A new endosymbiotic bacterium species associated with a nematode species of the genus Xiphinema (Nematoda, Longidoridae)

2021 virtual edition of the Conference Microscopy at the Frontiers of Science, september 29 and October 1st.Nematodes are the third largest group of metazoans; among them, the Family Longidoridae comprises two main genera of plant parasitic nematodes, Xiphinema and Longidorus, which contain several virus-vector species, e.g. the species X. index, the vector of grape fanleaf virus (GFLV), a serious pathogen of grapes. Bacterial endosymbionts of plant-parasitic nematodes represent a field of research that has become active in recent years. In this work we present a detailed characterization of the endosymbiont bacterium found in the nematode X. pachtaicum from the rhizosphere of sour orange trees (Citrus x aurantium L.) from Cordoba, Spain, and, based on morphological, phylogenetic and genomic characteristics propose a novel candidate genus and species for this uncultured bacterium (strain IAST). An intracellular bacterium, strain IAST, was observed to infect several species of the plant-parasitic nematode genus Xiphinema (X. astaregiense, X. incertum, X. madeirense, X. pachtaicum, X. parapachydermum and X. vallense). The bacterium could not be recovered on axenic medium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8–1.2 μm wide and 2.5–6.0 μm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAST as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAST is therefore proposed as representing ‘Candidatus Xiphinematincola pachtaicus’ gen. nov., sp. nov

‘Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov., an endosymbiotic bacterium associated with nematode species of the genus Xiphinema (Nematoda, Longidoridae)

An intracellular bacterium, strain IAST , was observed to infect several species of the plant-parasitic nematode genus Xiphinema (Xiphinema astaregiense, Xiphinema incertum, Xiphinema madeirense, Xiphinema pachtaicum, Xiphinema parapachydermum and Xiphinema vallense). The bacterium could not be recovered on axenic medium. The 16S rRNA gene sequence of IAST was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EBT (92.9% sequence identity) and ‘Candidatus Glomeribacter gigasporarum’ BEG34 (89.8% identity) are the closest taxa and form a separate phylogenetic clade inside Burkholderiaceae. Other genes (atpD, lepA and recA) also separated this species from its closest relatives using a multilocus sequence analysis approach. These genes were obtained using a partial genome of this bacterium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8–1.2µm wide and 2.5–6.0µm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAST as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAST is therefore proposed as representing ‘Candidatus Xiphinematincola pachtaicus’ gen. nov., sp. nov

The mature and immature spermatozoa of the free-living marine nematode Daptonema sp. (Nematoda: Monhysterida: Xyalidae)

The immature spermatozoa stored in the testis and female postvulval uterine sac (PUS), and mature spermatozoa from the uterus of the free-living marine nematode Daptonema sp. (Monhysterida: Monhysterina: Xyalidae) were studied. The spermatozoa have a nucleus without a nuclear envelope. The central cytoplasm of the immature spermatozoa from the testis and the distal part of the PUS is occupied by a mass of pale fibrous bodies (FB) surrounded by mitochondria and osmiophilic membranous organelles (MO). The spermatozoa in the proximal half of PUS have a wide peripheral layer of electron lucent filamentous cytoplasm. The uterus lumen contains an aggregation of mature spermatozoa of which the periphery is transformed into pseudopods. The FB replaced by a voluminous electron-lucent halo bounded by a continuous layer of MO, mitochondria and fibrous matter. The MO may be intact, but numerous MO are fused with the plasma membrane, having been transformed into transparent pouches, each one opening to the exterior via a pore. Ultrastructural data showed that the activation is regulated in the female when spermatozoa migrate toward the uterus and transform into amoeboid mature spermatozoa. In general, the spermatozoa of Daptonema sp. and some other Monhysterina closely resemble those of the taxa belonging to the order Rhabditida. However, the `rhabditid' pattern of spermatozoon structure and development is most likely the plesiomorphic state in Rhabditida and close sister groups

Dry Ice Self-Pressurised Rapid Freezing (DryIce SPRF): all you need to cool for cryofixation of nematodes is dry ice

Cryofixation immediately arrests all biochemical, physiological and dynamic processes underway in the sample in their present state, resulting in both excellent preservation of the specimen's ultrastructure and its antigenicity. Cryofixation involves extremely rapid cooling of specimens, creating an amorphous, or 'non-crystalline', state of water containing no detectable ice crystals, a process dependent on pressure, medium composition and temperature. Self-Pressurised Rapid Freezing (SPRF) employs plunge freezing of specimens in a sealed copper tube into a cryogen such as nitrogen slush (-210 degrees C),liquid nitrogen (-196 degrees C), ethane (-183 degrees C) or propane (-120 degrees C). In this study we have explored the use of SPRF with cooled acetone on dry ice (-80 degrees C) as the cryogen, a method named Drylce SPRF. Although with this relatively high temperature amorphous water cannot be formed, we have demonstrated that the ultrastructural and antigenicity results after Drylce SPRF on Caenorhabditis elegans are perfectly comparable with those achieved using High Pressure Freezing and SPRF. Thus, with sufficient pressure optimal results, with ice crystals below the resolution of transmission electron microscopy. can be achieved even at -78 degrees C. Furthermore, a huge advantage of Drylce SPRF over other techniques is its use of affordable, easily available and safe products

Electron microscopy techniques

This chapter provides information on the various procedures and techniques used for the preparation of plant parasitic and soil nematode samples for transmission and scanning electron microscopy studies regarding their ultrastructural information, morphogenesis, nematode-host interactions, responses to biotic and abiotic effects and as an important adjunct to taxonomic information

Self-pressurised rapid freezing (SPRF) : an easy-to-use and low-cost alternative cryo-fixation method for nematodes

Self-Pressurised Rapid Freezing (SPRF), an easy-to-use and low-cost alternative cryo-fixationmethod, was evaluated based on a comparative analysis of the ultrastructure of spermatozoa of the nematodes Acrobeles complexus and Caenorhabditis elegans. Sealed copper tubes, packed with active nematodes in water, were plunged into nitrogen slush, a semi-solid form of nitrogen. The water inside the capillary copper tube expands upon cooling due to the formation of hexagonal ice, thereby generating high pressure intrinsically for cryo-fixation of the sample. For sperm cells cryo-fixed by SPRF, the preservation of the ultrastructure was comparable to that achieved with high pressure freezing. This was evidenced by the clear details in mitochondria, membranous organelles and cytoskeleton in the pseudopod. It was demonstrated that SPRF fixation did not destroy antigenicity, based on the results of the immunolocalisation of the major sperm protein in both species. In conclusion, SPRF is a low-cost alternative cryo-fixation method for nematodes

Ultrastructure of sperm development in the genus Ditylenchus (Nematoda: Anguinidae)

Spermatogenesis in Ditylenchus arachis and D. dipsaci was studied using transmission electron microscopy. Spermatogenesis includes the formation of complexes of fibrous bodies (FB) and membranous organelles (MO) in the spermatocytes, which dissociate in separated MO and FB in the spermatids. Immature spermatozoa are unpolarised cells with separate FB and MO. Mature spermatozoa are arranged in chains. Ditylenchus dipsaci is unique in having MO that have already fused with the outer membrane in immature spermatozoa and have mature spermatozoa in the male testis, proving that not only insemination plays a role in spermiogenesis. Contrary to what has been described before, spermatogenesis in Ditylenchus, and other early diverging Tylenchomorpha, follow the typical 'rhabditid' pattern, while the absence of MO within Tylenchomorpha appears to be an apomorphic trait for the molecular defined clade of tylenchids that exclusively parasitise higher plants. This confirms the value of traits related to spermatogenesis in nematode phylogeny