	+
Family: Chrysomelidae	+			+	+
Family: Coccinellidae				+
Family: Cryptophagidae Atomaria sp.		+
Family: Curculionidae Isochnus flagellum (Erichson, 1902)		+			+
Family: Dermestidae	+	+
Dermestes depressus (Gebler, 1830)	+
Family: Hydraenidae**
Hydraena riparia (Kugelann, 1794)	+	+	+
Hydrobius fuscipes (Linnaeus, 1758)						+
Limnebius parvulus (Hydrophilus parvulus) (Herbst, 1797)						+
Family: Latridiidae		+
Family: Nitidulidae Epuraea sp.						+
Family: Rhynchitidae Deporaus betulae (Linnaeus, 1758)		+
Family: Scoletidae		+
Family: Staphylinidae	+	+	+		+	+	+
Order: Lepidоptera
Family: Crambidae		+	+				+
Family: Erebidae				+			+
Family: Geometridae			+	+
Family: Noctuidae			+	+
Family: Pieridae Aporia crataegi (Linnaeus, 1758)		+
Family: Drepanidae Drepana curvatula (Borkhausen, 1790)							+
Order: Neuroptera
Family: Hemerobiidae			+		+
Order: Psocoptera
Family: Psocidae		+	+		+		+
Order: Thysanoptera
Family: Phlaeothripidae	+	+
Family: Thripidae	+					+
Class: Arachnida Order: Araneae
Family: Araneidae			+	+		+	+
Family: Clubionidae	+	+					+
Family: Linyphiidae		+	+	+	+	+	+
Family: Lycosidae** Pirata piraticus (Clerck, 1757)		+
Family: Lycosidae			+			+	+
Family: Philodromidae
Family: Philodromidae Tibellus sp.				+
Family: Salticidae			+
Family: Tetragnathidae	+
Family: Thomisidae		+				+
Order: Opiliones
Family: Phalangida				+	+
Subclass: Acari
Superorder: Parasitiformes							+
Class: Entognatha
Subclass: Collembola	+	+	+	+	+	+	+
Subtype: Myriapoda
Order: Lithobiomorpha				+	+
* – amphibious insects
** – semi-aquatic arthropods

Table S6

The import of microarthropods (mg (DW)·m^− 2·day^− 1), and production of EPA, n-3 PUFA, and n-6 PUFA (mg·m^− 2·day^− 1) by microarthropods in the Krutaya Kacha River in 2022
Month	Import	EPA	n-3 PUFA	n-6 PUFA
July	12.336	0.007	0.020	0.036
July	14.956	0.008	0.024	0.044
July	10.511	0.006	0.017	0.031
August	6.411	0.004	0.010	0.019
August	9.389	0.005	0.015	0.028
August	18.603	0.010	0.030	0.055
September	12.530	0.007	0.020	0.037
September	10.458	0.006	0.017	0.031
September	10.892	0.006	0.018	0.032
September	3.659	0.002	0.006	0.011

The production of EPA by macrozoobenthos varied from 0.039 ± 0.017 mg·m^− 2·day^− 1 in June to 0.214 ± 0.028 mg·m^− 2·day^− 1 in August and averaged 0.109 ± 0.023 mg·m^− 2·day^− 1 (Table 1, Table S1). The production of n-3 PUFA by macrozoobenthos ranged from 0.087 ± 0.032 mg·m^− 2·day^− 1 in June to 0.509 ± 0.075 mg·m^− 2·day^− 1 in August and averaged 0.246 ± 0.052 mg·m^− 2·day^− 1. The production of n-6 PUFA by macrozoobenthos was lower by almost a factor of 2 than n-3 PUFA production and ranged from 0.046 ± 0.013 mg·m^− 2·day^− 1 in June to 0.298 ± 0.063 mg·m^− 2·day^− 1 in August, averaging 0.141 ± 0.029 mg·m^− 2·day^− 1. The values of the production of EPA, n-3 and n-6 PUFA in 2023 (June and July) were within the range of 2022 values. (Table S1).

Export of biomass and PUFA with emergence of amphibious insects

The study began before the insects started to emerge, when the first measurement of emergence was zero, and ended when emergence dropped to a value of 0.34 mg (DW)·m^− 2·day^− 1. During the field season, the export of biomass varied slightly: from 2.98 ± 1.55 mg (DW)·m^− 2·day^− 1 in May to 5.06 ± 0.96 mg (DW)·m^− 2·day^− 1 in June, and on average amounted to 4.28 ± 0.52 mg (DW)·m^− 2·day^− 1 (Fig. 3, Table 1, Table S3). The total production for the entire season (year) was 0.6 g (DW)·m^− 2·yr^− 1. The maximum value in 2022 was 15.1 mg (DW)·m^− 2·day^− 1. In 2023, all measurements except one were within the range of the 2022 values. One sample was found to contain a very large caddisfly, Hydatophylax variabilis, which increased the values to 27.2 mg (DW)·m^− 2·day^− 1 (Fig. 3). The emergence of taxa was uneven (Fig. 3). Plecoptera had two peaks of emergence: May and August. Moreover, different species emerged in different months: Megarcys pseudochracea, Paraleuctra zapekinae, and Nemoura cinerea in May; Leuctra fusca in August. Trichoptera emerged from June to September with peaks in July and September. In July, Agapetus jakutorum dominated, and in September, Chaetopteryx villosa dominated. Ephemeroptera emerged from June to September. Small species of the genus Baetis (B. bicaudatus and B. vernus) dominated throughout the Ephemeroptera emergence period. In addition, Ecdyonurus abracadabrus and Centroptilum luteolum dominated in July and August, and Epeorus pellucidus dominated in June and August. Diptera emerged throughout the season, peaking in June (Fig. 3). This taxon was represented by the greatest number of species, which were mainly representatives of the Orthocladiinae and Chironominae subfamilies. Diptera dominated in density in each month, and only in September their numbers decreased and Ephemeroptera became the subdominant taxon (Fig. 3). In 2023, spring was colder (ice breakup in rivers occurred later) than in the previous year, and the emergence began later but was faster. As a result, the data for June 2023 were more consistent with May 2022 (Fig. 3). The export of biomass did not vary significantly by month (Kruskal-Wallis test H = 3.043502, p = 0.5506).

The export of EPA with amphibious insects varied from 0.022 ± 0.009 mg·m^− 2·day^− 1 in May to 0.056 ± 0.015 mg·m^− 2·day^− 1 in June and averaged 0.039 ± 0.005 mg·m^− 2·day^− 1 (Table 1, Table S3). The export of n-3 PUFA varied from 0.056 ± 0.029 mg·m^− 2·day^− 1 in May to 0.104 ± 0.034 mg·m^− 2·day^− 1 in June and averaged 0.082 ± 0.011 mg·m^− 2·day^− 1. The export of n-6 PUFA was lower by a factor of two compared to the export of n-3 PUFA and varied from 0.024 ± 0.007 mg·m^− 2·day^− 1 in September to 0.077 ± 0.042 mg·m^− 2·day^− 1 in June, averaging 0.044 ± 0.007 mg·m^− 2·day^− 1. The highest values were recorded in July 2023 during the emergence of H. variabilis: 0.184, 0.505 and 0.197 mg·m^− 2·day^− 1 of EPA, n-3 PUFA, and n-6 PUFA, respectively.

The export of EPA, n-3 and n-6 PUFA with amphibious insects did not vary significantly by month (EPA Kruskal-Wallis test H = 5.707604, p = 0.2221; n-3 PUFA Kruskal-Wallis test H = 3.053917, p = 0.5488; n-6 PUFA Kruskal-Wallis test H = 5.485023, p = 0.2411) (Fig. 4).

Figure 4 The import (brown bars) and the export (blue bars) of EPA, n-3 and n-6 PUFA (mg·m^− 2·day^− 1) by month during the 2022 field season in the Krutaya Kacha River. Means labelled with the different letters are significantly different at P < 0.05 according to the non-parametric Kruskal–Wallis H-test

Import of biomass and PUFA with terrestrial and aquatic invertebrates

Throughout the season, the import of macroarthropods varied from 29.5 ± 6.8 mg (DW)·m^− 2·day^− 1 in September to 79.4 ± 21.7 mg (DW)·m^− 2·day^− 1 in June and averaged 45.2 ± 5.5 mg (DW)·m^− 2·day^− 1 (Fig. 5, Table 1, Table S4). The total production for the entire season (year) was 6.8 g (DW)·m^− 2·yr^− 1. The highest value of the import was recorded in mid-June: 171.2 mg (DW)·m^− 2·day^− 1. The production values measured in June and July 2023 were within the 2022 range (Fig. 5). The imported macroarthropods were mainly terrestrial invertebrates, the proportion of biomass of which varied from 67% in September to 94% in May and averaged 82% (Fig. 5). Amphibious insects accounted for 18% of the imported macroarthropods, corresponding to 8.2 ± 1.4 mg (DW)·m^− 2·day^− 1. The complete taxonomic composition is shown in Table S5. Among terrestrial invertebrates, Homoptera, Diptera, Hymenoptera, and Araneae dominated in density (Fig. 6). The import of biomass did not vary significantly by month (Kruskal-Wallis test H = 7.830756, p = 0.098) but tended to decrease towards autumn (Fig. 4).

The import of EPA with macroarthropods varied from 0.028 ± 0.003 mg·m^− 2·day^− 1 in August to 0.238 ± 0.077 mg·m^− 2·day^− 1 in May and averaged 0.122 ± 0.018 mg·m^− 2·day^− 1 (Table 1, Table S4). The import of n-3 PUFA varied from 0.128 ± 0.014 mg·m^− 2·day^− 1 in August to 1.021 ± 0.280 mg·m^− 2·day^− 1 in June and averaged 0.532 ± 0.08 mg·m^− 2·day^− 1. The import of n-6 PUFA varied from 0.451 ± 0.104 mg·m^− 2·day^− 1 in September to 1.198 ± 0.328 mg·m^− 2·day^− 1 in June and averaged 0.877 ± 0.091 mg·m^− 2·day^− 1, which was one and a half times higher than the import of n-3 PUFA. The imports of EPA, n-3 and n-6 PUFA measured in June and July 2023 were within the 2022 range (Table S4).

The imports of EPA and n-3 PUFA were the lowest in August but the import of n-6 PUFA was the lowest in September (Fig. 4). The import of EPA was significantly lower in August compared to May and July (Kruskal-Wallis test H = 19.43736, p = 0.0006), and the import of n-3 PUFA was significantly lower in August compared to June and July (Kruskal-Wallis test H = 19.97685, p = 0.0005) (Fig. 4A, B).

The import of microarthropods was on average lower by a factor of 4 than that of macroarthropods and reached a maximum value of 18.6 mg (DW)·m^− 2·day^− 1 in August. The microarthropods were dominated by coastal and surface dwellers, namely Collembola (44.5%), Diptera (23%), Homoptera and Psocoptera (16%). The import of EPA, n-3 and n-6 PUFA with microarthropods was lower by a factor of 20–30 compared with macroarthropods (Table 1, Table S6).

The production of macroarthropods was significantly higher than that of all other studied groups (Table 1). The import of biomass into the river was more than 10 times higher than the export from it. The values of production of EPA by zoobenthos and by imported macroarthropods did not differ significantly from each other, but was significantly higher than the production of EPA exported from the river with the emergence of amphibious insects. The production of EPA by imported microarthropods was the lowest and differed significantly from all groups. The values of production of n-3 PUFA by zoobenthos and imported macroarthropods did not differ significantly from each other, but were significantly higher than the production of n-3 PUFA by the other groups. The production of n-6 PUFA by imported macroarthropods was the highest and differed significantly from all groups (Table 1).

Figure 6 Percentages of taxonomic groups of terrestrial invertebrates by density in the import flux into the Krutaya Kacha River

The content (mg·g ^− 1 (DW)) of EPA, n-3 and n-6 PUFA in zoobenthos and exported and imported invertebrates

The contents of EPA and n-3 PUFA in zoobenthos and exported invertebrates did not differ significantly from each other and were significantly higher than in imported invertebrates (Table 1). The content of EPA in zoobenthos varied within the range 6.0–12.3 mg·g^− 1 (DW), in exported insects 5.9–16.6 mg·g^− 1 (DW), in imported macroarthropods 0–7.6 mg·g^− 1 (DW), and in imported microarthropods 0.2–2.7 mg·g^− 1 (DW) (Table S1, S3, S4, S6). The content of n-3 PUFA in zoobenthos varied within the range 14.6–25.8 mg·g^− 1 (DW), in exported insects 11.0–34.1 mg·g^− 1 (DW), in imported macroarthropods 0.8–23.6 mg·g^− 1 (DW), and in imported microarthropods 1.6–4.1 mg·g^− 1 (DW) (Table S1, S3, S4, S6).

The content of n-6 PUFA in imported macroarthropods was highest, and it significantly differed from that in exported insects and imported microarthropods but did not differ from that in zoobenthos (Table 1). The lowest values were found in imported microarthropods. The content of n-6 PUFA varied in imported macroarthropods within the range 5.6–48.2 mg·g^− 1 (DW), in zoobenthos 9.5–13.9 mg·g^− 1 (DW), in exported insects 7.5–21.0 mg·g^− 1 (DW), and in imported microarthropods 2.2–13.2 mg·g^− 1 (DW) (Table S1, S3, S4, S6).

In the small shaded river (typical for the boreal forest in the North Temperate Zone), whose surface received PAR levels lower by a factor of 4 than open terrestrial areas, the subsidies of terrestrial invertebrates significantly (≈ 4 times) exceeded the production of benthic invertebrates in the river. Obviously, these subsidies could make a considerable contribution to fish diet. Indeed, our previous study showed that the proportion of terrestrial invertebrates in the diet of grayling from the Krutaya Kacha River was about 40%, which was significantly higher compared to the diet of grayling from large unshaded rivers (Makhutova et al. 2024; Mashonskaya et al. (in press)). The large contribution of terrestrial invertebrates to the diet of fish living under conditions of low primary production was shown in other studies (Milardi et al. 2016; Carassou et al. 2017; Correa and Winemiller 2018). The contribution of microarthropods (< 1 mm) was similar to the zoobenthos production, but in terms of FA it was negligible, since the values of production of EPA and n-3 PUFA were lower by factors of 18 and 14, respectively. The values of biomass and FA import obtained in the present study were within the range of values obtained in other studies (Erős et al. 2012; Moyo et al. 2017). In particular, in previous works, the range of biomass subsidy was established as 8.5–679 mg (DW)·m^− 2·day^− 1, and in the present work we obtained 7.5–171.2 mg (DW)·m^− 2·day^− 1. The total import of n-3 and n-6 PUFA found in our study, 0.2–4.8 mg·m^− 2·day^− 1, was also similar to that found previously by other authors, 0.3–3 mg·m^− 2·day^− 1 (Erős et al. 2012; Moyo et al. 2017).

The amount of FA import from terrestrial ecosystems exceeded the FA amount produced in the river (2.2 times for n-3 PUFA and 6.4 times for n-6 PUFA). However, in terms of biochemical quality, allochthonous and autochthonous invertebrates differed greatly. The main feature of the imported biomass was a much higher n-6/n-3 PUFA ratio, with the PUFA composition being dominated by shorter-chain PUFAs, mostly containing 18 carbon atoms. Parmar et al. (2022) also showed that in terrestrial invertebrates the PUFA content is lower by a factor of almost 2 than in aquatic and semi-aquatic ones and can be 16.8 ± 11.8 mg·g^− 1 (DW), and EPA is only 1.1 ± 1.5 mg·g^− 1 (DW), which is lower by a factor of 10 than in aquatic invertebrates and by a factor of 5 than in semi-aquatic ones. Additionally, the imported biomass was biochemically heterogeneous, because it did not differ by month, but imported EPA and n-3 PUFA varied considerably and had the lowest values in August. It is obvious that fish consuming the same volume of food will receive less n-3 PUFA, specifically, smaller amounts of physiologically valuable EPA, from terrestrial invertebrates than from zoobenthos. Moreover, another n-3 PUFA that is physiologically valuable for fish, DHA (Tocher 2010; Makhutova and Gladyshev 2020; Závorka et al. 2023), was completely absent from the imported biomass, while gammarids (Gammarus lacustris), which are able to contain considerable amounts of this acid, were found in zoobenthos (Makhutova et al. 2016, 2018). A high proportion of terrestrial invertebrates in the diet of fish can lead to a decrease in the nutritional value of fish for consumers, which is especially important in the case of commercial fish. Indeed, in grayling from the Krutaya Kacha River and other shaded rivers studied, the contents of EPA and DHA in muscle tissue were significantly lower than in grayling whose diets were based on zoobenthos containing both n-3 LC-PUFA (Makhutova et al. 2024; Mashonskaya et al. (in press)). Scharnweber et al. (2024) also noted a deterioration in the physiological and biochemical characteristics of fish fed a diet based on terrestrial invertebrates.

The taxonomic composition of terrestrial invertebrates was dominated by the same groups as those found in other studies. For example, in the study by Erős et al. (2012), the dominant taxa were Diptera, Araneida, Coleoptera, and Hymenoptera, which accounted for 73.3–80.6% of the total invertebrate biomass. In our present study, the composition of dominants was supplemented by representatives of Homoptera.

The taxonomic composition of emerging insects was typical for streams (Ohler et al. 2024). The export of amphibious insects from the Krutaya Kacha River was rather low. The annual production value (0.6 g (DW)·m^− 2·yr^− 1) was close to the minimum known for streams, which is 0.4 g (DW)·m^− 2·yr^− 1 (Gladyshev et al. 2009). The annual export of PUFA (18.6 mg (DW)·m^− 2·yr^− 1) was comparable with the values reported in previous studies and even exceeded that obtained for some rivers located in forested sites (Gladyshev et al. 2009; Kowarik et al. 2023; Ohler et al. 2024). The export of PUFAs with emerging amphibious insects is influenced by many factors, such as the percentage of shading and pool habitats, agricultural and other land-use of adjacent territories leading to the release of pesticides, metals, and other toxic substances into aquatic ecosystems (Pietz et al. 2023; Ohler et al. 2024). It has been shown that small forested shaded rivers can export more PUFA (but not EPA) than rivers in open agricultural areas (Ohler et al. 2024), which may be related to the taxonomic composition of both producers and invertebrates.

Emerging insects supply consumers of terrestrial ecosystems with n-3 PUFA, including EPA (Borisova et al. 2016; Martin-Creuzburg et al. 2017; Popova et al. 2017; Gladyshev et al. 2019; Moyo et al. 2022). Some invertebrates and vertebrates depend on this emergence. For example, the abundance of spiders in the coastal zone is closely related to the emergence of aquatic insects. Thus, within the active bed of a stream, their density and biomass were highest, decreasing, though, by a factor of more than three at a distance of 25 m from the edge of the stream (Sanzone et al. 2003). The extent to which coastal spiders utilize aquatic PUFA may vary seasonally (Kowarik et al. 2021). The spiders Tigrosa georgicola from wetlands have higher levels of PUFA than spiders from mountainous areas (Fritz et al. 2017). Ohler et al. (2023) found the influence of PUFA profiles of the basic food sources on the weight and body condition of spiders (Tetragnatha sp.). Twining et al. (2016) demonstrated that for swallow (Tachycineta bicolor) chick performance, fatty acid composition was more important than food quantity. High LC-PUFA diets caused an increase in the growth rate and improvement in the body condition of the chicks compared with chicks on low LC-PUFA diets.

According to our measurements, one third of the zoobenthos production left the river when amphibious insects emerged. However, the emergent adult insects returned back to the river. The abundance of amphibious insects returning to the river obtained in the current study is substantially greater than the values reported in other studies (Gray 1989; Ballinger and Lake 2006; Dreyer et al. 2015). The technique of adding surfactants to traps may have led to an overestimate of the import of allochthonous invertebrates and amphibious adult insects returning to lay eggs, since once an invertebrate touched the water in the trap, it could not escape. In natural conditions, invertebrates can reach the riverbank, a rock or a snag and leave the water surface. On the other hand, without surfactants, predators (spiders, water striders) will not drown in the traps and will be able to feed on the trapped invertebrates, reducing their import. Surfactants were also used in other studies (Erős et al. 2012; Jonsson et al. 2013; Moyo et al. 2017; Moyo et al. 2022). Gray (1989) and Dreyer et al. (2015) used other methods (collecting insects in downstream nets and in traps suspended at 1 m, respectively), which also had shortcomings. Obviously, a method needs to be developed that will overcome the shortcomings of existing methods, and large amounts of field data need to be accumulated to assess invertebrate fluxes between aquatic and terrestrial ecosystems.

In our study, calculations of the production of all invertebrate groups, namely zoobenthos and imported and exported arthropods, allowed us to determine the ratios of these fluxes and showed the importance of imported invertebrates for the food webs of small shaded rivers of boreal forests. A large excess of terrestrial invertebrate flux substantially alters the diet of benthivorous fish, leading to a change in the biochemical composition of their muscle tissue. In our previous study, we found that the n-3/n-6 ratio in grayling from the Krutaya Kacha River was significantly lower than in grayling from the Yenisei River (large unshaded river), whose diet consisted exclusively of autochthonous invertebrates (n-3 and n-6 were 3.6 and 9.3, respectively) (Mashonskaya et al. (in press)). Quantitative data on food resource fluxes in rivers are needed to improve our understanding of the River Continuum Concept, which is still in its development stage (Sánchez-Hernández 2023).

CRediT authorship contribution statement

ONM: Conceptualization, Supervision, Data curation, Methodology, Project administration, Investigation, Formal analysis, Visualization, Software, Resources, Writing – original draft. YOM: Investigation, Formal analysis, Writing – original draft. EVB: Conceptualization, Methodology, Investigation, Formal analysis, Writing – review & editing. NIK: Investigation, Formal analysis. SPS: Investigation, Methodology, Formal analysis.

Ethical approval

Not applicable

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Author Contribution

ONM carried out study onceptualization, supervision, data curation, methodology, project administration, investigation, formal analysis, visualization, software, resources, and writing – original draft. YOM carried out study investigation, formal analysis, and writing – original draft. EVB carried out study conceptualization, methodology, investigation, formal analysis, writing – review and editing. NIK carried out study investigation and formal analysis. SPS carried out study investigation, methodology, and formal analysis.

Acknowledgements

The authors would like to thank Mikhail I. Gladyshev for participating in the preliminary discussion of the study, Alexander P. Tolomeev for providing traps for collecting emerging amphibious insects, Pavel P. Kormilets for creating illustrations, and Elena L. Krasova for linguistic check and improvements.

Data availability

The data in this study are available on request.

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No competing interests reported.

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Quantitative assessment of omega-3 and omega-6 polyunsaturated fatty acid fluxes between aquatic and terrestrial ecosystems of a small shaded river

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