Possible mediation of Cladocera species by a researcher's chest wader

Mediation of aquatic species has become an increasing problem for the last few decades. With the increasing commercial import, species’ direct or indirect spread can gain more space. There are several ways for them to land in their new home and spread through the country. Most of the aquatic species are spread by waterways, boats, vehicles, or even with the help of humans. Cladocerans have a good dispersal ability, thanks to their small size, additionally they possess good adaptation, and mechanisms to develop resting eggs. Benthic or littoral species can be mediated much more easily due to their living space, and with the help of human activities (e.g., scientists, anglers and people working in water bodies) they have a higher chance to colonize new habitats. Our goal was to explore if Cladocera species might be mediated by a scientist chest wader, while sampling in similar-sized, close-to-each other lakes, with different utilization. Most of the species were found in abandoned fishing lakes, followed by oxbow lakes (protected), and ultimately in intensively fished lakes. NMDS showed that samples from lakes with the same utilization are similar to each other. Differently utilized lakes can have various Cladocera species, even though they are closely related to each other. Based on the results, scientists can mediate species on their chest wader from lake to lake and may deteriorate the results. We recommend a necessary chest wader cleaning after every sampling process, especially when samples are taken from differently utilized lakes.


Introduction
Areas with untouched conditions of conservation can be highly disturbed by human activities, which can deteriorate the preservation of these conditions [1]. Humans have migrated across the oceans for thousands of years [2,3]. Domesticated animals and plants were taken to help them in their new home, but unwillingly other organisms that were attached to their boats, clothes etc., were transported, representing the first long-distance dispersals by humans [4].
In the last half-century, commercial transport has increased significantly. This increase in transport has had an extraordinary effect

Materials and methods
The studied lakes are in the N and N-E Hungary (Fig. 1.) representing the micro-region of the Great Plain. During the experiment (2021 summer) we collected samples from 13 different standing waterbodies with three different types of utilization. The oxbow lakes (green marking) are functioning as a natural habitat, with naturally existing fish stock, but without fish installation and fishing activity. Lakes with red markings are utilized as fishing lakes with intensive fish installation (and intensive fishing), while lakes with blue markings are abandoned fishing lakes (Fig. 1). These lakes were once used intensively for fishing but dried out several times during the last decade and without a proper water supply, operators left them behind.

The mediation experiment
From each of the above-mentioned lakes, we collected samples with three repetitions. Before the sampling, all the researchers cleaned their chest wader with a mixture of soap and water and brushed it with a strong bristle brush. After the cleaning, the waders were rinsed well with de-ionized water. The researchers went into all the lakes, until the point where the water level reached the chest wader up most part, around 1 cm from the top section. The distance from the bank of the lakes is around 10 m. Based on previous samplingsthis time was necessary to took a sampleso the researchers stayed exactly 10 min at the same place, and after the time limit, they left the lakes and immediately stepped into a clean plastic container, with a 10 L capacity. In the plastic container, they washed the chest waders down with de-ionized water, to avoid Cladocera species as contamination from a different source than the lake itself and used a soft bristle brush to gently remove all the particles attached to the wader. From the container, with the use of a 35 μm sized mesh, we sieved the samples into 50 mL centrifuge tubes and preserved them with 96% Patosolv alcohol. The process of cleaning the chest wader was repeated before and after at each sampling site.

Laboratorial work and processing of Cladocera samples
In the laboratory, we treated the samples with 100 mL of 10% KOH solution according to the standard method of Korhola and Rautio [41]. These mixtures were placed into plastic bakers and heated for 30 min at 70 • C in a Stuart SWB6D laboratorial water bath.
After the treatment, all the samples were filtered through a 35 μm sieve to eliminate bigger particles. The prepared samples were also A. Hajredini et al. preserved with the above-mentioned 96% Patosolv alcohol to avoid fungal growth, and a few drops of Safranin-glycerine were added to dye the chitinized particles, to help the identification. We used an Olympus BX53 microscope and an Olympus DP26 digital camera for identification. During the identification process, we used 100 μL of samples per slide, and a total of 1 mL of samples were counted.

Data analysis
During the statistical analysis, we used LogX transformation to normalise our data because our abundance data are strongly rightskewed. Based on Levene's test, the resulting p-value is smaller than 0.05 (p < 0.001), so we rejected the null hypothesis of equal variance and concluded that there is a variance between the assemblage differences. For the calculation of the diversity indices (Shannon-Wiener index and Simpson index), we used PAST ver. 3.17c (PAlaeontological STatistcis) software package [45]. Non-metric multidimensional scaling (NMDS) was used to represent the similarities and dissimilarities between the sampled lakes. To represent the pairwise dissimilarity between lakes in a low-dimensional space we used NMDS with the Bray-Curtis similarity index, based on the occurrence of the individual species. Map including the sampled lakes were generated by QGIS 3.16.10 programme.

Results
From the sampled 13 lakes, Cladocera specimens were found in all lakes except Kék víz. We found twenty-seven distinct species in the twelve lakes where Cladocerans were sampled. The Cladocera found includes the whole specimen, an ephippium, its remains or part of a remain. A high number of Cladocera species were found in oxbow lakes, most of these lakes were natural, protected water bodies, while the lowest number of species were found in intensively fished lakes, most of them manmade. There was a group of abandoned fishing lakes, which were not used for intensive fishing for the last 2-3 years, where the amount of Cladocera was remarkably high. In two oxbow lakes, the number of species was not at the same level compared to the others in this group, mostly because of long dry-out periods. From the intensively fished lakes, in Kék víz we did not find any Cladocera, while in Lake Vekeri-tó (VE) we found the highest amount of Cladocera, 1220 individuals. From oxbow lakes, the highest number of Cladocera was found in samples from Szabolcsi Holt-Tisza (SZ1) with 660 individuals, whereas the lowest number of Cladocera was found in Kis-Morotva tó (KMT) with 90 individuals (Table 1).
From the total of 27 species, 24 distinct species were found in oxbow lakes, while 25 different species were found in abandoned fishing lakes and 24 different species were found in intensively fished lakes. Monospilus dispar (G. O. Sars, 1861) was found only in one oxbow lake while missing on the other types of lakes studied, although other species were found at least in 2 differently utilized lakes. Because the Kék víz did not have any species of Cladocera, we were excluded from our statistical data.
The number of distinct species of Cladocera found in all 12 lakes studied was 27, belonging to 4 different families: Bosminidae, Chydoridae, Daphniidae, and Sididae. Kerek-erdő (KE) and Vekeri-tó (VE) had the highest richness with 22 distinct species, while Sziki tó (SZ2) and Sáska-tó (S) had the lowest richness with 3 different species, respectively. The dominant species in our samples was Bosmina longirostris (O. F. Müller, 1776) found in 11 of 12 lakes. Chydorus sphaericus (O. F. Müller, 1785) was found in the highest numbers in all our samples with 1030 individuals while M. dispar was found only once in one of the oxbow lakes. The dominant species in oxbow lakes were C. sphaericus found in 4 of 5 lakes sampled with 210 individuals. In abandoned fishing lakes C. sphaericus was again the dominant species with 400 individuals found in all 3 lakes sampled. In the intensively fished lakes again C. sphaericus was the dominant species with 220 individuals in 3 of 5 lakes sampled.
The Simpson diversity index tells us that the highest average diversity is found in abandoned fishing lakes followed by oxbow lakes and the least diversity was found in intensively fished lakes. The lowest value of the Simpson-index was found in lakes S (Sáska-tó) and SZ2 (Szíki-tó), intensively fished lakes with 0.6250, while the highest value was found on lake TI (Tímári Holt-Tisza) oxbow lake, with a value of 0.9199 (Table 1).
Although the Shannon diversity index shows the same pattern. The highest diversity was found in abandoned fishing lakes and the lowest diversity was foundin intensively fished lakes. Oxbow lakes had moderate diversity. The lowest diversity was in lake S (Sáskató) and SZ2 (Szíki-tó) with a value of 1.0400 while the highest is found in lake TI (Tímári Holt-Tisza) with a value of 2.6740. The tables down below show the species of Cladocera found on each of the lakes sampled during our study (Table 2).
Oxbow lakes were all-natural water bodies, they were protected, but lakes SZ1 (Szabolcsi Holt-Bodrog) and KMT (Kis-Morotva-tó) Table 1 The number of taxa and the number of individuals of Cladocera that were found in each of the lakes studied. Dominance, Simpson-, and Shannonindex are shown as well. Red color represents oxbow lakes, green color represents abandoned fishing lakes while blue color represents intensive fished lakes. Abbreviations are as following: TI (Tímári Holt-Tisza); SZO (Szögi Holt-Bodrog); KMT (Kis-Morotva tó); SZ1 (Szabolcsi Holt-Tisza); KBM (Keleti Holt-Bodrog); R (Rókás-tó); KTP (Kenu-pálya); VE (Vekeri-tó); L (Látóképi víztározó); KE (Kerek-erdei-tó); SZ2 (Szíki-tó) and S (Sáska-tó). Taxa  17  17  6  14  9  16  21  22  11  22  3  3  Individuals  310  590  90  660  140  520  760  1220  150  1060  40  have a higher water level fluctuation, also they can dry out. We can see this in Tables 2 and 3. C. sphaericus was the most dominant species while M. dispar was found only once. From all the abandoned and intensively fished lakes that were studied in this experiment, only lakes SZ2 (Szíki-tó) and KE (Kerekerdei-tó) were natural water bodies, while the others were artificial, and all of them have water level fluctuation. As we can see in Table 3.     were found only once. The NMDS shows the orientation of the lakes, based on Cladocera individual numbers from each lake taken from the chest waders after getting out from each lake (Fig. 2). Abandoned fishing lakes are grouped quite close to each other because the data from that group of lakes were very similar, while oxbow lakes were grouped close to each other except KMT and KBM. Intensively fished lakes are distributed in different areas, far from the other two groups except, lake KE. All five of the studied oxbow lakes are protected water bodies but they did not give us the same orientation. Lakes Ti, SZO, and SZ1 gave us equivalent results. Lake KMT and KBM are not in the same area as other oxbow lakes because they can easily dry out, while lake KBM can also be used for recreational fishing.
Abandoned fishing lakes were grouped in the same area, all three of them: KTP, R, and VE had a high number of Cladocera in the samples, and they are not used for fishing for the last three or more years. Lakes used for intensive fishing are the ones in which we found the lowest number of Cladocera specimens. Lakes S, SZ and L were positioned nearly the same way but not close to each other, while lake KE was orientated in the same way as abandoned fishing lakes, because of the high number of Cladocera found in the sample, due to natural fish stock without fish installation.

Discussion
There are a lot of natural vectors that can help the aquatic invertebrates be dispersed, and humans nowadays are one of the biggest factors influencing dispersal. It can make a willingly or unwillingly dispersal of aquatic organisms. Today there are thousands of cases where humans are the main reason for the dispersal of lots of distinct species. Over the last half-century, commercial transport has increased significantly, which had an extraordinary effect on the dispersal of species, with or without intent. Research on the human role, in dispersal, has increased in the last decades [5].
Every day, scientists around the world take samples from water bodies, for their daily or scientific research. Although taking samples on different water bodies in a short interval of time makes it somehow harder for changing or cleaning chest wader, this is a problem when the sampling sites differ from one another in terms of utilization type or other conditions. The number of aquatic species that can be attached to the chest wader of a scientist is high. Most of them can resist different conditions and stay attached easily to different objects, when they get in contact with new water bodies, they can thrive there without any problem.
Sometimes sampling process in limnology and paleolimnology approaches must be made by walking into the lake. This includes cases when renting or owning a boat is not reachable, when the water body is too shallow, or when sampling a high-mountain lake. There were many cases when the boats carry different aquatic organisms from one water body and disperse them to new aquatic habitats, even in the seas and oceans, different aquatic organisms are dispersed by boats and ships [4]. To prevent this, the small boats need to be cleaned after each sample-taking process. We wanted to know when the sampling is made by just walking into the water body and do the chest wader need to be cleaned before each sampling process. During walking into the lake until the sampling point is reached, our chest waders will be in constant contact with different aquatic organisms that can be attached to them and stay there for a period. Most of our Cladocera found during our study were remains, while we also found ephippia and whole organisms. After they die, Cladoceran remains are deposited in the sediment, during the sample-taking process the sediment is attached to the boots. The number of subfossil Cladocera is commonly higher than the number of contemporary Cladocera [46]. So, a good cleaning process is necessary to avoid false data in the samples. Attachment of different aquatic organisms found on the boots of scientists and then dispersal of this organism over long distances was described by Valls et al. [37]. When Candona furtosae (Teeter 1980) species, found only in Florida, was found hatched in Spain, from sediment by the scientists participating in an international biogeography meeting.
In this study, we tried to elaborate on the effect of scientist chest wader on the dispersal of Cladocera species. For the experiments we used three different utilization types of lakes: oxbow lakes, intensively fished lakes, and abandoned fishing lakes. Seven of the lakes, Tímári Holt-Tisza, Szögi Holt-bodrog, Kis-Morotva-tó, Szabolcsi Holt-Tisza, Keleti Holt-Bodrog, Kerek-erdei-tó and Szíki-tó, are natural water bodies while the others are man-made lakes. We wanted to know if the results depend on the utilization type of the lake, as well as if data can deteriorate if the chest waders are not cleaned between sampling processes and by this if scientists can have a role in the dispersal of Cladocera from lake to lake. In 12 of the 13 lakes sampled, we found at least one species of Cladocera, although only one our sample from lake Kék-Víz we could not find any species of Cladocera. The biggest factor of this Cladocera species missing is the high periods of drying out of this lake, which is accompanied by intensive fishing during periods of abundant water. Cladocera composition and density can rapidly change with the change in environmental conditions or climate change [47].
In oxbow lakes, we found a high diversity of species and a high number of Cladocera, because the conditions on these water bodies are favourable, and they have very small or no anthropogenic activities which explains our results. We also found species that were not found in other utilization types. High numbers of Cladocera species were found also in abandoned fishing lakes, with no fish introduction and low anthropogenic activities during the last years, so the Cladocera species had time to thrive. Many researchers found that zooplankton biomass will increase when fish biomass is decreased [48][49][50]. However in the investigated lakes that have a constant fish introduction, use and high human activities in intensively fished lakes the number and species stock of Cladocera in them were low. Boersma et al. [51] explained the great influence of fish on Cladocera species distribution. Species of contemporary Cladocera decrease in number because of fish predation as suggested by Berta et al. [52]. Apart from lake KE had quite a high amount of Cladocera found in the samples compared to other intensively fished lakes, even though is used for intensive fishing, there is no fishing introduced in this water body. The number of Cladocera species found in Hungarian water bodies is 98. We found 27 of them which is around 27.5% of all Cladocera species of Hungarian water bodies. C. sphaericus was the species that was found in the highest numbers in all 3 types of lake utilization. It is a species that can be found in a wide range of water bodies and ecosystems. It is also resistant to harsh environmental circumstances, with pH ranging from 3.7 to 9.9 in the finding sites and conductivity ranging from 0.4 to 957 mS/m, i.e., brackish water [53][54][55][56]. Whereas M. dispar was found in one of the oxbow lakes only once, by reason that it was protected by water body. It can be found from near sea level to 663 m above sea level. The species is absent from the smallest ponds (less than 1 ha), although it is found in 15% of lakes larger than 1000 ha. It is a fairly acid-sensitive species that can be found in both vegetative and stony substrates. The discovered sites had conductivity ranging from 1.5 mS/m to 53 mS/m [55][56][57]. In this case, if we take the sample from this protected lake where M. dispar is usually found, and then we go to take sample to another lake which has a different characteristic where M. dispar normally cannot be found, there is a high possibility that we produce false data, because we may have carried the species on our chest wader, which is not properly cleaned.
Abandoned fishing lakes had higher values on average than oxbow lakes, while intensive fishing lakes had a low value on average, according to Simpsons and Shannon diversity indexes. The high diversity of abandoned fishing lakes is derived from the lack of fish introduction and low anthropogenic pressure on these lakes. Predation has the biggest impact on zooplankton communities compared to other biotic and abiotic factors [58]. In our survey, it was clear that during sampling in lakes, Cladocera as well as a big number of other aquatic organisms can easily be attached to our chest wader. Similar results were given by other studies on aquatic invertebrates' dispersal [12,59].
Our study showed that when you go sampling on the same day to several water bodies, which of course differs in terms of utilization and protection from anthropogenic activities, a considered amount of Cladocera going to attach to our chest wader in each of the lakes. If we do not clean them after each sampling, there is a high chance that this can deteriorate our data and gives us false data on the species stock.

Conclusion
The number of Cladocera species in our samples was different among the utilization types. There have been distinct species of Cladocera found in the differently utilized lake, so the species stock varies based on the type of lake utilization. The sampling process needs to be performed with a clean chest wader to prevent mediation of Cladocera species and remains from one lake to another. Our data showed that scientists can disperse Cladocera with their chest wader, so a good cleaning process is a must-have before sampling, to get more accurate data and prevent deterioration of the results. To avoid the spread of aquatic species from one water type to another, we propose cleaning chest waders, scientists' clothes, anglers's clothes, and other human activity equipment used in natural water bodies.
When there are conditions and possibilities, it is necessary to clean the chest wader. We devised a figure to prevent deterioration when sampling in lakes with varying utilization (Fig. 3). When we sample intensively fished lakes first, we have the least chance of deteriorating our sample, especially if we go to other utilization type without cleaning our chest wader, therefore cleaning is recommended in this case. If we sample in an abandoned fishing lake, we must clean our chest waders before moving into a differently utilized lake, otherwise, our samples will deteriorate. Cleaning is recommended in case when after sampling an oxbow lake we go to an intensively fished lake type, whereas cleaning is required if we go to an abandoned fishing lake to prevent deterioration. The green arrow (Fig. 3) indicates that cleaning is recommended, the yellow arrow indicates that cleaning should be done, and the red arrow indicates that cleaning of the chest wader is required.

Author contribution statement
Arber Hajredini: Performed the experiments; Wrote the paper. Florent Demelezi; Imre Somlyai: Analyzed and interpreted the data. István Grigorszky: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data. Csaba Berta: Conceived and designed the experiments; Performed the experiments; Wrote the paper.

Data availability statement
Data will be made available on request.

Declaration of competing interest
The authors declare no conflict of interest.