Steroidogenesis is an indispensable process that is indirectly associated with spermatogenesis in the Leydig cell (LC) to utilize the lipid droplets (LDs) that are critical to maintaining normal testosterone synthesis. The regulation of LD mobilization, known as lipophagy, in the LC is still largely unknown. In the present study, the LC of the Chinese soft-shelled turtle was investigated to identify the steroidogenic activity and lipophagy during the annual reproductive cycle by light microscopy, immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM). The LC showed a dynamic steroidogenic function with strong activity of 3β-HSD, vimentin and tubular ER during hibernation by IHC and TEM. The tubulo-vesicular ER had a weak immunopositive reaction for 3β-HSD in the LC during reproductive phase, suggesting persistent steroidogenic activity. ORO staining and TEM demonstrated that a larger number of LDs had accumulated in the LC during hibernation than in the reproductive phase. These LDs existed in close association with mitochondria and lysosomes by being dynamically surrounded by intermediate filaments to facilitate LD utilization. Lysosomes were found directly attached to large LDs, forming an autophagic tube and engulfing LDs, suggesting that micro-lipophagy occurs during hibernation. Furthermore, the IHC of ATG7 (Autophagy Related Gene 7) and the IF of the LC3 (Microtubule-associated protein light chain 3), p62 (Sequestosome-1 (SQSTM1) and LAMP1(Lysosomal-associated membrane protein 1) results demonstrated strong expression, and further confirmation by TEM showed the existence of an autophagosome and an autolysosome and their fusion during the hibernation season. In conclusion, the present study provides clear evidence of LD consumption in the LC by lipophagy, lysosome and mitochondria during the hibernation period, which is a key aspect of steroidogenesis in the Chinese soft-shelled turtle.

Duck Tembusu virus (DTMUV) is a novel member of Flavivirus. The isolated and purified DTMUV strain XZ-2012 was used as a strain model, to intramuscularly inject the six-month egg-laying shelducks with the infective dose of 104TCID50. The dynamic distribution of the virus in spleen at different time post-infection (pi) was studied using RT-PCR, RT-qPCR, ELISA, immunofluorescence and transmission electron microscopy (TEM). The results showed that the virus occurred in the spleen after 2 hpi and lasted up to 18 dpi. The registered viral load increased from 2 hpi to 3 dpi, and then it diminished from 6 dpi to 18 dpi with a slight rise at 12 dpi. From 2 hpi to 6 dpi the DTMUV particles were mostly distributed in the periellipsoidal lymphatic sheath (PELS) of spleen white pulp, few being found in the sheathed capillary. From 9 dpi to 18 dpi, the DTMUV particles were migrating into periarterial lymphatic sheaths (PALS) around the central artery through the red pulp. Under TEM, the virus particles could be observed mostly in lymphocytes and macrophages. It was suggested that DTMUV invaded lymphocytes and macrophages of the spleen at 2 hpi and replicated significantly from 1 dpi to 3 dpi, being eliminated from 9 dpi to 18 dpi. This is the first study on the dynamic distribution of DTMUV from invasion to elimination in duck spleen conducted by molecular and morphological methods. It could provide theoretical basis for the occurrence, development and detoxification of the virus in the organs of the immune system.

Autophagy is a universal catabolic process preserved in eukaryotes from yeast to plants and mammals. The main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol during nutrient stress. Here, Fusarium oxysporum was utilized as a model organism, and we found that autophagy assumes an imperative job in affecting the morphology, development, improvement and pathogenicity of F. oxysporum. The search of autophagy pathway components from the F. oxysporum genome database recognized putative orthologs of 16 core autophagy-related (ATG) genes of yeast, which additionally incorporate the ubiquitin-like protein atg22. Present study elucidates the unreported role of Foatg22 in formation of autophagosomes. The deletion mutant of Foatg22 did not demonstrate positive monodansylcadaverine (MDC) staining, which exposed that Foatg22 is required for autophagy in F. oxysporum. Moreover, the ∆Foatg22 strains exhibited a decrease in hyphal development and conidiation, and reduction in pathogenicity on potato tubers and leaves of potato plant. The hyphae of ∆Foatg22 mutants were less dense when contrasted with wild-type (WT) and overexpression (OE) mutants. Our perceptions demonstrated that Foatg22 might be a key regulator for the control of dry rot disease in tuber and root crops during postharvest stage.

The ductuli efferentes (DE) form a transit passage for the passage of spermatozoa from the rete testis to the epididymis. After spermiation, various epithelial secretory proteins are transferred via extracellular vesicles (EVs) to the spermatozoa for their maturation and long-term viability. The aim of the present study was to investigate the distribution, classification, and source of multivesicular bodies (MVBs) and their EVs in the epithelia of the efferentes duct in a turtle species, the soft-shelled freshwater turtle Pelodiscus sinensis by using light and transmission electron microscopy. The results showed that CD63 as a classical exosome marker was strongly immunolocalized within the apical and lateral cytoplasm of the ciliated cells (CC) and moderate to weak in the non-ciliated cells (NCC) of DE. The ultrastructure revealed that early endosome was present at the basement membrane and perinuclear cytoplasm of both CC and NCC, whereas MVBs were located over the nucleus in the cytoplasm of NCC and adjacent to the basal bodies of cilia within the CC. Many EVs, as sources of MVBs, were located within the blebs that were attached to the cilia of CC, within the apical blebs from NCC, and the lateral spaces of CC and NCC. There was ultrastructure evidence of EVs associated with spermatozoa in the lumens of DE. Collectively, the present study provides cytological evidence that the DE epithelium secreted EVs to the lumen by (1) apical blebs, (2) ciliary blebs, and (3) from the basolateral region. These EVs were associated with spermatozoa in the DE lumen of this turtle. Characterization and cellular distribution of these EVs in the DE of a turtle may provide a study model to further investigate the transferring of micromolecules via EVs to the spermatozoa.