This study aims to assess the effects induced by 24 hr exposure to a subtoxic copper concentration on the reproductive system (gonads, spermatozoa, and protamine-like[PL] proteins) of Mytilus galloprovincialis. Inductively coupled plasma-mass spectrometry indicated accumulation of this metal in gonads, spermatozoa, and PL proteins of exposed mussels. Further, real-time polymerase chain reaction analyses showed altered expression levels of mt10 and PL proteins genes in spermatozoa and gonads, respectively, of exposed mussels. Protamine-like proteins, which represent the main basic component of sperm chromatin of this organism, showed a higher DNA binding affinity and a different DNA binding mode in exposed mussels. Moreover, an increased amount of NaCl was required for the release from sperm nuclei of PL-III, the main PL protein component. Finally, PL proteins extracted from exposed mussels promoted DNA oxidative damage in the presence of H 2 O 2. These results demonstrate that the tolerable copper amount could also affect the properties of PL proteins and determine the negative effects on the reproductive system of this organism. These analyses could be useful to develop quick and efficient chromatin-based genotoxicity tests for pollution biomonitoring programs.

We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.

The development of the mouth in animals has fascinated researchers for decades, and a recent study proposed the modern view of recurrent evolution of protostomy and deuterostomy. Here we expanded our knowledge about conserved traits of mouth formation in chordates, testing the hypothesis that nitric oxide (NO) is a potential regulator of this process. In the present work we show for the first time that NO is an essential cell signaling molecule for cephalochordate mouth formation, as previously shown for vertebrates, indicating its conserved ancestral role in chordates. The experimental decrease of NO during early amphioxus Branchiostoma lanceolatum development impaired the formation of the mouth and gill slits, demonstrating that it is a prerequisite in pharyngeal morphogenesis. Our results represent the first step in the understanding of NO physiology in non-vertebrate chordates, opening new evolutionary perspectives into the ancestral importance of NO homeostasis and acquisition of novel biological roles during evolution.

Molecular research on the evolution of extraocular photoreception has drawn attention to photosensitive animals lacking proper eye organs. Outside of vertebrates, little is known about this type of sensory system in any other deuterostome. In this study, we investigate such an extraocular photoreceptor cell (PRC) system in developmental stages of the sea urchin Paracentrotus lividus. We provide a general overview of the cell type families present at the mature rudiment stage using single-cell transcriptomics, while emphasizing the PRCs complexity. We show that three neuronal and one muscle-like PRC type families express retinal genes prior to metamorphosis. Two of the three neuronal PRC type families express a rhabdomeric opsin as well as an echinoderm-specific opsin (echinopsin), and their genetic wiring includes sea urchin orthologs of key retinal genes such as hlf, pp2ab56e, barh, otx, ac/sc, brn3, six1/2, pax6, six3, neuroD, irxA, isl and ato. Using qPCR, in situ hybridization, and immunohistochemical analysis, we found that the expressed retinal gene composition becomes more complex from mature rudiment to juvenile stage. The majority of retinal genes are expressed dominantly in the animals’ podia, and in addition to the genes already expressed in the mature rudiment, the juvenile podia express a ciliary opsin, another echinopsin, and two Go-opsins. The expression of a core of vertebrate retinal gene orthologs indicates that sea urchins have an evolutionarily conserved gene regulatory toolkit that controls photoreceptor specification and function, and that their podia are photosensory organs.