Trophic Position of the White Worm (Enchytraeus albidus) in the Context of Digestive Enzyme Genes Revealed by Transcriptomics Analysis.

To assess the impact of Enchytraeidae (potworms) on the functioning of the decomposer system, knowledge of the feeding preferences of enchytraeid species is required. Different food preferences can be explained by variations in enzymatic activities among different enchytraeid species, as there are no significant differences in the morphology or anatomy of their alimentary tracts. However, it is crucial to distinguish between the contribution of microbial enzymes and the animal's digestive capacity. Here, we computationally analyzed the endogenous digestive enzyme genes in Enchytraeus albidus. The analysis was based on RNA-Seq of COI-monohaplotype culture (PL-A strain) specimens, utilizing transcriptome profiling to determine the trophic position of the species. We also corroborated the results obtained using transcriptomics data from genetically heterogeneous freeze-tolerant strains. Our results revealed that E. albidus expresses a wide range of glycosidases, including GH9 cellulases and a specific digestive SH3b-domain-containing i-type lysozyme, previously described in the earthworm Eisenia andrei. Therefore, E. albidus combines traits of both primary decomposers (primary saprophytophages) and secondary decomposers (sapro-microphytophages/microbivores) and can be defined as an intermediate decomposer. Based on assemblies of publicly available RNA-Seq reads, we found close homologs for these cellulases and i-type lysozymes in various clitellate taxa, including Crassiclitellata and Enchytraeidae.

Anti-Inflammatory and Antioxidant Pyrrolo[3,4-d]pyridazinone Derivatives Interact with DNA and Bind to Plasma Proteins-Spectroscopic and In Silico Studies.

From the point of view of the search for new pharmaceuticals, pyridazinone derivatives are a very promising group of compounds. In our previous works, we have proved that newly synthesized ligands from this group have desirable biological and pharmacokinetic properties. Therefore, we decided to continue the research evaluating the activity of pyrrolo[3,4-dpyridazinone derivatives. In this work, we focused on the interactions of five pyridazinone derivatives with the following biomolecules: DNA and two plasma proteins: orosomucoid and gamma globulin. Using several of spectroscopic methods, such as UV-Vis, CD, and fluorescence spectroscopy, we proved that the tested compounds form stable complexes with all biomacromolecules selected for analysis. These findings were also confirmed by the results obtained by molecular modeling. All tested pyridazinone derivatives bind to the ctDNA molecule via groove binding mechanisms. All these molecules can also be bound and transported by the tested plasma proteins; however, the stability of the complexes formed is lower than those formed with serum albumin.

Discovery and characterization of the α-amylases cDNAs from Enchytraeus albidus shed light on the evolution of "Enchytraeus-Eisenia type" Amy homologs in Annelida.

Although enchytraeids have gained popularity in scientific research, fundamental questions regarding their feeding ecology and biology remain largely unexplored. This study investigates α-amylases, major digestive enzymes responsible for hydrolyzing starch and similar polysaccharides into sugars, in Enchytraeus albidus. Genetic data related to α-amylases is currently lacking for the family Enchytraeidae but also for the entire Annelida. To detect and identify coding sequences of the expressed α-amylase genes in COI-monohaplotype culture (PL-A strain) of E. albidus, we used classical "gene fishing" and transcriptomic approaches. We also compared coding sequence variants of α-amylase retrieved from transcriptomic data related to freeze-tolerant strains. Our results reveal that E. albidus possesses two distinct α-amylase genes (Amy I and Amy II) that are homologs to earthworm Eisenia fetida Ef-Amy genes. Different strains of E. albidus possess distinctive alleles of α-amylases with unique SNP patterns specific to a particular strain. Unlike Amy II, Amy I seems to be a highly polymorphic and multicopy gene. The domain architecture of the putative Amy proteins was found the same as for classical animal α-amylases with ABC-domains. A characteristic feature of Amy II is the lack of GHGA motif in the flexible loop region, similarly to many insect amylases. We identified "Enchytraeus-Eisenia type" α-amylase homologs in other clitellates and polychaetes, indicating the ancestral origin of Amy I/II proteins in Annelida. This study provides the first insight into the endogenous non-proteolytic digestive enzyme genes in potworms, discusses the evolution of Amy α-amylases in Annelida, and explores phylogenetic implications.

Do Cuticular Gaps Make It Possible to Study the Composition of the Cell Walls in the Glands of Drosophyllum lusitanicum?

Carnivorous plants can survive in poor habitats because they have the ability to attract, capture, and digest prey and absorb animal nutrients using modified organs that are equipped with glands. These glands have terminal cells with permeable cuticles. Cuticular discontinuities allow both secretion and endocytosis. In Drosophyllum lusitanicum, these emergences have glandular cells with cuticular discontinuities in the form of cuticular gaps. In this study, we determined whether these specific cuticular discontinuities were permeable enough to antibodies to show the occurrence of the cell wall polymers in the glands. Scanning transmission electron microscopy was used to show the structure of the cuticle. Fluorescence microscopy revealed the localization of the carbohydrate epitopes that are associated with the major cell wall polysaccharides and glycoproteins. We showed that Drosophyllum leaf epidermal cells have a continuous and well-developed cuticle, which helps the plant inhibit water loss and live in a dry environment. The cuticular gaps only partially allow us to study the composition of cell walls in the glands of Drosophyllum. We recoded arabinogalactan proteins, some homogalacturonans, and hemicelluloses. However, antibody penetration was only limited to the cell wall surface. The localization of the wall components in the cell wall ingrowths was missing. The use of enzymatic digestion improves the labeling of hemicelluloses in Drosophyllum glands.

Fungal symbionts impact cyanobacterial biofilm durability and photosynthetic efficiency.

Cyanobacteria contribute to over 25% of the world's net primary photosynthetic production and are pivotal in mitigating greenhouse gas emissions.1 This study unveils a previously unobserved symbiotic relationship between benthic cyanobacteria and fungi that have also adapted to life as a plant endophyte. The interaction suggests an initial phase of lichenization. We isolated Leptolyngbya frigida from the Naracauli stream, which emanates from abandoned Zn industrial waste in Sardinia. Seasonally, L. frigida participates in a biomineralization processes, mitigating the Zn transfer to rivers and, subsequently, the sea.2,3,4L. frigida is a benthic cyanobacterium that establishes a biofilm on the stream bed. Notably, the area predominantly features Juncus acutus. From these roots, endophytic fungi were predominantly isolated as Clonostachys rosea, a fungus recognized for its biocontrol capabilities against plant pathogens. An intriguing observation was made when L. frigida was cultured with C. rosea on a low-carbohydrate agar medium: the fungal mycelium transformed into wall-less forms, a phenomenon not documented previously. In liquid environments, the resulting biofilm first settled at the container's bottom. Even upon rising to the surface, this biofilm remained pigment rich. Concurrently, a secondary biofilm began its formation at the bottom. These fungal-integrated biofilms displayed enhanced resilience and superior photosynthetic performance compared to those without fungal presence. Moreover, the symbiotic relationship significantly amplified O2 emission and CO2 sequestration by the biofilm.

Differences in the Occurrence of Cell Wall Components between Distinct Cell Types in Glands of Drosophyllum lusitanicum.

Carnivorous plants are mixotrophs that have developed the ability to lure, trap, and digest small organisms and utilize components of the digested bodies. Leaves of Drosophyllum lusitanicum have two kinds of glands (emergences): stalked mucilage glands and sessile digestive glands. The stalked mucilage glands perform the primary role in prey lure and trapping. Apart from their role in carnivory, they absorb water condensed from oceanic fog; thus, plants can survive in arid conditions. To better understand the function of carnivorous plant emergences, the molecular composition of their cell walls was investigated using immunocytochemical methods. In this research, Drosophyllum lusitanicum was used as a study system to determine whether cell wall immunocytochemistry differs between the mucilage and digestive glands of other carnivorous plant species. Light and electron microscopy were used to observe gland structure. Fluorescence microscopy revealed the localization of carbohydrate epitopes associated with the major cell wall polysaccharides and glycoproteins. The mucilage gland (emergence) consists of a glandular head, a connecting neck zone, and stalk. The gland head is formed by an outer and inner layer of glandular (secretory) cells and supported by a layer of endodermoid (barrier) cells. The endodermoid cells have contact with a core of spongy tracheids with spiral-shaped thickenings. Lateral tracheids are surrounded by epidermal and parenchymal neck cells. Different patterns of cell wall components were found in the various cell types of the glands. Cell walls of glandular cells generally are poor in both low and highly esterified homogalacturonans (HGs) but enriched with hemicelluloses. Cell walls of inner glandular cells are especially rich in arabinogalactan proteins (AGPs). The cell wall ingrowths in glandular cells are significantly enriched with hemicelluloses and AGPs. In the case of cell wall components, the glandular cells of Drosophyllum lusitanicum mucilage glands are similar to the glandular cells of the digestive glands of Aldrovanda vesiculosa and Dionaea muscipula.

New Schiff bases derived from dimethylpyridine-1,2,4-triazole hybrid as cytotoxic agents targeting gastrointestinal cancers: Design, synthesis, biological evaluation and molecular docking studies.

In this research, a series of novel hybrid structures of dimethylpyridine-1,2,4-triazole Schiff bases were designed, synthesized, and evaluated for their in vitro cytotoxic potency on several human gastrointestinal cancer cells (EPG, Caco-2, LoVo, LoVo/Dx, HT29) and normal colonic epithelial cells (CCD 841 CoN). Schiff base 4h was the most potent compound against gastric EPG cancer cells (CC50 = 12.10 ± 3.10 µM), being 9- and 21-fold more cytotoxic than 5-FU and cisplatin, respectively. Moreover, it was not toxic to normal cells. Regarding the cytotoxicity against colorectal cancer cells, compounds 4d and 4l exhibited good activity against HT29 cells (CC50 = 52.80 ± 2.80 µM and 61.40 ± 10.70 µM, respectively), and were comparable to or more potent than cisplatin and 5-FU. Also, they were less toxic to normal cells with a higher selectivity index (SI, CCD 841 CoN/HT29 = 4.20 and 2.85, respectively) than reference drugs (SI, CCD 841 CoN/HT29 < 1). Selected Schiff bases were subjected to the P-glycoprotein inhibition assay. Schiff bases 4d, 4e, and 4l influenced P-gp efflux function, significantly increasing the accumulation of rhodamine 123 in colon cancer cell lines. Further mechanistic studies showed that compound 4l induced apoptotic cell death through a caspase-dependent mechanism and by regulating the p53-MDM2 signaling pathway in HT29 cells. Also, physicochemical predictions of compounds 4d, 4e, 4h, and 4i were examined in silico. The results revealed that the compounds possessed promising drug-likeness profiles.

Ovary organization and ultrastructure in six species of Amynthas and Metaphire earthworms (Annelida, Crassiclitellata, Megascolecidae).

Ovaries in earthworms belonging to the family Megascolecidae are paired structures attached to the septum in the anterior part of the XIII segment. They are fan to rosette shaped with numerous rows of growing oocytes, known as egg strings, radiating from the ovary center towards the segmental cavity. The histological and ultrastructural ovary organization in megascolecids and the course of oogenesis remain unknown. The paper presents the results of light and electron microscopy analyses of ovaries in six megascolecid species, three from the genus Amynthas and three from Metaphire. Both parthenogenetic and sexually reproducing species were included in the study. The organization and ultrastructure of ovaries in all studied species are broadly similar. Considering the histological organization of ovaries, they could be divided into two zones. Zone I (proximal, close to the connection with the septum) is tightly packed with germline and somatic cells. Germ cells are interconnected via intercellular bridges and thin strands of the central cytoplasm (known as cytophore) and form syncytial cysts. Cysts unite oogonia, early meiotic cells (till diplotene), and clustering cells develop synchronously. During diplotene, interconnected cells lose developmental synchrony; most probably, one cell per cyst grows faster than others, detaches from the cysts, and becomes an oocyte. The remaining cells grow slightly and are still interconnected via the thin and reticular cytophore; these cells are considered nurse cells. Zone II has a form of egg strings where growing oocytes are isolated one from another by thin somatic cells and form short cords. We present the ultrastructural details of germline and somatic cells. We propose the term "Amynthas" type of ovaries for this ovary organization. We suppose that such ovaries are characteristic of other megascolecids and related families.

The three-dimensional conformation and activity of mitochondria in syncytial male germ line-cysts of medicinal leeches.

We studied the spatial conformation and activity of mitochondria in the developing syncytial male germline cysts during spermatogenesis of the medicinal leeches using light, fluorescent, transmission electron microscopy, and serial block-face scanning electron microscopy. In cysts with spermatogonia and spermatocytes, mitochondria form networks and are in a dynamic hyperfusion state, while in cysts with spermatids, a single huge mitochondrion is observed. As spermiogenesis progresses, this huge mitochondrion is finally located in the future midpiece. The highest activity, in terms of membrane potential, of the mitochondria in H. medicinalis germline cysts was observed in cysts with spermatocytes; the lowest was in cysts with late elongated spermatids.

Synthesis, Biological, Spectroscopic and Computational Investigations of Novel N-Acylhydrazone Derivatives of Pyrrolo[3,4-d]pyridazinone as Dual COX/LOX Inhibitors.

Secure and efficient treatment of diverse pain and inflammatory disorders is continually challenging. Although NSAIDs and other painkillers are well-known and commonly available, they are sometimes insufficient and can cause dangerous adverse effects. As yet reported, derivatives of pyrrolo[3,4-d]pyridazinone are potent COX-2 inhibitors with a COX-2/COX-1 selectivity index better than meloxicam. Considering that N-acylhydrazone (NAH) moiety is a privileged structure occurring in many promising drug candidates, we decided to introduce this pharmacophore into new series of pyrrolo[3,4-d]pyridazinone derivatives. The current paper presents the synthesis and in vitro, spectroscopic, and in silico studies evaluating the biological and physicochemical properties of NAH derivatives of pyrrolo[3,4-d]pyridazinone. Novel compounds 5a-c-7a-c were received with high purity and good yields and did not show cytotoxicity in the MTT assay. Their COX-1, COX-2, and 15-LOX inhibitory activities were estimated using enzymatic tests and molecular docking studies. The title N-acylhydrazones appeared to be promising dual COX/LOX inhibitors. Moreover, spectroscopic and computational methods revealed that new compounds form stable complexes with the most abundant plasma proteins-AAG and HSA, but do not destabilize their secondary structure. Additionally, predicted pharmacokinetic and drug-likeness properties of investigated molecules suggest their potentially good membrane permeability and satisfactory bioavailability.

Placobdella nabeulensis sp. nov. (Hirudinea: Glossiphoniidae), a new glossiphoniiform leech from Palearctic North Africa.

BACKGROUND: Herein, we describe a new species of turtle blood-feeding leech, Placobdella nabeulensis sp. nov. from Palearctic North Africa (Tunisia and Algeria). The new species is described based on detailed morphological analyses using light and scanning electron microscopes. RESULTS: Apart from the detailed morphology of the atrium, morphological features alone do not sufficiently separate the species from congeners due to the absence of distinct diagnostic characters. Therefore, we turned to molecular data to better distinguish this new species from other members of the genus and establish a basis for its genetic separation. Four DNA fragments were successfully amplified, including mitochondrial COI and 12S rDNA, as well as nuclear 28S rDNA and histone H3. We then provided the molecular descriptor of the taxon, based on redundant diagnostic nucleotide combinations in DNA sequence alignment within the Folmer region. Results of the phylogenetic analysis and species delimitation methods (ABGD, ASAP, and bPTP) based on the COI locus support the species rank of the Tunisian-Algerian Placobdella. CONCLUSIONS: The new species is most closely related to the European species Placobdella costata (Fr. Müller, 1846) and the present study indicates that Placobdella nabeulensis sp. nov. has likely been confused with the European counterpart in several previous studies. This article is registered at www.zoobank.org under urn:lsid:zoobank.org:pub:4A4B9C1D-2556-430F-8E4B-0CE99F2012F5.

Phylogenetical Position versus Pollination Syndromes: Floral Trichomes of Central American and Mexican Pinguicula.

Central American and Mexican Pinguicula species are characterized by enormous divergence in size and color of flowers and are pollinated by butterflies, flies, bees, and hummingbirds. It is known that floral trichomes are key characters in plant-pollinator interaction. The main aim of our study was to verify our hypothesis that the distribution and diversity of non-glandular and glandular trichomes are related to the pollinator syndromes rather than the phylogenetic relationships. The studied sample consisted of Central American and Mexican species. In our study, we relied on light microscopy and scanning electron microscopy with a phylogenetic perspective based on ITS DNA sequences. The flower morphology of species pollinated by butterflies and hummingbirds was similar in contrast to species pollinated by flies and bees. Species pollinated by butterflies and hummingbirds contained low diversity of non-glandular trichomes, which occurred mostly in the tube and basal part of the spur. Surprisingly, in P. esseriana and P. mesophytica, non-glandular trichomes also occurred at the base of lower lip petals. In the case of species pollinated by flies/bees, we observed a high variety of non-glandular trichomes, which occurred on the surface of corolla petals, in the tube, and at the entrance to the spur. Furthermore, we did not identify any non-glandular trichomes in the spur. The capitate glandular trichomes were of similar morphology in all examined species. There were minor differences in the shape of the trichome head, as well as the length and the number of stalk cells. The distribution and the diversity of non-glandular and glandular trichomes and pollinator syndromes were mapped onto a phylogenetic reconstruction of the genus. Most micromorphological characters appear to be associated more with floral adaptation to pollinators and less with phylogeny.

Ovary micromorphology in hormogastrid earthworms with a particular emphasis on the organization of the germline cysts.

There is a gap in our knowledge of microorganization and the functioning of ovaries in earthworms (Crassiclitellata) and allied taxa. Recent analyses of ovaries in microdriles and leech-like taxa revealed that they are composed of syncytial germline cysts accompanied by somatic cells. Although the pattern of cyst organization is conserved across Clitellata - each cell is connected via one intercellular bridge (ring canal) to the central and anuclear cytoplasmic mass termed the cytophore - this system shows high evolutionary plasticity. In Crassiclitellata, only the gross morphology of ovaries and their segmental localization is well known, whereas ultrastructural data are limited to lumbricids like Dendrobaena veneta. Here we present the first report about ovarian histology and ultrastructure in Hormogastridae, a small family of earthworms inhabiting the western parts of the Mediterranean sea basin. We analyzed three species from three different genera and showed that the pattern of ovary organization is the same within this taxon. Ovaries are cone-like, with a broad part connected to the septum and a narrow distal end forming an egg string. Ovaries are composed of numerous cysts uniting a small number of cells, eight in Carpetania matritensis. There is a gradient of cysts development along the long ovary axis, and three zones can be distinguished. In zone I, cysts develop in complete synchrony and unite oogonia and early meiotic cells (till diplotene). Then (zone II), the synchrony is lost, and one cell (prospective oocyte) grows faster than the rest (prospective nurse cells). In zone III, oocytes pass the growth phase and gather nutrients; at this time, their contact with the cytophore is lost. Nurse cells grow slightly, eventually die via apoptosis, and are removed by coelomocytes. The most characteristic feature of hormogastrid germ cysts is the inconspicuous cytophore in the form of thread-like thin cytoplasmic strands (reticular cytophore). We found that the ovary organization in studied hormogastrids is very similar to that described for D. veneta and propose the term "Dendrobaena" type of ovaries. We expect the same microorganization of ovaries will be found in other hormogastrids and lumbricids.

Immunocytochemical Analysis of Bifid Trichomes in Aldrovanda vesiculosa L. Traps.

The two-armed bifids (bifid trichomes) occur on the external (abaxial) trap surface, petiole, and stem of the aquatic carnivorous plant Aldrovanda vesiculosa (Droseracee). These trichomes play the role of mucilage trichomes. This study aimed to fill the gap in the literature concerning the immunocytochemistry of the bifid trichomes and compare them with digestive trichomes. Light and electron microscopy was used to show the trichome structure. Fluorescence microscopy revealed the localization of carbohydrate epitopes associated with the major cell wall polysaccharides and glycoproteins. The stalk cells and the basal cells of the trichomes were differentiated as endodermal cells. Cell wall ingrowths occurred in all cell types of the bifid trichomes. Trichome cells differed in the composition of their cell walls. The cell walls of the head cells and stalk cells were enriched with arabinogalactan proteins (AGPs); however, they were generally poor in both low- and highly-esterified homogalacturonans (HGs). The cell walls in the trichome cells were rich in hemicelluloses: xyloglucan and galactoxyloglucan. The cell wall ingrowths in the basal cells were significantly enriched with hemicelluloses. The presence of endodermal cells and transfer cells supports the idea that bifid trichomes actively transport solutes, which are polysaccharide in nature. The presence of AGPs (which are considered plant signaling molecules) in the cell walls in these trichome cells indicates the active and important role of these trichomes in plant function. Future research should focus on the question of how the molecular architecture of trap cell walls changes in cells during trap development and prey capture and digestion in A. vesiculosa and other carnivorous plants.

Synthesis, Anticancer Activity and Molecular Docking Studies of Novel N-Mannich Bases of 1,3,4-Oxadiazole Based on 4,6-Dimethylpyridine Scaffold.

Cancer is one of the greatest challenges in modern medicine today. Difficult and long-term treatment, the many side effects of the drugs used and the growing resistance to treatment of neoplastic cells necessitate new approaches to therapy. A very promising targeted therapy is based on direct impact only on cancer cells. As a continuation of our research on new biologically active molecules, we report herein the design, synthesis and anticancer evaluation of a new series of N-Mannich-base-type hybrid compounds containing morfoline or different substituted piperazines moieties, a 1,3,4-oxadiazole ring and a 4,6-dimethylpyridine core. All compounds were tested for their potential cytotoxicity against five human cancer cell lines, A375, C32, SNB-19, MCF-7/WT and MCF-7/DX. Two of the active N-Mannich bases (compounds 5 and 6) were further evaluated for growth inhibition effects in melanoma (A375 and C32), and normal (HaCaT) cell lines using clonogenic assay and a population doubling time test. The apoptosis was determined with the neutral version of comet assay. The confocal microscopy method enabled the visualization of F-actin reorganization. The obtained results demonstrated that compounds 5 and 6 have cytotoxic and proapoptotic effects on melanoma cells and are capable of inducing F-actin depolarization in a dose-dependent manner. Moreover, computational chemistry approaches, molecular docking and electrostatic potential were employed to study non-covalent interactions of the investigated compounds with four receptors. It was found that all the examined molecules exhibit a similar binding affinity with respect to the chosen reference drugs.

Targeting Lineage-Specific Transcription Factors and Cytokines of the Th17/Treg Axis by Novel 1,3,4-Oxadiazole Derivatives of Pyrrolo[3,4-d]pyridazinone Attenuates TNBS-Induced Experimental Colitis.

The pharmacotherapy of inflammatory bowel disease (IBD) is still not fully effective and safe. Attempts to search for new IBD drugs remain an incessant research aim. One of the novel approaches is targeting the developmental pathway molecules and effector cytokines of Th17/Treg axis. This study aimed to elucidate the impact of new pyrrolo[3,4-d]pyridazinone derivatives, compounds 7b, 10b, or 13b, on the course of experimental colitis in rats and to assess whether these new compounds may influence Th17/Treg axis. Rats were pretreated with studied compounds intragastrically before intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid used for colitis induction. Body weight loss, disease activity index, colon index, and colon tissue damage were analyzed to evaluate the severity of colitis. The colonic levels of RORγt, STAT3, CCR6, Foxp3, IL-6, IL-10, IL-17, TNF-α, IL-23, and PGE2 were assessed. Pretreatment with compounds 7b and 13b alleviated the severity of colitis and concomitantly counteracted the increased levels of RORγt, STAT3, CCR6, IL-6, IL-17, IL-23, TNF-α, and PGE2. The beneficial effect of compounds 7b and 13b may be due to the decrease in the levels of Th17-specific transcription factors and cytokines. The studied compounds might therefore constitute a promising therapeutic strategy in Th17/Treg imbalance-driven inflammatory conditions such as IBD.

Immunocytochemical Analysis of the Wall Ingrowths in the Digestive Gland Transfer Cells in Aldrovanda vesiculosa L. (Droseraceae).

Carnivorous plants are unique due to their ability to attract small animals or protozoa, retain them in specialized traps, digest them, and absorb nutrients from the dissolved prey material; however, to this end, these plants need a special secretion-digestive system (glands). A common trait of the digestive glands of carnivorous plants is the presence of transfer cells. Using the aquatic carnivorous species Aldrovanda vesiculosa, we showed carnivorous plants as a model for studies of wall ingrowths/transfer cells. We addressed the following questions: Is the cell wall ingrowth composition the same between carnivorous plant glands and other plant system models? Is there a difference in the cell wall ingrowth composition between various types of gland cells (glandular versus endodermoid cells)? Fluorescence microscopy and immunogold electron microscopy were employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. The cell wall ingrowths were enriched with arabinogalactan proteins (AGPs) localized with the JIM8, JIM13, and JIM14 epitopes. Both methylesterified and de-esterified homogalacturonans (HGs) were absent or weakly present in the wall ingrowths in transfer cells (stalk cells and head cells of the gland). Both the cell walls and the cell wall ingrowths in the transfer cells were rich in hemicelluloses: xyloglucan (LM15) and galactoxyloglucan (LM25). There were differences in the composition between the cell wall ingrowths and the primary cell walls in A. vesiculosa secretory gland cells in the case of the absence or inaccessibility of pectins (JIM5, LM19, JIM7, LM5, LM6 epitopes); thus, the wall ingrowths are specific cell wall microdomains. Even in the same organ (gland), transfer cells may differ in the composition of the cell wall ingrowths (glandular versus endodermoid cells). We found both similarities and differences in the composition of the cell wall ingrowths between the A. vesiculosa transfer cells and transfer cells of other plant species.

Arabinogalactan Proteins in the Digestive Glands of Dionaea muscipula J.Ellis Traps.

The arabinogalactan proteins (AGP) play important roles in plant growth and developmental processes. However, to the best of our knowledge, there is no information on the spatial distribution of AGP in the plant organs and tissues of carnivorous plants during their carnivorous cycle. The Dionaea muscipula trap forms an "external stomach" and is equipped with an effective digestive-absorbing system. Because its digestive glands are composed of specialized cells, the hypothesis that their cell walls are also very specialized in terms of their composition (AGP) compared to the cell wall of the trap epidermal and parenchyma cells was tested. Another aim of this study was to determine whether there is a spatio-temporal distribution of the AGP in the digestive glands during the secretory cycle of D. muscipula. Antibodies that act against AGPs, including JIM8, JIM13 and JIM14, were used. The localization of the examined compounds was determined using immunohistochemistry techniques and immunogold labeling. In both the un-fed and fed traps, there was an accumulation of AGP in the cell walls of the gland secretory cells. The epitope, which is recognized by JIM14, was a useful marker of the digestive glands. The secretory cells of the D. muscipula digestive glands are transfer cells and an accumulation of specific AGP was at the site where the cell wall labyrinth occurred. Immunogold labeling confirmed an occurrence of AGP in the cell wall ingrowths. There were differences in the AGP occurrence (labeled with JIM8 and JIM13) in the cell walls of the gland secretory cells between the unfed and fed traps.

All for one: changes in mitochondrial morphology and activity during syncytial oogenesis†.

The syncytial groups of germ cells (germ-line cysts) forming in ovaries of clitellate annelids are an attractive model to study mitochondrial stage-specific changes. Using transmission electron microscopy, serial block-face scanning electron microscopy, and fluorescent microscopy, we analyzed the mitochondria distribution and morphology and the state of membrane potential in female cysts in Enchytraeus albidus. We visualized in 3D at the ultrastructural level mitochondria in cysts at successive stages: 2-celled, 4-celled, 16-celled cysts, and cyst in advanced oogenesis. We found that mitochondria form extensive aggregates-they are fused and connected into large and branched mitochondrial networks. The most extensive networks are formed with up to 10 000 fused mitochondria, whereas individual organelles represent up to 2% of the total mitochondrial volume. We classify such a morphology of mitochondria as a dynamic hyperfusion state and suggest that this can maintain their high activity and intensify the process of cellular respiration within the syncytial cysts. We found some individual mitochondria undergoing degradation, which implies that damaged mitochondria are removed from networks for their final elimination. As growing oocytes were shown to possess less active mitochondria than the nurse cells, the high activity of mitochondria in the nurse cells and their dynamic hyperfusion state are attributed to serve the needs of the growing oocyte. In addition, we measured by calorimetry the total antioxidant capacity of germ-line cysts in comparison with somatic tissue, and it suggests that antioxidative defense systems, together with mitochondrial networks, can effectively protect germ-line mitochondria from damage.

Ovary micromorphology and oogenesis in a rhyacodriline oligochaete (Clitellata: Naididae, Rhyacodrilinae).

The main goal of the article is to describe the ovary organization and oogenesis in Peristodrilus montanus, an aquatic oligochaete of the subfamily Rhyacodrilinae. The presented analysis will not only enrich the knowledge about how eggs are formed but, because of the suggested conservatism of ovary organization in clitellate annelids, can contribute to disentangling the complex phylogenetic relationships of the rhyacodrilines within Naididae. The paired, conically shaped ovaries are located in segment XI. They are composed of a dozen or so syncytial germ-line cysts, which are associated with somatic cells. Each germ cell in a cyst has one intercellular bridge that joins it to a central and anuclear cytoplasmic mass, the cytophore. This pattern of cyst organization is typical for all clitellates that have been studied to date. Initially, the germ cells in a cyst undergo a synchronous development, however, there is no synchrony between cysts, and therefore there is a developmental gradient (oogonia, pre-diplotene germ cells, germ cells in diplotene) of oogenesis along the long ovary axis. The cysts are composed of a maximum of 32 cells. Cysts with cells in diplotene detach from the ovaries and the extraovarian phase of oogenesis begins. The developmental synchrony is lost, one cell (an oocyte) per cyst starts to gather cell components and yolk and grows considerably. The remaining cells grow to some extent and function as nurse cells. Like in other microdriles, P. montanus oocytes are rich in yolk; other features of oogenesis are also similar to those that are known from other microdrile taxa. The system of ovary organization found in the studied species is broadly similar to the corresponding features known from Naidinae and Phreodrilidae and, to some extent, in Enchytraeidae. However, this system is different from the one that is known in Tubificinae, Limnodriloidinae and Branchiurinae.