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Nisin (NIS) Z was incorporated (0.05 %, 0.1 %, 0.2 %) into edible films based on chitosan lactate (CHL) and 75/25 blends of polysaccharides (corn starch (CS), wheat starch (WS), oxidized potato starch (OPS), pullulan (PUL)) with CHL. The increase in the NIS/polymer ratio promoted the diffusion-driven release. Compared with the fully dissolvable CHL and PUL/CHL carriers, the starch/CHL films had limited solubility (≈27-37 %) and, consequently, ensured slower/incomplete release of NIS. The assayable NIS half-release times, determined in water, ranged from1 min to ∼13 h. Probably due to the similar pH (≈4.5), there were generally no large differences between the antibacterial activities of the formulations. The NIS-supplemented systems limited the growth of some pathogens (B. cereus, L. monocytogenes, S. aureus), phytopathogens (P. carotovorum), and bacterial starter cultures. The NIS improved the UV-blocking ability of the films, but the 0.2 % NIS addition weakened (by ≈17-32 %) the tensile strength of most of the films.

A new species of entomopathogenic nematodes, Steinernema sandneri n. sp., was recovered by baiting from Poland. Its morphological traits indicate that the new species is a member of the feltiae-kraussei group. A body length of 843 (708–965) μm, a more anterior position of excretory pore (56 μm), and the lower D% value (40 vs > 46) discriminate this species from most of the other group members. The first-generation males of S. sandneri n. sp. can be distinguished from the other clade members by a 60 μm long spicule, a relatively long gubernaculum (GS% = 79), and the position of the excretory pore (80 μm). Phylogenetic analysis of the ITS rDNA, D2D3 of 28 S rDNA, and cox1 sequences confirmed that S. sandneri n. sp. is a new species of the feltiae-kraussei group, closely related to S. kraussei and S. silvaticum.

Entomopathogenic bacteria from the genera Photorhabdus and Xenorhabdus are closely related Gram-negative bacilli from the family Enterobacteriaceae (γ-Proteobacteria). They establish obligate mutualistic associations with soil nematodes from the genera Steinernema and Heterorhabditis to facilitate insect pathogenesis. The research of these two bacterial genera is focused mainly on their unique interactions with two different animal hosts, i.e. nematodes and insects. So far, studies of the mutualistic bacteria of nematodes collected from around the world have contributed to an increase in the number of the described Xenorhabdus and Photorhabdus species. Recently, the classification system of entomopatogenic nematode microsymbionts has undergone profound revision and now 26 species of the genus Xenorhabdus and 19 species of the genus Photorhabdus have been identified. Despite their similar life style and close phylogenetic origin, Photorhabdus and Xenorhabdus bacterial species differ significantly in e.g. the nematode host range, symbiotic strategies for parasite success, and arrays of released antibiotics and insecticidal toxins. As the knowledge of the diversity of entomopathogenic nematode microsymbionts helps to enable the use thereof, assessment of the phylogenetic relationships of these astounding bacterial genera is now a major challenge for researchers. The present article summarizes the main information on the taxonomy and evolutionary history of Xenorhabdus and Photorhabdus, entomopathogenic nematode symbionts.

Root-feeding

The properties of binary blend films (100/0, 75/25, 50/50, 25/75 and 0/100) based on gelatin (GEL) and polysaccharides exhibiting emulsifying activity: gum Arabic (GAR), methylcellulose (MC), octenyl succinic anhydride modified starch (OSA) and water soluble soy polysaccharides (WSSP) were investigated. The micrographs showed that among the polysaccharides only MC was not miscible with GEL. Consequently, GEL/MC blend films were non-homogenous, opaque and susceptible to dissolution. The dynamic light scattering (DLS), microscopic, optical and mechanical measurements revealed that the polymer incompatibility tended to be the highest at mass ratio GEL:MC = 50:50. As verified by Fourier-transform infrared (FTIR) spectroscopy, OSA and WSSP interacted stronger with GEL than MC and GAR. Among the blend films, the MC-containing films had the most crystalline structure. Substituting part of GAR, MC and WSSP for GEL decreased the water vapor permeability of the blend films. Apart from MC, the increasing level of polysaccharides in the films resulted in gradual decrease of mechanical parameters. The most stretchable film was achieved by mixing of GEL with OSA at ratio of 25/75. This film, however, exhibited noticeable symptoms of recrystalization during storage.

Microsporidia Nosema are transferred among bees via the faecal-oral route. Nosema spp. spores have been detected on flowers and transferred to hives along with the bee pollen. The aim of the present study was to determine whether Nosema microsporidia are transferred by air in an apiary, in a control area (without the presence of bee colonies), and/or in a laboratory during cage experiments with artificially infected bees. The novel way of transmission by air was investigated by the volumetric method using a Hirst-type aerobiological sampler located on the ground in the apiary, in the Botanical Garden and on the laboratory floor. Concurrently, the mean rate of Nosema infections in the foragers in the apiary was estimated with the Bürker haemocytometer method. Spore-trapping tapes were imaged by means of light microscopy, Nomarski interference contrast microscopy and scanning electron microscopy. The highest concentration of Nosema spores per 1m3 of air (4.65) was recorded in August, while the lowest concentration (2.89) was noted in July. This was confirmed by a Real-Time PCR analysis. The presence of N. apis as well as N. ceranae was detected in each of the tested tapes from the apiary. The average copy number of N. apis was estimated at 14.4 × 104 copies per 1 cm2 of the tape; whereas the number of N. ceranae was 2.24 × 104 copies per tape per 1 cm2. The results indicate that Nosema microsporidia were transferred by the wind in the apiary, but not in the Botanical Garden and laboratory by air. This was confirmed by genetic analyses. DNA from immobilised biological material was isolated and subjected to a PCR to detect the Nosema species. A fragment of the 16S rRNA gene, characteristic of Nosema apis and N. ceranae, was detected. Our research adds knowledge about the transfer of Nosema spp. microsporidia in the natural environment and indicates the season associated with the greatest risk of a bee colony infection with Nosema spp.

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management. W badaniach skupiono się na potencjalnych związkach pomiędzy mikroflorą jelita środkowego larw chrabąszcza majowego Melolontha melolontha a ich opornością na infekcję przez nicienie entomopatogeniczne (EPN). Za pomocą sekwencjonowania nanoporowego genu 16S rRNA zbadaliśmy zgrupowania bakterii związane z owadami kontrolnymi oraz owadami niewrażliwymi na infekcję przez EPN. W złożonej i zmiennej mikroflorze jelita środkowego dzikich larw M. melolontha najliczniejszymi gatunkami bakterii były Firmicutes, Proteobacteria, Actinobacteria i Bacteroidetes. Stwierdzono, że podstawowa mikroflora obejmuje 82 rodzaje, które stanowiły 3,4% ogólnej liczby zidentyfikowanych rodzajów. Larwy owadów oporne na EPN różniły się znacznie od larw kontrolnych liczebnością wielu rodzajów należących do rzędu Actinomycetales, Rhizobiales i Clostridiales. Larwy oporne na EPN różniły się znacznie od larw kontrolnych liczebnością wielu rodzajów należących do rzędu Actinomycetales, Rhizobiales i Clostridiales. Analiza sieci mikroflory bakteryjnej wykazała ponadto różnice w zgrupowaniach kluczowych gatunków pomiędzy tymi dwiema grupami owadów, co wskazuje na różnice we wzajemnych interakcjach między bakteriami. Wśród bakterii zasiedlających jelito owadów wykryliśmy Xenorhabdus i Photorhabdus oraz różne gatunki bakterii wykazujące oddziaływania antagonistyczne względem tych entomopatogenów. Przeprowadzone badania torują drogę kolejnym, mającym na celu wyjaśnienie wpływu mikroflory jelitowej żywiciela na infekcję przez EPN, co może przyczynić się do zwiększenia skuteczności nicieni wykorzystywanych w biologicznych metodach ochrony roślin przed szkodnikami. NCN

Chapter discusses procedures of collection and preparation of gut samples for light and electron microscopy studies, and all further steps to get proper image. Approved and disapproved methods of influencing the microscopy image are briefly reviewed. Finally, the most often used methods of quantitative evaluation of the objects seen in the digital microscopy images were described.

The fertility and productive value of soil are closely related to the physical and chemical properties of the soil as well as its biological activity. This activity is related to the intensity of microbially catalysed processes of transformation of organic and mineral substances contained in the soil. These processes are closely correlated with the abundance and biodiversity of soil microorganisms, especially bacteria, and the activity of enzymes produced by them. In this article we have compared some physicochemical properties of soil derived from conventional and organic farms and microbial communities inhabiting these ecosystems. We aim to investigate whether the soil management regime affects the abundance and diversity of these environments in terms of bacteria. Some differences in microbial communities were observed, but the rhizosphere of plants from organic and conventional soils does not harbour separate microbiomes. Albeit, the method of fertilization influences the diversity of soil microorganisms. A greater diversity of bacteria was observed in soils from farms where organic fertilizers were applied. Soil pH and activity of some soil enzymes were also shown to differ between organic and conventional soil cropping systems. The Voivodship Fund for Environmental Protection and Water Management in Lublin

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus, have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus. Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB, rpoD, or recA gene sequences as Pseudomonas chlororaphis, Citrobacter murliniae, Acinetobacter calcoaceticus, Chryseobacterium lathyri, Chryseobacterium sp., Serratia liquefaciens, and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

In the present study, Lactobacillus plantarum strain was isolated and identified from spontaneous fermentation of Brassica oleracea var. capitata L. We used the Unweighted Pair Group Method with Arithmetic Mean Analysis (UPGMA) and Principal Component Analysis (PCA) to examine the variations in the functional properties of the isolates. Six functional traits were analyzed, i.e., viability at low pH, resistance to lysozyme and to SIF, auto- and coaggregation, and s-glucosidase activity. The present work is the first study in which the PCA and UPGMA statistical methods were used together to analyze data obtained from the same microbiological experiments. This provided information about the similarity of the examined isolates in terms of their functional traits. Additionally, the level of the analyzed functional traits within the particular groups of isolates was shown. The presented approach is the basis for choosing isolates that are most closely related to the reference strain isolated from pickled cabbage.

Four strains of entomopathogenic nematodes were isolated with a live trap method in southern Poland. The isolates were identified asSteinernema silvaticumbased on morphological, morphometric and molecular data. Infective juveniles of PolishS. silvaticumisolates differ in body length fromS. weiseri(951 vs 740 μm, respectively), and in the hyaline tail portion fromS. kraussei(48 vs 38%, respectively). First-generation males ofS. silvaticumare longer than those ofS. kraussei,S. weiseriandS. ichnusae(1829 vs 1400, 1180 and 1341 μm, respectively). Males ofS. silvaticumand a sister speciesS. krausseican be distinguished by the distance from the anterior end to the nerve ring (142 vs 105 μm), spicule (66 vs 49 μm) and gubernaculum length (45 vs 33 μm), and the presence of a mucron. The analysis of internal transcribed spacer (ITS), D2-D3 andcox1sequences of the tested nematodes revealed differences of 3–5%, 3% and 12–13%, respectively, fromS. krausseistrains. The phylogeny of both nuclear and mitochondrial genes indicated close relationships of the PolishS. silvaticumisolates withS. kraussei,S. oregonenseandS. cholashanense. The reproductive isolation of the studied isolates was confirmed by hybridization tests with other Europeanfeltiae–krausseigroup representatives. This study has supplemented the original description ofS. silvaticumwith morphological and morphometric characterization of the first-generation males and females. This is also the first molecular study of this species based on a multi-gene approach.

Entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) are a group of organisms capable of infecting larvae of insects living in soil, including representatives of the family Scarabaeidae. Their insecticidal activity is related to the presence of symbiotic bacteria Xenorhabdus spp. or Photorhabdus spp. in the alimentary tract, which are released into the insect body, leading to its death caused by bacterial toxins and septicemia. Although the antibacterial activities of symbionts of entomopathogenic nematodes have been well described, there is insufficient knowledge of the interactions between these bacteria and microorganisms that naturally inhabit the alimentary tract of insects infested by nematodes. In this study, 900 bacterial strains isolated from midgut samples of Amphimallon solstitiale larvae were tested for their antagonistic activity against the selected five Xenorhabdus and Photorhabdus species. Cross-streak tests showed significant antibacterial activity of 20 isolates. These bacteria were identified as Bacillus [Brevibacterium] frigoritolerans, Bacillus toyonensis, Bacillus wiedmannii, Chryseobacterium lathyri, Chryseobacterium sp., Citrobacter murliniae, Enterococcus malodoratus, Paenibacillus sp., Serratia marcescens and Serratia sp. Since some representatives of the intestinal microbiota of A. solstitiale are able to inhibit the growth of Xenorhabdus and Photorhrhabdus bacteria in vitro, it can be assumed that this type of bacterial interaction may occur at certain stages of insect infection by Steinernema or Heterorhabditis nematodes.

Single infective juveniles of Heterorhabditis bacteriophora, H. megidis (Nematoda: Heterorhabditidae), Steinernema arenarium, S. carpocapsae and S. feltiae (Nematoda: Steinernematidae) were used to infect single Galleria mellonella (Lepidoptera: Pyralidae) larvae. Four parameters of entomopathogenic nematodes pathogenicity were assessed: the mortality of insects, infectivity of nematodes, number of nematodes established per single G. mellonella, and degree of infective juveniles colonization (percent of infective juveniles which intestine was colonized by symbiotic bacteria). The accuracy, repeatability, and versatility for different species of EPNs in bioassay arenas were compared. Our modifications of the original methods yielded ~ 50% higher efficiency of infective juveniles in cell culture plates and > 20% higher efficiency in centrifuge test tubes. The efficiency of nematodes in cell culture plates (39–77%) was relatively low, especially in the case of Heterorhabditis spp. In the bioassay arena, infective juveniles migrated between cells. The results of our studies indicate that the pathogenicity of EPNs should be assessed in centrifuge test tubes. In these arenas, the infectivity of single IJs was ~ 90% for Heterorhabditis spp. and ~ 95% for Steinernema spp. The degree of colonization of the EPN isolates by symbiotic bacteria was in the range of 96–98%.

Polymer blending and incorporation of active substances offer a possibility of generation of novel packaging materials with interesting features. Astaxanthin is one of the most powerful antioxidants. Hence, in this study, water-soluble AstaSana astaxanthin (AST) was incorporated into 75/25 gum arabic/gelatin (GAR75/GEL25) and water-soluble soy polysaccharides/gelatin (WSSP75/GEL25) blend films in different concentrations (0, 0.25%, 0.5%, 1%). Microscope images showed good compatibility between the polysaccharides and GEL. Basing on time required for 50% release, the WSSP-based film exhibited an approximately four-fold slower release rate (t50% = 65.16–142.80 min) than the GAR-based film (t50% = 14.64–34.02 min). This result was mainly ascribed to the slower dissolution of the WSSP-based carrier. The faster release rate of the GAR-based films resulted in stronger antioxidant activity (quarter-scavenging time (t25%ABTS) = 0.22–7.51 min) in comparison to the WSSP-based films (t25%ABTS = 0.91–12.94 min). The increase in the AST concentration was accompanied by gradually reduced solubility and the release rate. It is possible that the increasing number of starch granules (from the AST formulation) acted as a dissolution blocking agent. In general, the WSSP75/GEL25 film displayed the most linear (the Zero-order similar) release profile. So, this carrier has potential for release of AST at a quasi-constant speed.

Three strains of symbiotic bacteria were isolated from an entomopathogenic nematode Steinernema poinari retrieved from soil in eastern Poland. Using 16S rDNA, recA, gltX, gyrB, and dnaN gene sequences for phylogenetic analysis, these strains were shown to belong to the species Xenorhabdus bovienii. The nucleotide identity between the studied S. poinari microsymbionts and other X. bovienii strains calculated for 16S rDNA and concatenated sequences of four protein-coding genes was 98.7–100% and 97.9–99.5%, respectively. The phenotypic properties of the isolates also supported their close phylogenetic relationship with X. bovienii. All three tested X. bovienii strains of different Steinernema clade origin supported the recovery of infective juveniles and subsequent development of the nematode population. However, the colonization degree of new infective juvenile generations was significantly affected by the bacterial host donor/recipient. The colonization degree of infective juveniles reared on bacterial symbionts deriving from a non-cognate clade of nematodes was extremely low, but proved the possible host-switching between non-related Steinernema species. Electronic supplementary material The online version of this article (10.1007/s00203-018-1544-9) contains supplementary material, which is available to authorized users.

Due to strong tissue hydration and complex architecture of the mucous membrane, appropriate preparation of inhomogeneous gastrointestinal tissues, especially from the intestine, for scanning electron microscopy is still a challenge and requires constant improvement of preparation techniques. In this article, we describe a simplified method of preparation of small intestinal mucosa tissues for observations in a scanning electron microscope. We emphasized the most important points in the preparation process that, when ignored, may result in formation of numerous artifacts and the inability to analyze the samples reliably. The developed technique facilitates proper animal tissue sampling in the field conditions, reducing the time of tissue collection and sample preparation as well as the total process costs. The fixative of choice, that is, buffered formalin, fixes, and stiffens the processed tissues properly, which is especially important in preservation of long, highly hydrated intestinal villi without shrinkage artifacts. The method described has been successfully used in comparative studies of the development of small intestines in mammals (pigs, mice, rats), reptiles, and birds (hens).

The purpose of this study was to compare the effects of increasing concentrations of ascorbate ions (AIs, 0-100mM) in the form of ascorbic acid (AA) and sodium ascorbate (SA) on the properties of edible oxidized potato starch films. The browning reactions were faster in the SA-added films than in those of AA-added. In, turn, AA recrystallized faster than its sodium salt. The highest concentration of SA increased the water vapor permeability of the films. The mechanical strength and stiffness of the films gradually decreased with the increase of AI content. Fourier-transform infrared spectroscopy spectra suggested that addition of SA provoked a more intensive structural changes in the films than AA. X-ray diffraction showed that 25 and 50mM AI-added films exhibited higher crystanility than the control. The films with AA and SA did not differ in terms of dissolving behavior, ability to release AI, and consequently, antioxidant activity.

The relationships between six bacterial symbionts of the entomopathogenic nematodes Heterorhabditis bacteriophora and Heterorhabditis megidis from Poland to species and subspecies of the genus Photorhabdus were evaluated. This study was based on phylogenetic analysis of sequence data of five genes: 16S rRNA, gyrB, recA, gltX, and dnaN. The bacteria were also characterized phenotypically by biochemical and physiological tests. Our results have revealed that the Photorhabdus strains isolated from H. megidis belong to P. temperata, subsp. temperata and subsp. cinerea. Isolates from H. bacteriophora represent P. luminescens subs. kayaii and P. temperata subs. cinerea. This study for the first time provides evidence for H. bacteriophora and P. temperata subsp. cinerea symbiotic association. In addition, we tested whether the microsymbionts of the Polish H. bacteriophora and H. megidis isolates support the development of non-native nematode host population and colonization of their infective juveniles. It has been shown that the studied Photorhabdus strains can readily swap their nematode host, both at intra- and interspecies level. It supports the hypothesis of different symbiotic associations in the Heterorhabditis–Photorhabdus lineage.

We report a case of granulomatous peritonitis due to Baylisascaris transfuga in a young male European brown bear (Ursus arctus). At necropsy, there were extensive abdominal adhesions and extensive granulomatous tissue on the peritoneum and liver capsule. In the gastrointestinal tract, there were 58 nematodes that were identified as Baylisascaris transfuga using light and scanning electron microscopy.

The unique feature of enterocytes in newborn mammals is the presence of an apical canalicular system (ACS) leading to production of large vacuoles, important for colostral macromolecule uptake. The vacuolated fetal-type enterocytes (VFE) enable transfer of colostral and milk proteins from the intestinal lumen across the epithelium without loosing their biological activity. First VFE are observed in the pig and lamb fetuses in the second trimester of pregnancy, located at the upper part of villi in the proximal region of the fetal small intestine and subsequently in the middle and distal regions. After birth the VFE are replaced with enterocytes lacking ACS. The present study aimed to investigate the depletion of VFE in the small intestine in the sow reared pig neonates during the first postnatal weeks using scanning electron microscopy (SEM). The SEM analysis demonstrated the gradual disappearance of vacuolated enterocytes in time. VFE remained in the jejunum for a few days after birth, whereas in the duodenum single VFE were present only at birth. In the proximal jejunum, the VFE were localized in the upper part of the villi, and disappeared until the day 3 of life. VFE were present in the mid and distal jejunum, and diminished gradually until day 14 of life. By the day 21 of life, the vacuolated cells were not observed neither in the jejunum nor ileum. In conclusion, morphology analysis of pig small intestinal mucosa suggests that replacement of fetal type vacuolated enterocytes is resumed within 21 days after birth.