Importance of clitellar tissue in the regeneration ability of earthworm Eudrilus eugeniae.

Among the annelids, earthworms are renowned for their phenomenal ability to regenerate the lost segments. The adult earthworm Eudrilus eugeniae contains 120 segments and the body segments of the earthworm are divided into pre-clitellar, clitellar and post-clitellar segments. The present study denoted that clitellum plays vital role in the successful regeneration of the species. We have performed histological studies to identify among the three skin layers of the earthworm, which cellular layer supports the blastema formation and regeneration of the species. The histological evidences denoted that the proliferation of the longitudinal cell layer at the amputation site is crucial for the successful regeneration of the earthworm and it takes place only in the presence of an intact clitellum. Besides we have performed clitellar transcriptome analysis of the earthworm Eudrilus eugeniae to monitor the key differentially expressed genes and their associated functions and pathways controlling the clitellar tissue changes during both anterior and posterior regeneration of the earthworm. A total of 4707 differentially expressed genes (DEGs) were identified between the control clitellum and clitellum of anterior regenerated earthworms and 4343 DEGs were detected between the control clitellum and clitellum of posterior regenerated earthworms. The functional enrichment analysis confirmed the genes regulating the muscle mass shape and structure were significantly downregulated and the genes associated with response to starvation and anterior-posterior axis specification were significantly upregulated in the clitellar tissue during both anterior and posterior regeneration of the earthworm. The RNA sequencing data of clitellum and the comparative transcriptomic analysis were helpful to understand the complex regeneration process of the earthworm.

Conserved lamin A protein expression in differentiated cells in the earthworm Eudrilus eugeniae.

Lamin A is an intermediate filament protein found in most of the differentiated vertebrate cells but absent in stem cells. It shapes the skeletal frame structure beneath the inner nuclear membrane of the cell nucleus. As there are few studies of the expression of lamin A in invertebrates, in the present work, we have analyzed the sequence, immunochemical conservation and expression pattern of lamin A protein in the earthworm Eudrilus eugeniae, a model organism for tissue regeneration. The expression of lamin A has been confirmed in E. eugeniae by immunoblot. Its localization in the nuclear membrane has been observed by immunohistochemistry using two different rabbit anti-sera raised against human lamin A peptides, which are located at the C-terminus of the lamin A protein. These two antibodies detected 70 kDa lamin A protein in mice and a single 65 kDa protein in the earthworm. The Oct-4 positive undifferentiated blastemal tissues of regenerating earthworm do not express lamin A, while the Oct-4 negative differentiated cells express lamin A. This pattern was also confirmed in the earthworm prostate gland. The present study is the first evidence for the immunochemical identification of lamin A and Oct-4 in the earthworm. Along with the partial sequence obtained from the earthworm genome, the present results suggest that lamin A protein and its expression pattern is conserved from the earthworm to humans.