The golden mussel Limnoperna fortunei is an Asian invasive bivalve that threats aquatic biodiversity and causes economic damage, especially to the hydroelectric sector in South America. Traditional control methods have been inefficient to stop the advance of the invasive mollusk, which currently is found in 40% of Brazilian hydroelectric power plants. In order to develop an effective strategy to stop golden mussel infestations, we need to better understand its reproductive and sexual mechanisms. In this study, we sequenced total RNA samples from male and female golden mussel gonads in the spawning stage. A transcriptome was assembled resulting in 200,185 contigs with 2,250 bp N50 and 99.3% completeness. Differential expression analysis identified 3,906 differentially expressed transcripts between the sexes. We searched for genes related to the sex determination/differentiation pathways in bivalves and model species and investigated their expression profiles in the transcriptome of the golden mussel gonads. From a total of 187 genes identified in the literature, 131 potential homologs were found in the L. fortunei transcriptome, of which 15 were overexpressed in males and four in females. To this group belong gene families relevant to sexual development in various organisms, from mammals to invertebrates, such as Dmrt (doublesex and mab3-related-transcription factor), Sox (SRY-related HMG-box) and Fox (forkhead box).

In invertebrates, a few studies have suggested apoptosis as the mechanism of choice to protect the retina after exposure to ultraviolet (UV) radiation. We demonstrated previously, by electron microscopy, that the retina and lamina ganglionaris (or lamina) cells of the crab Ucides cordatus displayed subcellular signs of apoptosis after exposure to UVB and UVC. Here, we first ascertained, by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, that UV irradiation indeed produced the previously reported results. We next tested, in the visual system of U. cordatus, whether the expression (as analyzed by immunohistochemistry and observed with laser scanning microscopy) and levels (as examined by Western blotting) of catalase, Bax, and p53 were affected by the same dose of UV radiation as that used previously. Our data revealed that the intensity of catalase, Bax, and p53 labeling was stronger in irradiated retina and lamina cells than in non-irradiated retina and lamina. However, no significant difference was observed in the concentrations of these proteins isolated from the whole optic lobe. The results thus suggest that UVB and UVC induce apoptosis in the crustacean retina and lamina by increasing catalase expression and activating the Bax- and p53-mediated apoptosis pathways.

The neural system appears before the vascular system in the phylogenetic tree. During evolution, vascular system generation takes advantage of the pre-existing vascular endothelial growth factor (VEGF) in order to form its networks. Nevertheless, the role of VEGF in neuronal and glial cells is not yet completely understood. In order to support the hypothesis of a neural role for VEGF, we searched for VEGF- and VEGF receptor (VEGFR)-like immunoreactivities (immunohisto/cytochemistry and Western blotting) in the eyestalk of the invertebrate Ucides cordatus (Crustacea, Brachyura, Ucididae). Our results showed that both neurons and glial cells expressed VEGF-immunoreactivity, and that VEGFR was evidenced in neural cells. This is the first report about the VEGF/VEGFR-like immunoreactivities in the nervous tissue of a crustacean, and enables U. cordatus to be included in the repertoire of animal models used for ascertaining the role of VEGF in the nervous system.

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Although there is a considerable demand for cell culture protocols from invertebrates for both basic and applied research, few attempts have been made to culture neural cells of crustaceans. We describe an in vitro method that permits the proliferation, growth and characterization of neural cells from the visual system of an adult decapod crustacean. We explain the coating of the culture plates with different adhesive substrates, and the adaptation of the medium to maintain viable neural cells for up to 7 days. Scanning electron microscopy allowed us to monitor the conditioned culture medium to assess cell morphology and cell damage. We quantified cells in the different substrates and performed statistical analyses. Of the most commonly used substrates, poly-L-ornithine was found to be the best for maintaining neural cells for 7 days. We characterized glial cells and neurons, and observed cell proliferation using immunocytochemical reactions with specific markers. This protocol was designed to aid in conducting investigations of adult crustacean neural cells in culture. We believe that an advantage of this method is the potential for adaptation to neural cells from other arthropods and even other groups of invertebrates.

Limnoperna fortuneipopularly known as the Golden musselis an aggressive invasive species that has been causing environmental damage and adversely affecting economic sectors dependent on freshwater ecosystems in South America. As a non-model species, knowledge about its biology is very limited, especially molecular mechanisms that contribute to its invasiveness, such as its high reproduction rate. Quantitative PCR (qPCR) is considered the gold standard technique to determine gene expression levels and its accuracy relies on the use of stably expressed reference genes for data normalization and to minimize technical variability. Our goal was to identify reliable reference genes to perform gene expression analysis on the gonads of L. fortunei. The stability of five candidate genes (RPS3, EF1ɑ, HS6ST3B, NAPA and UBE2F) in the gonads of male and female mussels was evaluated by using two algorithms, Bestkeeper and Genorm. Results show that NAPA, UBE2F and RPS3 genes are stable enough to compose a reliable normalization factor for gene expression analyses comparing both sexes. HS6ST3B and NAPA were found to be more stable in female gonads; thus, their application as a normalization factor is preferable for studies limited to female processes only.

Acellular liver scaffolds (ALS) produced by decellularization have been successfully explored for distinct regenerative purposes. To date, it is unknown whether transplanted ALSs are affected by cirrhotic livers, either becoming cirrhotic themselves or instead remaining as a robust template for healthy cell growth after transplantation into cirrhotic rats. Moreover, little is known about the clinical course of recipient cirrhotic livers after ALS transplantation. To address these questions, we transplanted ALSs into cirrhotic rats previously treated with the granulocyte colony-stimulating factor. Here, we report successful cellular engraftment within the transplanted ALSs at 7, 15, and 30 days after transplantation. Recellularization was orchestrated by liver tissue cell activation, resident hepatocytes and bile duct proliferation, and an immune response mediated by the granulocyte components. Furthermore, we showed that transplanted ALSs ensured a pro-regenerative and anti-inflammatory microenvironment, attracted vessels from the host cirrhotic tissue, and promoted progenitor cell recruitment. ALS transplantation induced cirrhotic liver regeneration and extracellular matrix remodeling. Moreover, the transplanted ALS sustained blood circulation and attenuated alterations in the ultrasonographic and biochemical parameters in cirrhotic rats. Taken together, our results confirm that transplanted ALSs are not affected by cirrhotic livers and remain a robust template for healthy cell growth and stimulated cirrhotic liver regeneration.

Background. Insufficient quantities of freshly harvested Limnoperna fortunei gametes and embryos constrain reproduction research in the laboratory. Cryopreservation would allow the accumulation and storage of gametes when they are available. The lack of available cryopreservation protocol for Limnoperna fortunei in the literature led our research group to undertake a study to establish which cryoprotective agents can be might be most useful for cryopreservation of this species’ sperm. Methods. 2%, 5%, and 10% concentration of ethylene glycol, dimethyl sulfoxide, glycerol, and polyethylene glycol 4000 as well as 0.2 M glucose, sucrose, and trehalose (mixed into the 10% concentration of the aforementioned agents) were tested in a 1:1 ratio with sperm, in 0.25 ml straws, frozen in liquid nitrogen. Results. After 48 hours the best survival rate was in the samples with 10% polyethylene glycol 4000, 36.1%, which also resulted in viable sperm after 7 and 15 days.