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. 2023 Apr 11;106(2):00368504231166956. doi: 10.1177/00368504231166956

Circadian (re)colonisation dynamics of macroinvertebrates in an isolated karst spring

Denis Bućan 1, Marko Miliša 2,✉
PMCID: PMC10358621  PMID: 37042031

Abstract

Freshwater ecosystems, especially springs, are highly sensitive to environmental changes. They are also excellent natural laboratories because of their stable conditions, reducing the number of variables to be considered in field studies. We examined the composition, dynamics and colonisation patterns of macroinvertebrates with respect to canopy coverage and time of day in which available areas are actively colonised. We used artificial substrates that mimicked the natural habitat structure at an isolated karst spring and recovered exposed substrates every 12 h. Physico-chemical parameters of water did not differ significantly regardless of canopy cover. The most numerous representatives and the pioneering champions were larvae of Baetidae (Ephemeroptera) and Chironomidae (Diptera). Simuliidae were also among the most successful pioneering species. Most observed groups more actively colonised substrates in the closed canopy area. Oligochaeta and Gammarus fossarum were more numerous on substrates in the open canopy area. Individuals of all analysed groups showed day-night migration patterns and were more active at night. Coleoptera (Elmis sp.) were the poorest (re)colonisers among the analysed taxa.

Keywords: Benthos, moss mats, artificial substrates, pioneer colonisers, Croatia

Introduction

Karst springs are a unique habitat, as complex ecosystems that do not conform to the physico-chemical, biological and hydromorphological succession of downstream sections.1,2 Carbonate rocks are the base that forms karst ecosystems, which are comprised of morphological, hydrological and hydrogeological terrain features built of water-soluble sedimentary rocks. 3 Karst is characterised by a lack of surface water, and numerous cracks formed by tectonic or water dissolution through which most water seeps underground. Any significant changes can seriously disrupt its biology. The organisms dwelling in such landscapes are usually stenovalent and not adapted to environmental changes.4–6 Springs, therefore, are very good natural laboratories because of the stability of abiotic conditions within, especially cold water rheocrenes, where water surfaces create a stable lotic habitat. 7 Experimenting in such a habitat reduces the number of variables to be considered, and the relatively low species diversity and abundance enable more focused research.2,8 Since springs are isolated habitats (rarely are two or more springs near enough for organisms to migrate between them), endemism is common.9,10

Among the freshwater organisms, macroinvertebrates are the most common focus in bioassessment given their vulnerability to changes in environmental conditions, and since they are relatively long-lived, certain environmental changes drive the composition and structure of their assemblages. 11 Also, the stationary lifestyle, limited mobility 8 and inability to quickly translocate to another stream in case of pollution make macroinvertebrates important bioindicators in freshwaters.12–14 Flow velocity, substrate composition, food supply and the physico-chemical properties are among the most important factors affecting macroinvertebrate communities.15,16

Natural physical disturbances are an essential factor in the stability of lotic communities as they can lead to migrations of organisms that then colonise new habitat. Colonisation movements can be multidirectional, for example, upstream and downstream, vertical (movement into (and through) interstices or lateral (from faster to slower flows).17–19 Vertical movements are an important ethological mechanism to avoid predatory pressure and other unfavourable conditions. In this way, deeper substrate layers become refuges and a source for recolonisation. 20 The deeper layers can shelter younger individuals that would otherwise be able to tolerate the predatory and competition pressure of surface layers. Further, larger individuals usually cannot occupy small interstitial spaces.16,20,21 Transport and drift of organic matter affect the dynamics of benthic communities. 22 Horizontal movement (drift) in streams is mostly downstream, with the passive transport of organisms, 17 usually under the influence of flow force. Drift also includes active movements of macroinvertebrates, enabling organisms to escape an unsuitable habitat and be transported to one with more beneficial conditions, lesser predatory pressure and more stable environmental conditions. 23 Organisms seeking out better conditions in their habitat can move upstream and sideways. The important pathway of colonisation for certain groups, mainly poor swimming larve (e.g. Simuliidae) is by drift. 24 In the case of aquatic insects, the main colonisation mechanisms depend on the presence of winged adult stages. Winged images play an important role in the colonisation of newly beaver-created ponds 25 or regulated river sections. 26 In the case of mussels, the transport of glochids by fish also plays an important role in the colonisation of new habitats. 27

Many studies have shown that macroinvertebrates recover quickly (in less than a month) in areas affected by extreme natural and anthropogenic disturbances.20,24,28 Colonisation depends on the mobility of individuals, type of substrate, amount of food, life cycle, season, competition and predatory pressures. 17 These factors, in addition to pollutants, can result in significant disturbances or the disappearance of biocenosis structures, leading to recolonisation. 16 In affected habitats, macroinvertebrates that move faster (e.g. Ephemeroptera compared to Gastropoda) are able to more quickly colonise disturbed empty areas. Recolonisation will also develop much faster if unfavourable conditions occur after the macroinvertebrates have finished breeding. The larger the disturbed area, the longer the recolonisation period will be. During recolonisation, macroinvertebrates will more quickly colonise areas where there already is a certain community of microorganisms that create biofilm (bacteria, protozoa and algae).29,30

Artificial substrates are often used in research on the effects of stream regulation on invertebrates, 31 and in the research of migratory and colonisation processes.32–34 They simulate the desired natural substrate (e.g. moss mats), and placing a larger number of artificial substrates in similar habitats will minimise subjectivity, unlike in sampling natural habitats. 35 These substrates are small, light and inexpensive, with a known surface area. 36 Furthermore, sampling with artificial substrates can certainly be considered an advantage in reducing habitat destruction. 37

The aim of this study was to determine: (i) which macroinvertebrates are champion pioneers in a karst spring of the Jankovac stream; (ii) macroinvertebrate propensity towards colonising open versus closed canopy areas; (iii) migration potential of macroinvertebrates regarding time of day; and (iv) macroinvertebrate composition of the spring area. We hypothesised that macroinvertebrate colonisation is more pronounced on artificial substrates in open canopy areas of the stream, with more individuals during the day. Depending on their characteristics, some macroinvertebrates prefer open while others prefer closed canopy areas, though in general, greater abundances will be found in closed canopy areas due to the higher number of available habitats and refugia and therefore resources. Since colonisers are mostly larvae with poor eyesight, they come to the substrates to feed during the night and hide from predators during the day. We expected that actively moving taxa (e.g. Ephemeroptera, Amhipoda) would be the pioneering colonisers, while less mobile taxa would require more time to colonise the substrates. This study will improve riparian vegetation management and improve our knowledge of the human impacts that can alter canopy coverage of spring areas, especially due to the high biodiversity found in these small areas.

Materials and methods

The experiment on the macroinvertebrate community was conducted in the spring area of the Jankovac stream, located in Papuk Nature Park, in continental Croatia (45°31′05″N, 17°41′14″E). The study area (length 60 m, mean width 3 m, slope 3.8°) is near pristine, meaning that the anthropogenic effect is reduced to a minimum. The stream bed is covered with cobbles, pebbles, dense clusters of bryophyte cover and present tufa deposition. The moss genera Cratoneuron and Eurhynchium are present in the stream, 38 with beech (Fagus sylvatica L.) and hornbeam (Carpinus betulus L.) comprising the riparian vegetation. In the upper spring area of the stream, the closed canopy is composed of deciduous forest, while the lower area is the open canopy. Jankovac spring is located in an isolated karst area, while the surrounding area is made of metamorphic and igneous rocks at an elevation of about 520 m.

Using electronic probes, the standard physico-chemical water parameters were measured: electrical conductivity, pH, temperature, concentration of dissolved oxygen and oxygen saturation (Hach sensION, WTW pH 330i, WTW Oxi 95, respectively). Parameters were measured five times (once per day in the afternoon) during each sampling event.

We placed 112 plastic artificial substrates in the stream on the first day of the experiment and collected them after 12-h periods over the course of four consecutive days in June. Artificial substrates structurally imitated moss mats, the dominant biological microhabitat in the spring area. Substrates were plastic wire scourers, weaved in the shape of tubes, with dimensions: 40 × 6 cm (diameter), which after wrapping (rolling) made a test substrate with dimensions: 8 × 2 cm. The thread diameter of the substrate was 1.2 mm, and mesh size 1.5 × 3 mm. The mean total volume of the substrate was 21.9 cm3 ± 3%. Substrates were soaked in distilled water for 7 days and rinsed before the start of the experiment.

Experimental habitats differed in canopy: half of the substrates were placed in closed canopy areas of the spring, nearly completely shaded with riparian vegetation, while the second half were in open canopy areas in a random manner. In addition to these properties, day-night migrations of macroinvertebrates were monitored during the experiment, where morning samples were considered night migrations and colonisation, while evening samples were considered day migrations and colonisation.

Samples were collected in a container with 70% ethanol in the morning (at 8 AM) and evening (at 8 PM) over four consecutive days, with a total of seven samplings with replicates for each combination. Substrates were rapidly extracted from the stream to reduce the potential losses of individuals from the substrates. We also took four replicate reference samples from the surrounding natural substrate, in both the open and closed canopy areas to determine the natural community structure. Natural samples were collected with a Surber net (20 × 20 cm aperture and mesh size 500 μm).

From the collected samples, we separated all organisms and identified individuals using a stereo microscope (Zeiss Stemi 2000) and available keys for the determination of species for certain orders of insects: Ephemeroptera, 39 Plecoptera, 40 Coleoptera 41 and Trichoptera. 42 We calculated the mean number of individuals colonising the artificial substrates from the number of individuals collected from the substrates, that is, replicates. We used the mean number of individuals to compare colonisation dynamics regarding canopy and the time of day of certain specimens.

The Kruskal–Wallis test analysis of variance was used to determine whether there were statistically significant differences in the physico-chemical water parameters between the open and closed canopy sections of the stream. To analyse the differences among abundances of a combination of environmental variables (canopy × time of day), we further used Multiple comparisons of means analysis (post hoc comparison after Kruskal–Wallis tests43,44). All statistical analyses were performed in Statistica 12.0 (Dell Inc. 2015). The figures were prepared in Grapher 16.2 (Golden Software, LLC) and tables in Microsoft Excel 2016 (Microsoft Corporation, 2016).

Results

Physico-chemical parameters

Water temperature in the study area fluctuated negligibly during the study period. Water temperatures in the open canopy area were higher than in the closed canopy area (Table 1). Oxygen saturation of water was also higher in the open canopy area. The difference between the mean oxygen saturation values was highest on the final measurement day. pH values were higher in the open canopy area during all measurements (Table 1). Mean values of concentrated dissolved oxygen and electrical conductivity did not significantly differ (Mann–Whitney U test) between the open and closed canopy areas (Table 1).

Table 1.

Characteristics of the sampling sites in the spring area of Jankovac stream.

Site OC CC
Water temperature (°C) Mean 9.80 9.67
SD 0.16 0.20
O2 (mg dm−3) Mean 10.61 10.52
SD 0.14 0.12
O2 (%) Mean 99.27 98.30
SD 1.71 1.17
pH Mean 8.06 7.95
SD 0.09 0.07
Conductivity (μS cm−1) Mean 506.25 506.75
SD 7.54 5.96
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OC: open canopy area; CC, closed canopy area.

Macroinvertebrates

Community composition

Macroinvertebrate community, in open and closed canopy area, consisted mostly of Turbellaria, Gastropoda, Oligochaeta, Hydrachnidia, Amphipoda and Insect larvae (Ephemeroptera, Plecoptera, Trichoptera, Diptera (mostly Chironomidae and Simuliidae)) and larvae and adults of Coleoptera. Also, one representative of fish and amphibians each was found in the substrates. The most numerous were insect larvae and Amphipoda (Gammarus fossarum) (Table 2).

Table 2.

Macroinvertebrates found on artificial substrates in the Jankovac stream.

Taxa N %
Turbellaria Crenobia alpina 53 0.99%
Nematoda Nematoda 11 0.21%
Gastropoda Graziana papukensis 7 0.13%
Oligochaeta Oligochaeta 135 2.53%
Arachnida Hydrachnidia 37 0.69%
Amphipoda Gammarus fossarum 416 7.81%
Collembola Collembola 40 0.75%
Ephemeroptera Acentrella sinaica 21 0.39%
Baetopus tenellus 5 0.09%
Electrogena sp. 3 0.06%
Baetidae juv. non det. 1414 26.54%
Ephemeroptera (total) 1443 27.08%
Odonata Odonata 4 0.08%
Plecoptera Isoperla sp. 5 0.09%
Leuctra sp. 10 0.19%
Protonemura sp. 61 1.14%
Plecoptera juv. non det. 3 0.06%
Plecoptera (total) 79 1.48%
Hemiptera Hemiptera 5 0.09%
Coleoptera Elmis sp. 58 1.09%
Elmis sp. (imago) 11 0.21%
Limnius sp. 4 0.08%
Limnius sp. (imago) 1 0.02%
Dytiscidae (imago) 3 0.06%
Ochthebius sp. (imago) 1 0.02%
Coleoptera juv. non det. 12 0.23%
Coleoptera (total) 90 1.69%
Trichoptera Chaetopteryx sp. 6 0.11%
Drusus muelleri 1 0.02%
Glossosoma conformis 2 0.04%
Micropterna sequax 14 0.26%
Stenophylax permistus 4 0.08%
Synagapetus dubitans 1 0.02%
Rhyacophila laevis 4 0.08%
Trichoptera (imago) 2 0.04%
Trichoptera juv. non det. 14 0.26%
Trichoptera (total) 48 0.90%
Diptera Chironomidae 2628 49.32%
Simuliidae 322 6.04%
Diptera (imago) 2 0.04%
Diptera juv. non det. 4 0.08%
Pupa non det. 2 0.04%
Diptera (total) 2958 55.52%
Pisces Pisces 1 0.02%
Amphibia Amphibia 1 0.02%
Total 5328 100.00%
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Protonemura was the dominant genus representative of Plecoptera, though specimens of the genera Isoperla and Leuctra were also present. Juvenile larval stages of the family Baetidae were the most abundant Ephemeroptera, though their age prevented confident identification to the genus level. Older larval stages identified were representatives of the taxa Electrogena sp., Acentrella sinaica and Baetopus tenellus. The most abundant representative of Coleoptera was Elmis sp. We also found representatives of the taxa Esolus sp., Limnius sp., Riolus sp. and Ochtebius sp., mostly larvae. Within the order Trichoptera, only the species Micropterna sequax stood out numerically; however, its abundance and all trichopterans in total were quite low. In the study area, the only representative of the class Turbellaria was the species Crenobia alpina (Table 2).

Colonisation dynamics

We further analysed the colonisation patterns of the most numerous groups, Oligochaeta (2.53%), Amphipoda (Gammarus fossarum) (7.81%) and insect larvae: juvenile Ephemeroptera, Baetidae (27.02%), and two Diptera family Chironomidae (49.32%) and Simuliidae (6.04%) (Table 2).

By comparing the total number of taxa on substrates in the open versus closed canopy areas of the stream, most groups were found to be better at colonising substrates in the closed canopy area (Figure 1). The most prominent phenomenon was noted with the representatives of Baetidae and Simuliidae, which were 3–4 times more numerous on substrates in the closed canopy than in open canopy areas. However, the number of Oligochaeta and Gammarus fossarum was greater on substrates in the open canopy area than in closed canopy areas of the stream. Chironomidae individuals, which were the most numerous, equally colonised substrates in closed and open canopy areas of the stream (Figure 1).

Figure 1.

Figure 1.

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The mean number of individuals found on artificial substrates in the open canopy (left) and closed canopy (right) area of the stream Jankovac. Time of day: ☼ - day, - night.

We compared the mean values of individual taxa over time in the open and closed canopy areas. We noted an increase and decrease in the numbers of all analysed groups every 12 h. Substrates collected during the morning hours (night activity) had a higher number of individuals than substrates collected in the evening (day activity).

There was a higher number of Baetidae larvae colonising the artificial substrate in open and closed canopy areas during the night. Abundances increased with increased exposure to the substrates, with a peak abundance after three days. The Chironomidae abundance showed regular day-night migration activity during all four days of the experiment, in both open and closed canopy areas of the stream. Colonisation of Gammarus fossarum in the open canopy area was higher during the night. Despite the lower number of Simuliidae and Oligochaeta, both daily and nightly movements were present (Figure 1).

We compared the mean number of analysed taxa found on natural and artificial substrates and almost all taxa achieved equilibrium, that is, natural levels of abundances after only 12 h in both open and closed canopy areas. Individuals of the Gammarus fossarum reached natural abundances after 84 h in the closed canopy area. The number of Baetidae, Chironomidae and Simuliidae individuals was noticeably higher on artificial than natural substrates in the open canopy area of the stream, while in the closed canopy area, the number of Gammarus fossarum was higher on artificial than on natural substrates. The high number of Trichoptera and Coleoptera individuals found on natural substrates and the low number of artificial substrates indicate that they are not good colonisers (Table 3).

Table 3.

Macroinvertebrates found in reference samples from the natural substrate in Jankovac stream per m2.

Number of specimens
Taxa OC OC (M) CC CC (M)
Turbellaria Crenobia alpina 250 250 200 50
Gastropoda Graziana papukensis 125 75 2125
Ancylus fluviatilis 25 275
Gastropoda non det. 25
Oligochaeta Oligochaeta 575 800 150 150
Arachnida Hydracarina 50
Amphipoda Gammarus fossarum 1775 875 2750 675
Collembola Collembola 50 75
Ephemeroptera Acantrella sinaica 100 200 100
Baetopus tenellus 25
Electrogena sp. 75
Heptagenia sp. 150
Heptagenidae 50
Epeorus assimilis 25
Baetidae juv. Non det. 225 50 675 2625
Ephemeroptera (total) 500 50 1025 2725
Plecoptera Isoperla sp. 50
Leuctra sp. 50 50 225
Protonemura sp. 25 100
Plecoptera (total) 100 50 25 325
Coleoptera Elmis sp. 325 1500 950
Elmis sp. (imago) 575 25
Esolus sp. (imago) 25
Limnius sp. 175 25 525
Limnius sp. (imago) 650
Riolus sp. 25 25 25
Riolus sp. (imago) 25
Coleoptera juv. Non det. 225 525 900
Coleoptera (total) 550 300 3800 1875
Trichoptera Chaetopteryx sp. 25 25
Drusus muelleri 250
Micropterna sequax 100
Allogamus unctatus 25
Synagapetus dubitans 350 275
Rhyacophila laevis 25
Goera pilosa 25
Trichoptera juv. Non det. 250 25 750
Trichoptera (total) 650 25 1450 0
Diptera Chironomidae 1100 1275 400 5675
Simuliidae 625
Aphidae 25
Pupa non det. 175 25 50
Diptera (total) 1300 1300 400 6350
Total 5850 3725 12275 12275
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OC: open canopy area; OC (M): open canopy area (moss); CC: closed canopy area; CC (M): closed canopy area (moss).

In analysing the colonisation dynamics on artificial substrates, individuals of the family Baetidae and Chironomidae were the most numerous on the substrates collected early on. Chironomidae abundance declined during the day in the open canopy area, and slightly increased in the closed canopy area, unlike Baetidae, whose numbers increased during the experiment in both open and closed canopy area. The number of Simuliidae was higher at the beginning of the experiment and gradually decreased with time in both the open and closed canopy areas. A number of specimens of Oligochaeta had a more or less constant abundance during the experiment, regardless of canopy cover. The abundance of Gammarus fossarum declined slightly during the day in the open canopy area, while in the closed canopy area, they increased over time with the highest intensity after three days (Figure 1).

Multiple comparison analysis showed that the abundance of Oligochaeta individuals was significantly higher during the night in the open canopy area (R: 35.192) than during both night (R: 11.792, p < 0.01) and day (R: 12.850, p < 0.01) in the closed canopy area of the stream. Furthermore, a statistically significantly lower number of Oligochaeta individuals was observed during the night in the closed canopy (R: 11.792) than during both night (R: 35.192, p < 0.01) and day (R: 30.750, p < 0.01) in the open canopy area. An abundance of Oligochaeta was statistically significantly higher during day in the open canopy area (R: 30.75) than during the night (R: 11.792, p < 0.01) and day (R: 12.850, p < 0.05) in the closed canopy area. As well, Oligochaeta individuals were significantly better at colonising substrates during the night (R: 35.192, p < 0.01) and day (R: 30.750, p < 0.05) in the open canopy area than during the day in the closed canopy area (R: 12.850). Statistical significance was also confirmed with the Kruskal–Wallis test (H (3, N = 45) = 30.42857; p < 0.01).

Multiple comparison analysis of the abundance of the Gammarus fossarum individuals showed a higher abundance during the night in the open canopy area (R: 33.885) than night (R: 14.958, p < 0.01) and day (R: 12.350, p < 0.01) in the closed canopy area. Also, Gammarus fossarum statistically significantly better colonised substrates during the night (R: 33.885, p < 0.01) and day (R: 29.150, p < 0.05) in the open canopy area than substrates during the day in the closed canopy area (R: 12.350). The Kruskal–Wallis test showed the difference was statistically significant (H (3, N = 45) = 22.35111; p < 0.01).

Multiple comparison analysis of the Baetidae individuals showed higher abundance during the night in the closed canopy area (R: 36.500) than during night (R: 19.269, p < 0.01) and day (R: 8.900, p < 0.01) in the open canopy area. Accordingly, statistically significantly higher values of Baetidae individuals were recorded on substrates during night (R: 36.500, p < 0.01) and day (R: 25.750, p < 0.05) in the closed canopy area than during day in the open canopy area (R: 8.900).

Multiple comparison analysis showed that there were no statistically significant differences in abundances, regarding canopy, for the families Chironomidae and Simuliidae. Abundances of Chironomidae and Simuliidae individuals had a tendency to better colonising the substrates during the night throughout the experiment in both the open and closed canopy areas (Figure 1).

Discussion

Despite only a slightly higher mean water temperature of 0.2°C in the open canopy area compared to the closed canopy area, this could be an important difference in spring habitats that normally have a very small range of diurnal and annual temperatures.2,8 Insect life cycles, for example, laying eggs, embryonic development, larvae growth rate, adult individual size and time of emergence, are highly dependent on temperature.7,12 An increase in temperature also increases feeding, metabolism and respiration and accelerates the time of recovery.24,45 Although oxygen concentrations were nearly equal in both microhabitats, oxygen saturation was higher in the open canopy area of the stream due to autotrophic oxygen production by algae and mosses and the greater light availability in the open canopy.

Electrical conductivity was high, as expected in streams with limestone substrates that enrich the water with calcium and bicarbonate ions,38,46 and values were similar in both open and closed canopy areas. Mean pH values were 0.1 higher in the open than closed canopy area. Values ranged around 8 and did not fluctuate, which is also a result of the direct influence of the karstic carbonate substrates (known as good buffer waters with insignificant pH changes). 45

Rheophile species were dominant on the experimental substrates and natural habitats. 22 They are able to take advantage of water flow above the moss mats for feeding and gas exchange. 15 A large number of Diptera can be found in karst spring areas, especially Chironomidae. Diptera usually accounts for about one-third of the total biomass, but more than half the total species richness.8,47 Representatives of the family Chironomidae were the most numerous macroinvertebrates, comprising nearly half the total macroinvertebrate abundance. Ephemeroptera accounted for nearly one-third of the total macroinvertebrates. According to the dynamics and magnitude of colonising artificial substrates, we can conclude that Baetidae and Chironomidae are especially good pioneer species and can recover within days.14,28

Unlike previous research, a noticeably higher number of Diptera and Ephemeroptera was found in the present study.38,46,48 Although the abundance of Baetidae and Chironomidae was very high in natural samples, they were much more abundant in artificial substrates, that is, they colonised artificial substrates very well. A good colonisation pattern in the open canopy area was also seen for Simuliidae individuals. Despite the different substrates, Oligochaeta individuals equally colonised natural and artificial substrates. Gammarus fossarum individuals were more numerous in the non-moss natural microhabitat in the closed canopy area, so the artificial moss-like substrates did not suit them. Although a lower abundance of Baetidae, Chironomidae and Simuliidae individuals was recorded on natural substrates, their strong ability to colonise new artificial substrates makes them the best pioneering colonisers. Only a small number of Plecoptera and Trichoptera individuals were found, which can be assumed to be due to their emergence that appeared to have taken place shortly before the study was conducted. We found a small number of Leuctra specimens on artificial substrates since they live in interstitial habitats and the moss-like substrates were not a preferable option for them. 49 In addition to the macrolithal, moss was also a preferred habitat for Protonemura, which explains the higher number of individuals found on artificial substates in comparison to the natural habitat. 50 The spring area of the Jankovac stream had a large abundance of Trichoptera,38,48 as confirmed with the number of individuals found on the natural substrate, but only a small number on artificial substrates. We conclude that their body size and cases make them slow colonisers51,52 The order Coleoptera are slow colonisers, achieving maximum values during winter, which could explain the relatively low abundance found on the artificial substrates.21,51,53 A small number on the artificial substrates as opposed to the higher number on natural substrates confirms that Coleoptera is slow colonisers. The genus Elmis best colonised artificial substrates and were the most abundant in the study area.38,54 An endemic snail species (Graziana papukensis Radoman 1975 55 ) was also attached and collected from the artificial substrates from the spring of the Jankovac stream. The artificial substrates that imitated moss proved to be a good substrate, as shown by the total number of individuals, compared to the abundance in nearby natural habitats. Drift can also affect plants, create strong resistance to the water that then uproots them and carries them away, and with them the organisms inhabiting the surrounding areas. The artificial substrates we used are inclined to roll, due to their partial fixation with stone, but could not be washed away, and thus we reduced the possibility of drift of the benthic community colonising the substrate. 23

We analysed Oligochaeta, Amphipoda (Gammarus fossarum), Ephemeroptera (Baetidae) and Diptera (Chironomidae and Simuliidae), as the most numerous taxa found on the artificial substrates. Gammarus fossarum and Baetidae are good migrators (upstream and lateral) and prefer moss habitats.51,56,57 Furthermore, comparing the colonisation of certain groups that were most abundant in the most samples gave interesting and unexpected information. According to the literature, photoperiod has a positive effect on the reproductive cycle of macroinvertebrates.58–60 Specimens of Oligochaeta and Gammarus fossarum were more numerous on substrates in the open canopy area, indicating that they choose habitats with more light.59,61 Juvenile Baetidae and Simuliidae colonised artificial substrates in the closed canopy area of the stream more (up to four times more) than in the open canopy area. We assume the reason for this is a preference toward a darker environment as protection from visual predators (fish and amphibians), which are the dominant predators in this stream because of their small body size. The closed and open canopy reaches were connected, and thus if the abundance of Baetidae mayfly was lower in the open canopy reach, populations in the closed canopy reach could be regarded as source populations that enhance colonisation of the taxa in open canopy habitats. Chironomidae were equally numerous on substrates in both the open and closed canopy areas. Although photoperiod has an influence on the Diptera life cycle and they prefer open canopy areas, Chironomidae and Simuliidae (at least several species within the families) are good colonisers and can adapt to the most suitable habitats.8,17,60,62

Macroinvertebrates were numerous on the substrates collected in the morning (two to three times more) because they are more active during the night when feeding than during the day when hiding from potential predators.63,64

The population of Baetidae increased continuously on substrates in open and closed canopy areas, presumably for better protection of juvenile individuals and frequent swimming to find a favourable habitat for life. 64 Previous research has confirmed that shallow sediment often acts as a nursery habitat and protection from unfavourable situations for early life stages.18,65 Multiple comparison of means analysis also confirmed a higher number of Baetidae on substrates during the night. By the end of the study, representatives of Gammarus fossarum and Baetidae reached their maximum on substrates in closed canopy areas. The opposite situation was noted with Chironomidae and Simuliidae, whose numbers decreased with time. Presumably, the reason is the constant migration of individuals and the lookout for more favourable living conditions.64,66 Chironomidae are good colonisers because of their small body size and short life cycle. 67 Although multiple comparison of means of Chironomidae and Simuliidae did not confirm a statistically significant difference, the colonisation dynamics clearly showed higher values during the night. The number of Oligochaeta was higher on substrates during the night,68,69 as like Baetidae, they take shelter from predators during the night and come out to feed during the day.18,33

We concluded that all the analysed groups were successful at colonising the artificial substrates as seen by the abundances found on the natural substrates. All analysed groups reach an equilibrium in the open canopy area after only 12 h of substrate exposure. In the closed canopy area, all taxa reached equilibrium after 12 h of substrate exposure, with the exception of Gammarus fossarum, which needed 84 h. The new substrate, though artificial, was a favourable new habitat and food source for macroinvertebrates. The large numbers of Chironomidae and Simuliidae point to the increased, mostly passive mobility of these organisms, which is enabled by their small, non-chitinous and wormlike body, as noted elsewhere.28,64 As mentioned before, these taxa are rheophilic and are often found in drift, enabling their spread to new areas.16,23

During this four-day study at the spring area of the Jankovac stream, nearly all groups of macroinvertebrates found on natural substrates were also found on the artificial substrates. Macroinvertebrates showed a colonisation dynamic with expressed day-night migrations, indicating the importance of considering the time of day in community composition research. The type of artificial substrates used proved to be good for macroinvertebrate colonisation and displayed that all the analysed groups were excellent champion colonisers. To get a more complete picture of migration and macroinvertebrate colonisation, this research should be continued. More individuals of all analysed groups were recorded in samples collected in the morning, indicating higher nocturnal activity, while during the day, they take shelter as protection from predatory pressures. All analysed groups proved to be very good champion colonisers. These findings can lead to applicable conclusions, such as minimising riparian vegetation management and similar human impacts that could alter canopy coverage of the spring areas, especially due to their high biodiversity in small areas.

Author biographies

Denis Bućan is a PhD student at the Croatian Natural History Museum. His expertise in Freshwater ecology focusing on recovery processes of macroinvertebrate communities.

Marko Miliša is a professor of Invertebrate biology. His area of research is Freshwater ecology focusing on macroinvertebrates in karst waters.

Footnotes

Author contributions: Conceptualization, all authors; methodology, M.M.; validation, all authors; formal analysis, all authors; investigation, all authors; resources, M.M.; data curation, D.B.; writing – original draft preparation, D.B.; writing – review and editing, M.M.; visualization, all authors; supervision, M.M.; project administration, M.M.; All authors have read and agreed to the published version of the manuscript.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.

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