Identification of a cryptic functional apolipophorin-III domain within the Prominin-1 gene of Litopenaeus vannamei.

The Apolipophorin-III (apoLp-III) is reported as an essential protein element in lipids transport and incorporation in lepidopterans. Structurally, apoLp-III has an α-helix bundle structure composed of five α-helices. Interestingly, classic studies proposed a structural switch triggered by its interaction with lipids, where the α-helix bundle opens. Currently, the study of the apoLp-III has been limited to insects, with no homologs identified in other arthropods. By implementing a structure-based search with the Phyre2 algorithm surveying the shrimp Litopenaeus vannamei's transcriptome, we identified a putative apoLp-III in this farmed penaeid (LvApoLp-III). Unlike canonical apoLp-III, the LvApoLp-III was identified as an internal domain within the transmembrane protein Prominin-1. Structural modeling using the template-based Phyre2 and template-free AlphaFold algorithms rendered two distinct structural topologies: the α-helix bundle and a coiled-coil structure. Notably, the secondary structure composition on both models was alike, with differences in the orientation and distribution of the α-helices and hydrophobic moieties. Both models provide insights into the classical structural switch induced by lipids in apoLp-III. To corroborate structure/function inferences, we cloned the synthetic LvApoLp-III domain, overexpressed, and purified the recombinant protein. Circular dichroism measurements with the recombinant LvApoLp-III agreed with the structural models. In vitro liposome interaction demonstrated that the apoLp-III domain within the PROM1 of L.vannamei associated similarly to exchangeable apolipoproteins. Altogether, this work reports the presence of an apolipophorin-III domain in crustaceans for the first time and opens questions regarding its function and importance in lipid metabolism or the immune system.

Distribution of PAHs and trace elements in Spartina densiflora and associated sediments from low to highly contaminated South American estuarine saltmarshes.

Estuarine saltmarshes from South America are exposed to several anthropogenic impacts due to diverse human activities that occur in both Atlantic/Pacific coastal environments. Primarily, chemical and petrochemical industries negatively impact saltmarshes generating inputs/deposition of non-essential trace elements (NTEs) and polycyclic aromatic hydrocarbons (PAHs) in sediments. The native cordgrass Spartina densiflora inhabits a wide range of environments, from non-impacted to highly impacted areas. It is important to know its performance towards pollution in different environmental settings in South America. The content of Cd, Hg, Pb, and PAHs was determined in the roots and leaves of S. densiflora, bulk sediments (Bs), and rhizosediments (Rs) of estuaries from Argentina, Brazil, and Chile. Differences in NTEs and PAHs levels were observed between Bs, Rs, and Spartina tissues from different saltmarsh areas. Differences in Rs/Bs (RHICF; rhizosediments concentration factors), roots/Bs (RCF; roots concentration factors) and leaves/roots (TF; translocation factors) factors were also found. In terms of NTEs, S. densiflora showed a high capability to increase levels in their Rs (RHICF>1) and bioconcentrate Cd in roots (RCF > 1), while no general translocation (TF < 1) was observed. Conversely, in cordgrass tissues, Bs and Rs, PAHs contents showed RCF and TF > 1, which was in line with lower levels in Rs related to Bs (RHICF<1) in most sites. These findings showed the S. densiflora capacity to retain, remove and/or translocate priority contaminants depending on intrinsic chemical characteristics and the level of contamination. The present study enables future considerations regarding the biomonitoring and phytoremediation/stabilization capabilities of Spartina in coastal environments.

Selection and optimization of reference genes for RT-qPCR normalization: A case study in Solanum lycopersicum exposed to UV-B.

Quantitative RT- PCR is one of the most common methods to study gene expression in response to stress. Therefore, it is crucial to have suitable reference genes (RGs) for result normalization. Although several reports describe UV-B-modulated gene expression in Solanum lycopersicum, there are no suitable RGs identified until now. The aim of this work was to evaluate the suitability of seven traditional genes: actin (ACT), tubulin (TUB), ubiquitin (UBI), glyceraldehyde- 3 phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), phosphatase 2A catalytic subunit (PP2A) and GAGA binding transcriptional activator (GAGA); and two non-traditional genes: thioredoxin h1 (TRX h1) and UV-B RESISTANCE LOCUS 8 (UVR8), as candidate RGs for their potential use as reliable internal controls in leaves, stems and roots of tomato seedlings exposed to acute and chronic UV-B. The stability of these genes expression was evaluated using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta Ct and ANOVA. Considering the comprehensive stability ranking, we recommend ACT+TUB as the best pair of RGs for leaves, PP2A+GAPDH+TRX h1 for stems and TUB+UVR8 for roots. The reliability of the selected RGs for each tissue was verified amplifying tomato chalcone synthase 1 (CHS1) and cyclobutane pyrimidine dimer (CPD) photolyase (PHR1-LIKE). Under UV-B treatment, CHS1 was upregulated in leaves, stems and roots whereas PHR1-LIKE was only upregulated in leaves and stems. This interpretation differs when the most and least stable RGs are chosen. This is the first report regarding suitable RGs selection for accurate normalization of gene expression in tomato seedlings exposed to UV-B irradiation.

Imidacloprid modifies the mitotic kinetics and causes both aneugenic and clastogenic effects in the macrophyte Bidens laevis L.

Imidacloprid (IMI) is a neonicotinoid insecticide widely used in agricultural activities all around the world. This compound is transported from croplands to surrounding freshwater ecosystems, producing adverse effects on non-target organisms. Because of the relevance of aquatic macrophytes in the above-mentioned environments and the lack of studies of potential effects of IMI on them, this work aimed to assess the mitotic process and potential genotoxicity in the aquatic macrophyte Bidens laevis L. Although the analysis of the Mitotic Index (MI) showed that IMI was not cytotoxic, the Cell Proliferation Kinetics (CPK) frequencies evidenced modifications in the kinetics of the mitotic process. Indeed, the anaphases ratio decreased at 10 and 100 µg/L IMI, while at 1000 µg/L an increase of prophases ratio and a decrease of metaphases ratio were observed. Regarding genotoxicity, IMI produced an increase of the abnormal metaphases frequency from 10 µg/L to 1000 µg/L as well as an increase in clastogenic anaphases-telophases frequency at 100 and 1000 µg/L. In addition, aneugenic anaphases-telophases and C-mitosis frequencies also increased at 1000 µg/L, confirming the effects on the mitotic spindle. Considering the genotoxic effects on B. laevis through two different mechanisms (aneugenic and clastogenic) and the wide spread use of IMI in agriculture, these mechanisms of toxicity on macrophytes should be considered among other recognized effects of this insecticide on aquatic biota.

Selection of reference genes for reverse transcription-qPCR analysis in the biomonitor macrophyte Bidens laevis L.

The RT-qPCR has been the method used to analyze gene expression in plants but its benefits have not been completely exploited in the field of plants ecotoxicology when used as molecular biomarkers. The correct use of RT-qPCR demands to establish a certain number of reference genes (RG) which are expected to be invariable in their expression although it does not always happen. The main goals of this work were to: (1) analyze the stability of six potential RG, (2) establish the optimum number of RG, (3) select the most suitable RG to be applied in Bidens laevis under different test conditions and tissues and (4) confirm its convenience by normalizing the expression of one gene of interest under three different challenges. When all data were pooled together, the geNorm algorithm pointed out beta-actin and beta-tubulin (TUB) as the optimal RG pair while NormFinder algorithm selected nicotinamide adenine dinucleotide dehydrogenase (NADHD) and histone 3 (H3) as possessing the most invariable levels of expression. On the other hand, when data were grouped by tissues, ANOVA test selected H3 and TUB, while data grouped by conditions indicated that H3 and NADHD were the most stable RG under this analysis. Therefore, for a general-purpose set of RG, the overall analysis showed that a set of three RG would be optimum, and H3, TUB and NADHD were the selected ones. On the other hand, as RG can vary depending on the tissues or conditions, results achieved with ANOVA would be more reliable. Thus, appropriate normalization process would clearly need more than one RG.

De novo assembly and transcriptome characterization of the freshwater prawn Palaemonetes argentinus: Implications for a detoxification response.

Palaemonetes argentinus, an abundant freshwater prawn species in the northern and central region of Argentina, has been used as a bioindicator of environmental pollutants as it displays a very high sensitivity to pollutants exposure. Despite their extraordinary ecological relevance, a lack of genomic information has hindered a more thorough understanding of the molecular mechanisms potentially involved in detoxification processes of this species. Thus, transcriptomic profiling studies represent a promising approach to overcome the limitations imposed by the lack of extensive genomic resources for P. argentinus, and may improve the understanding of its physiological and molecular response triggered by pollutants. This work represents the first comprehensive transcriptome-based characterization of the non-model species P. argentinus to generate functional genomic annotations and provides valuable resources for future genetic studies. Trinity de novo assembly consisted of 24,738 transcripts with high representation of detoxification (phase I and II), anti-oxidation, osmoregulation pathways and DNA replication and bioenergetics. This crustacean transcriptome provides valuable molecular information about detoxification and biochemical processes that could be applied as biomarkers in further ecotoxicology studies.

Genetic and biochemical biomarkers in the macrophyte Bidens laevis L. exposed to a commercial formulation of endosulfan.

Previous studies in the wetland macrophyte Bidens laevis L have demonstrated that the insecticide endosulfan induces a high frequency of somatic chromosome aberrations in anaphase-telophase (CAAT) but no DNA changes as determined by the single cell gel electrophoresis (Comet) assay. Thus, cytogenetic biomarkers appear to be more sensitive to the toxic effects of the insecticide than the DNA molecule in the studied species. For this reason, the goals of this study were to use cytogenetic biomarkers--CAAT and abnormal metaphase--and defense biomarkers such as the activity of the antioxidant enzymes--guaiacol peroxidases (POD), glutathione reductase, and microsomal and cytosolic (m- and c-) glutathione-S-transferase (GST)--to evaluate in B. laevis effects caused by a commercial formulation of endosulfan. The frequency of CAAT was increased at 5, 10, 50, and 100 µg/L endosulfan with respect to the negative controls by 3.1, 2.5, 2.5, and 3.2-fold, respectively while the frequency of abnormal metaphases was also increased at the same concentrations by 3.5, 2.8, 3.2, and 11.3-fold, respectively. In addition to these aneugenic effects, other abnormalities such as C-mitosis and chromosome clumping were observed at 10 µg/L endosulfan. On the other hand, POD induction at 0.02, 0.5, 5, and 10 µg/L and m-GST inhibition at 0.5, 10, and 50 µg/L in plants exposed during 24 h to endosulfan were observed but all of these responses were highly variable. In conclusion, only cytogenetic biomarkers like CAAT in B. laevis can serve potentially as early warning systems to detect environmentally relevant concentrations of endosulfan in aquatic ecosystems.