The cytochrome c oxidase subunit I (cox1) gene is the main mitochondrial molecular marker playing a pivotal role in phylogenetic research and is a crucial barcode sequence. Folmer’s “universal” primers designed to amplify this gene in metazoan invertebrates allowed quick and easy barcode and phylogenetic analysis. On the other hand, the increase in the number of studies on barcoding leads to more frequent publishing of incorrect sequences, due to amplification of non-target taxa, and insufficient analysis of the obtained sequences. Consequently, some sequences deposited in genetic databases are incorrectly described as obtained from invertebrates, while being in fact bacterial sequences. In our study, in which we used Folmer’s primers to amplify COI sequences of the crustacean fairy shrimp Branchipus schaefferi (Fischer 1834), we also obtained COI sequences of microbial contaminants from Aeromonas sp. However, when we searched the GenBank database for sequences closely matching these contaminations we found entries described as representatives of Gastrotricha and Mollusca. When these entries were compared with other sequences bearing the same names in the database, the genetic distance between the incorrect and correct sequences amplified from the same species was c.a. 65%. Although the responsibility for the correct molecular identification of species rests on researchers, the errors found in already published sequences data have not been re-evaluated so far. On the basis of the standard sampling technique we have estimated with 95% probability that the chances of finding incorrectly described metazoan sequences in the GenBank depend on the systematic group, and variety from less than 1% (Mollusca and Arthropoda) up to 6.9% (Gastrotricha). Consequently, the increasing popularity of DNA barcoding and metabarcoding analysis may lead to overestimation of species diversity. Finally, the study also discusses the sources of the problems with amplification of non-target sequences.

Bacterial endosymbionts of aquatic invertebrates remain poorly studied. This is at least partly due to a lack of suitable techniques and primers for their identification. We designed a pair of non-degenerate primers which enabled us to amplify a fragment of ca. 500 bp of the 16S rRNA gene from various known bacterial endosymbiont species. By using this approach, we identified four bacterial endosymbionts, two endoparasites and one uncultured bacterium in seven, taxonomically diverse, freshwater crustacean hosts from temporary waters across a wide geographical area. The overall efficiency of our new WOLBSL and WOLBSR primers for amplification of the bacterial 16S rRNA gene was 100%. However, if different bacterial species from one sample were amplified simultaneously, sequences were illegible, despite a good quality of PCR products. Therefore, we suggest using our primers at the first stage of bacterial endosymbiont identification. Subsequently, genus specific primers are recommended. Overall, in the era of next-generation sequencing our method can be used as a first simple and low-cost approach to identify potential microbial symbionts associated with freshwater crustaceans using simple Sanger sequencing. The potential to detected bacterial symbionts in various invertebrate hosts in such a way will facilitate studies on host-symbiont interactions and coevolution.

Abstract:We studied the diet of the American mink (Neovison vison) in small artificial watercourses located in a farmland area of the valley of the River Barycz in Poland. Rodents, mainly Microtus spp., were the most frequent prey identified, occurring in 88.3 % of all analyzed mink scat. Minks also fed willingly on fish, birds and amphibians, whereas insects, crayfish and reptiles accounted for only a small part of the biomass of food consumed. The food niche breadth of the mink's diet was wide, and varied significantly between seasons; in spring and autumn minks preyed mainly on rodents and fish, while the winter and summer diets consisted of a broad characterization of prey items. The proportion of mammals in the diet also decreased significantly during the summer months. These patterns differ from those previously reported in Europe, and demonstrate the plasticity of the mink diet across habitats.

a b s t r a c tA survey on temporary ponds has been conducted in search for large branchiopod crustaceans (Anostraca, Notostraca, Spinicaudata and Laevicaudata) in Wielkopolska province (western Poland). 728 pools have been studied and large branchiopods have been found in 221 of them. Seven species have been recorded, including three anostracans: Branchipus schaefferi, Chirocephalus shadini and Eubranchipus grubii; two notostracans: Lepidurus apus and Triops cancriformis; one spinicaudatan, Cyzicus tetracerus and one laevicaudatan, Lynceus brachyurus. According to the analysis of co-occurrence, the species form three groups, differing in habitat preferences and conservation status. The number of species shows that the diversity of globally threatened large branchiopods is still relatively high in the region. On the other hand, their conservation status is highly diverse and in most species unfavourable. Distribution of all species is highly clustered: large branchiopods have been generally found in 33 UTM squares (10 × 10 km) of 96 squares studied. However, only two species, i.e. E. grubii and L. apus occurred in more than five such squares and could be assessed as moderately widespread. Most water bodies inhabited by large branchiopods occur in groups forming patches of suitable habitats which are dispersed among prevailing seemingly unsuitable areas. Sustaining the existence of large metapopulations seems, therefore, to be essential for conservation of branchiopod species diversity. Field observations also bring some examples of human activities unintentionally supporting the branchiopod conservation.

The following paper presents the results on the determination of the diversity of species from the Phytophthora genus occurring in the declining oak stands in Krotoszyn Plateau in Poland. From the 50s of the last century, significant deterioration of oak health was observed in these stands, and Phytophthora species were suggested as one of the factors of the decline. In order to determine the presence of pathogenic organisms from the Phytophthora genus in these stands, 180 rhizosphere soil samples from three forest districts throughout the Krotoszyn Plateau were collected and subjected to the isolation method. Phytophthora species were consistently isolated from all the sampled stands, and 194 isolates from 111 positive samples were obtained. However, 150 (77%) and 44 (23%) isolates originated from the samples taken under the symptomatic and asymptomatic trees, respectively. All the obtained isolates were morphologically classified using the light and scanning electron microscopy and divided into morphological groups. Genomic DNA was isolated from selected isolates representing each group, ITS regions were amplified and sequence analyses were performed. In total, four different Phytophthora species were detected, including P. cactorum, P. plurivora, P. quercina and P. europaea. The most often isolated species were P. cactorum and P. plurivora. This is the first report of P. europaea in oak stands in Poland.

Filtration patterns in the fairy shrimp Eubranchipus grubii were analysed under laboratory conditions using water containing phytoplankton from their actual locality in western Poland. After 48 hours of feeding, we compared the structure of algal communities in water samples in treatments with males, females and control. The mean filtration rate for E. grubii was relatively high (14,488 mL h-1) in comparison to other crustacean filter feeders and was higher for females than for males. Fairy shrimps grazed effectively on all 11 dominant phytoplankton taxa and there was no sign of overall preferences with regard to taxon, cell volume, length or shape. Female fairy shrimps removed significantly more cells of three taxa (Cryptomonas erosa, C. ovata and Trachelomonas volvocina). The differences between sexes did not depend on the cell volume or length of particular phytoplankton taxa but were significantly related to their initial abundance, initial biomass and shape. The higher the abundance and biomass of the algal taxa, the more intensively it was grazed by females than by males. Females were also observed to graze more on spherical and elongated cells. In conclusion, our results show E. grubii to be an effective, generalist filter feeder capable of significantly influencing the phytoplankton community of a vernal pool. Possible implications of such grazing pressure are also discussed.

The morphology of resting eggs was studied in Polish populations of 7 large branchiopod species:Branchipus schaefferi,Chirocephalus shadini,Eubranchipus grubii(Anostraca),Lepidurus apus,Triops cancriformis(Notostraca),Lynceus brachyurus(Laevicaudata), andCyzicus tetracerus(Spinicaudata). Scanning electron microscope photographs for each species are also provided. In some species (especiallyE. grubii), the ornamentation and size of cysts from Polish populations significantly differ from literature data for populations from other parts of Europe. We also present a key to determination of the studied species based on cyst morphology and discuss whether those traits are useful in surveys of new localities of endangered large branchiopods.

Borysthenia naticina, recorded over the last fifty years in the Wielkopolska district (W. Poland). Their distribution is shown on UTM/MGRS grid maps, and habitat information is provided. Among the discussed species, B. tentaculata (447 sites) and V. contectus (210 sites) are the most frequent in the region. Two alien species-M. tuberculata (two sites) and L. naticoides (seven sites) and the native B. naticina, recorded from five sites, are the least frequent.