Environmental, anthropogenic, and spatial factors affecting species composition and species associations in helminth communities of water frogs (Pelophylax esculentus complex) in Latvia.

We investigated factors affecting species composition and patterns of species associations in parasite communities of water frogs (Pelophylax esculentus complex), applying the distance-based redundancy analysis for component communities (assemblages harboured by host populations) and Markov random fields modelling for infracommunities (assemblages harboured by individual hosts), respectively. We asked (a) What are the relative effects of variation in environmental, land use (i.e., anthropogenic), and spatial factors on the variation in the species composition of component communities (i.e., in a locality)? and (b) What is the dominant pattern of species associations in infracommunities (in a host individual), and how do these associations vary along environmental and/or anthropogenic gradients? In component communities, the greatest portion of variation in helminth species composition was explained by the combined effects of space, anthropogenic pressure, and NDVI, with the pure effect of the spatial predictor being much stronger than the effects of the remaining predictors. In infracommunities, the probability of occurrence of some, but not all, helminth species depended on the occurrence of another species, with the numbers of negative and positive co-occurrences being equal. The strength and/or sign of associations of some species pairs were spatially stable, whereas interactions between other species pairs varied along the gradient of the amount of green vegetation, from negative to positive and vice versa. We conclude that the processes in parasite infracommunities and component communities in frogs are intertwined, with both bottom-up and top-down effects acting at different hierarchical scales.

Comparison of the Benzanthrone Luminophores: They Are Not Equal for Rapid Examination of Parafasciolopsis fasciolaemorpha (Trematoda: Digenea).

Luminescent derivatives of benzanthrone are becoming more useful based on their light-absorbing and fluorescent-emitting properties. Our previous studies showed that luminescent staining properties of the same benzanthrone dye differ for variable parasite samples. Therefore, two types of benzanthrone dyes were prepared. One has a strongly basic amidine group and a halogen atom, and the other has an amide moiety and a tertiary amine group. Trematoda Parafasciolopsis fasciolaemorpha is a liver fluke of a moose (Alces alces) and has a significant influence on the health and abundance of the moose population. Staining protocols for parasite P. fasciolaemorpha specific organ or organ systems imaging are mostly time-consuming and labor-intensive. The study aimed to compare the fixation technique and the staining protocol by synthesized benzanthrone luminescent dyes to determine detailed morphology, anatomical arrangement of the organ systems and gross organization of the muscle layers of P. fasciolaemorpha using confocal laser scanning microscopy. Luminophores were tested for samples fixed in different fixatives. Developed dyes and staining protocol resulting in imaging of all parts of trematode without additional sample preparation procedures, which usually are required for parasite examination. Obtained results confirmed that the most qualitative results could be reached using 3-N-(2-piperidinylacetamido)benzanthrone dye which has amide moiety and a tertiary amine group. Based on obtained results, 3-N-(2-piperidinylacetamido)benzanthrone gave more qualitative parasite visualization than 2-bromo-3-N-(N',N'-dimethylformamidino)benzanthrone.

A Simple and Rapid Staining Technique for Sex Determination of Trichinella Larvae Parasites by Confocal Laser Scanning Microscopy.

The roundworms of Trichinella genus are worldwide distributed and their prevalence in nature is high. Trichinella genus parasites are the causative agents of foodborne zoonosis trichinellosis. The main prevention and control of the infection are meat inspection by the magnetic stirrer method for the detection of Trichinella larvae in muscle samples. The treatment can be effective if the parasite is discovered early in the intestinal phase. Once the Trichinella larva has reached the muscle tissue, the parasite remains therein and there is no treatment for this life cycle stage. The Trichinella species is dioecious with separate male and female individuals. The developed staining technique that uses confocal laser scanning microscopy (CLSM) displays sufficient results for Trichinella larvae examination and this protocol is applicable to study the internal and external structures and for the sex determination of T. britovi and T. spiralis larvae samples. In the present study, a luminescent derivative was synthesized and used for staining of T. spiralis and T. britovi larvae samples for the examination by CLSM. Various fixatives, such as AFA, 70% ethanol, and Bouin's and Carnoy's solutions were tested for sample preparation. The synthesized luminescent compound demonstrates best visualization results for samples fixed in Bouin's fixative.

Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy.

In the present study a new luminescent dye 3-N-(2-pyrrolidinylacetamido)benzanthrone (AZR) was synthesized. Spectroscopic measurements of the novel benzanthrone 3-aminoderivative were performed in seven organic solvents showing strong fluorescence. The capability of the prepared dye for visualization has been tested on flax, red clover and alfalfa to determinate the embryo in plant callus tissue cultures. Callus cells were stained with AZR and further analysed utilizing confocal laser scanning fluorescence microscopy. Performed experiments show high visualization effectiveness of newly synthesized fluorescent dye AZR that is efficient in fast and relatively inexpensive diagnostics of callus embryos that are problematic due to in vitro culture specificity.

Novel luminescent dyes for confocal laser scanning microscopy used in Trematoda parasite diagnostics.

Benzanthrone derivates are now widely used in many industrial and scientific applications as dyes for polymers and textiles. In biochemical, biomedical and diagnostics investigations benzanthrone dyes are used as a lipophilic fluorescent probe since many benzanthrone derivates demonstrate bright fluorescence and they have ability to intercalate between membrane lipids. The aim of research presented here was to assess the luminescence ability of benzanthrone derivatives using microscopic visualization of biological objects. Accordingly, specimens of freshwater trematodes: Diplostomum spathaceum, Diplodiscus subclavatus and Prosotocus confusus, were stained by novel benzanthrone dyes using different fixatives. The samples were examined under a confocal laser scanning microscope. All of the dyes tested demonstrated good results for digestive and reproductive system visualization. Based on obtained results we conclude that benzanthrone dyes could be used for internal and external structure confocal laser scanning microscopic imaging of trematode specimens.

DNA extraction from amoebal isolates and genotype determination of Acanthamoeba from tap water in Latvia.

Acanthamoeba are free-living amoebae found in various ecological environments, and they are major microbial predators. Some Acanthamoeba species can cause keratitis, granulomatous amoebic encephalitis, and infections of the organs. This is the first study in Latvia describing Acanthamoeba detection by molecular techniques and genotyping using tap water from different sources. An 18S rRNA gene investigation of the 20 Acanthamoeba isolates suggested that 19 belonged to the hazardous T4 genotype and one sample was T12. The results indicate that tap water in Latvia may be a source of Acanthamoeba that is potentially pathogenic to humans. All obtained sequences were submitted to the GenBank database under the following accession numbers: MG386295-MG386314.