Immunohistochemical analysis of the distribution of molecules involved in ionic and pH regulation in the lancelet Branchiostoma floridae (Hubbs, 1922).

The aim of present work is to analyse the distribution of carbonic anhydrase II (CAII), cystic fibrosis transmembrane regulator (CFTR), vacuolar-type H+-ATPase (V-H+-ATPase), Na+/K+ ATPase, Na+/H+ exchanger (NHE) and SLC26A6 (solute carrier family 26, member 6), also known as pendrin protein, in the lancelet Branchiostoma floridae in order to go in depth in the evolution of osmoregulation and pH regulation in Chordates. In view of their phylogenetic position, lancelets may indeed provide a critical point of reference for studies on osmoregulation evolution in Chordates. The results of present work demonstrated that, except to Na+/K+ ATPase that is strongly expressed in nephridia only, all the other studied molecules are abundantly present in skin, coelomic epithelium, renal papillae and nephridia and hepatic coecum. Thus, it is possible to hypothesize that also in lancelet, as in fish, these organs are involved in pH control and ionic regulation. In the digestive tract of B. floridae, the intestine epithelium was weakly immune-reactive to all tested antibodies, while the hepatic coecum showed an intense immunoreactivity to all molecules. Since in amphioxus the hepatic coecum functions simultaneously as stomach, liver and pancreas, these immunohistochemical results proved the secretion of H+ and HCO3- ions, typical of digestive process. Colocalization studies indicated a co-expression of the studied proteins in all considered organs, excluding NHE and pendrin for renal papillae, since some renal papillae are NHE immunopositive only.

Fantastic animals as an experimental model to teach animal adaptation.

BACKGROUND: Science curricula and teachers should emphasize evolution in a manner commensurate with its importance as a unifying concept in science. The concept of adaptation represents a first step to understand the results of natural selection. We settled an experimental project of alternative didactic to improve knowledge of organism adaptation. Students were involved and stimulated in learning processes by creative activities. To set adaptation in a historic frame, fossil records as evidence of past life and evolution were considered. RESULTS: The experimental project is schematized in nine phases: review of previous knowledge; lesson on fossils; lesson on fantastic animals; planning an imaginary world; creation of an imaginary animal; revision of the imaginary animals; adaptations of real animals; adaptations of fossil animals; and public exposition. A rubric to evaluate the student's performances is reported. The project involved professors and students of the University of Modena and Reggio Emilia and of the "G. Marconi" Secondary School of First Degree (Modena, Italy). CONCLUSION: The educational objectives of the project are in line with the National Indications of the Italian Ministry of Public Instruction: knowledge of the characteristics of living beings, the meanings of the term "adaptation", the meaning of fossils, the definition of ecosystem, and the particularity of the different biomes. At the end of the project, students will be able to grasp particular adaptations of real organisms and to deduce information about the environment in which the organism evolved. This project allows students to review previous knowledge and to form their personalities.

The avian community of the Karen Mogensen Reserve, a wealth of biodiversity within the poorly investigated and threatened environments of northwestern Costa Rica.

Despite being characterized by some of the most threatened forest ecosystems of Mesoamerica, the Nicoya Peninsula is among the least known regions of neotropical Costa Rica in terms of its birdlife. Within this region, in the framework of an ongoing international cooperation program between Italy and Costa Rica, we had the opportunity to investigate the Karen Mogensen Reserve, a protected area distinguished by the presence of a variety of habitats, including tropical dry forest and moist forest. Species richness in the Reserve was relatively high compared with similar areas in northwestern Costa Rica. A series of surveys carried out over a 20-year period documented an avian community consisting of 207 species, of which 115 were breeding in the zone and another 14 were potentially breeding. We recorded five IUCN globally Vulnerable or Near-Threatened species, along with six species reported for the first time from the Nicoya Peninsula, each representing range extension of more than 100 km. Twenty-six species, mostly breeding in the area, are at their southernmost range borders, and are likely susceptible to global environmental alterations, such as the effects of climate change. Furthermore, our study revealed the presence of two species endemic to a restricted area of Central America and four subspecies endemic to Costa Rica, along with breeding populations of two species that are geographically isolated from the main ones. The present analysis led to the ecological characterization of the resident avian community, showing that 65% of the species are strictly associated with forested environments, and especially with the understory or middle tree level, hence more vulnerable to environmental change (climatic, anthropogenic, etc.) and susceptible to local extinction. These results underscore the importance of the Karen Mogensen Reserve for bird conservation within a vulnerable environmental context, and warrant the continuation of periodic bird surveys, taxonomic study of isolated populations or endemic taxa, and improvement of local conservation measures. The data collected will be an important tool for future studies aimed at evaluating the consequences of habitat fragmentation and to monitor the effects of climate change on the resident avifauna. We exhort the creation of programs that integrate bird monitoring, ecological research, conservation initiatives, and the involvement of the local communities, by promoting environmental education, capacity-building, and income generation. To this purpose, the Karen Mogensen Reserve may represent a convincing model and valuable example to apply in similar neotropical contexts.

The early stress responses in fish larvae.

During the life cycle of fish the larval stages are the most interesting and variable. Teleost larvae undergo a daily increase in adaptability and many organs differentiate and become active. These processes are concerted and require an early neuro-immune-endocrine integration. In larvae communication among the nervous, endocrine and immune systems utilizes several known signal molecule families which could be different from those of the adult fish. The immune-neuroendocrine system was studied in several fish species, among which in particular the sea bass (Dicentrarchus labrax), that is a species of great commercial interest, very important in aquaculture and thus highly studied. Indeed the immune system of this species is the best known among marine teleosts. In this review the data on main signal molecules of stress carried out on larvae of fish are considered and discussed. For sea bass active roles in the early immunological responses of some well-known molecules involved in the stress, such as ACTH, nitric oxide, CRF, HSP-70 and cortisol have been proposed. These molecules and/or their receptors are biologically active mainly in the gut before complete differentiation of gut-associated lymphoid tissue (GALT), probably acting in an autocrine/paracrine way. An intriguing idea emerges from all results of these researches; the molecules involved in stress responses, expressed in the adult cells of the hypothalamic-pituitary axis, during the larval life of fish are present in several other localizations, where they perform probably the same role. It may be hypothesized that the functions performed by hypothalamic-pituitary system are particularly important for the survival of the larva and therefore they comprises several other localizations of body. Indeed the larval stages of fish are very crucial phases that include many physiological changes and several possible stress both internal and environmental.

Expression of carbonic anhydrase, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase in the lancelet Branchiostoma lanceolatum (Pallas, 1774).

Sequencing of the amphioxus genome revealed that it contains a basic set of chordate genes involved in development and cell signaling. Despite the availability of genomic data, up till now no studies have been addressed on the comprehension of the amphioxus osmoregulation. Using primers designed on Branchiostoma floridae carbonic anhydrase (CA) II, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase, a 100bp long region, containing the protein region recognized by the respective antibodies, has been amplified and sequenced in B. lanceolatum indicating the presence of hortologous V-ATPase, CFTR and carbonic anhydrase II genes in Branchiostoma lanceolatum. Immunohistochemical results showed that all three transporting proteins are expressed in almost 90% of epithelial cells of the skin in B. lanceolatum adults with a different degree of positivity in different regions of body wall and with a different localization in the cells. The comparison of results between young and adult lancelets showed that the distribution of these transporters is quite different. Indeed, in the young specimens the expression pattern of all tested molecules appears concentrated at the gut level, whereas in adult the gut loses its key role that is mostly supported by skin.

Immunolocalization of corticotropin-releasing factor (CRF) and corticotropin-releasing factor receptor 2 (CRF-R2) in the developing gut of the sea bass (Dicentrarchus labrax L.).

Our previous data indicated an important role for adrenocorticotropic (ACTH)-like molecules co-operating with macrophages to control the modifications in body homeostasis during the first period of the life of sea bass (up to 30 days post-hatching) before the lymphoid cells have reached complete maturation. The aim of the study was to determine the immunolocalization of corticotropin-releasing factor (CRF), which is a very important mediator of stress-related responses. Our data showed that immunostaining for CRF is localized already at 8 days after hatching in nerve fibers of the gastrointestinal tract wall from the pharynx to the anterior gut, when the larvae are still feeding on yolk. This pattern of immunolocalization appeared similar to that in 24-day-old larvae, but at this stage there were also large cells immunopositive to CRF located in the wall of the midgut and hindgut. Lipopolysaccharide (LPS) treatment, which is a known stimulator of stress hormone responses, did not modify the CRF immunostaining pattern, though it did affect the immunolocalization of the peripheral CRF receptor, i.e. CRF-R2. Immunolocalization of CRF-R2 appeared in nerve fibers of the gut wall in larvae fixed 1h after the end of lipopolysaccharide (LPS) treatment. The present results suggest that CRF plays important autocrine and/or paracrine roles in the early immune responses at the gut level in the larval stages of sea bass (Dicentrarchus labrax L.) as already proposed for ACTH. Moreover, our studies taken together with other research on fish, in comparison with mammals, suggest a phylogenetically old role of CRF in immune-endocrine interactions.

ACTH response to LPS in the first stages of development of the fish Dicentrarchus labrax L.

ACTH and ACTH receptor-like molecules were found at the examined stages of development (2, 4, 8, 12, 18, and 24 days post-hatching) in yolk sac, pronephros tubules, interrenal tissue, thymus, liver, spleen, cardinal veins, and skin of the teleost fish Dicentrarchus labrax. ACTH and the related receptor-like molecules show a similar distribution. LPS treatment at two different stages (8 and 24 days post-hatching) provoked both a release and an induction of ACTH-like molecules, suggesting an important role of this peptide to control the modifications in body homeostasis during the first period of the sea bass' life, i.e., 30 days post-hatching, before the lymphoid cells have reached complete maturation.

Xanthophore migration from the dermis to the epidermis and dermal remodeling during Salamandra salamandra salamandra (L.) larval development.

During larval development of Salamandra salamandra salamandra chromatophores organize to form the definitive pigment pattern constituted by a black background with yellow patches that are characterized by epidermal xanthophores and dermal iridophores. Simultaneously the dermis undergoes remodeling from the larval stage to that typical of the adult. In the present study we ultrastucturally and immunocytochemically examined skin fragments of S. s. salamandra larvae and juveniles in order to investigate the modalities of xanthophore migration and differentiation in the context of dermal remodeling from the larval to adult stage. Semithin and thin sections showed that the dermis in newly born larvae consists of a compact connective tissue (basement lamella), to which fibroblasts and xanthophores adhere, and of a loose deep collagen layer. As larval development proceeds, fibroblasts and xanthophores invade the basement lamella, skin glands develop and the adult dermis forms. At metamorphosis, xanthophores reach the epidermis crossing through the basal lamina. We examined immunocytochemically the expression of signal molecules, such as fibronectin, vitronectin, beta1-integrin, chondroitin sulfate, E-cadherin, N-cadherin and plasminogen activator, which are known to be involved in regulating morphogenetic events. Their role in dermal remodeling and in pigment pattern formation is discussed.

Occurrence of ACTH- and enkephalin-like peptides in the developing gut of Dicentrarchus labrax L.

We carried out immunohistochemical tests in the developing gut of the sea bass Dicentrarchus labrax to follow the appearance and distribution of the immunoreactivity (IR) to antibodies against POMC-derived, adrenocorticotropic hormone (ACTH), alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin (beta-End), and against two enkephalins, with the aim to study a possible involvement of these molecules in the early neuro-immune-endocrine integration. Our data show that IR to antibodies against some molecules involved in the stress response, such as ACTH and enkephalins, are present in the sea bass gut from an early larval stage (4 days after hatching), before transition to the exotrophic feeding. Moreover, the present study demonstrates for the first time the presence of ACTH-like immunoreactive material in developing gut of a fish. The possible roles of tested molecules are discussed.

Structure-function relationships in the integument of Salamandra salamandra during ontogenetic development.

Morphological, cytological and transport properties of the integument of Salamandra salamandra were investigated during natural ontogenetic development, from birth to adult. Three stages were operationally defined: I, larvae, from birth to metamorphosis; II, metamorphosis (judged externally by the colour change and loss of the gills); and III, post-metamorphosis to adult. Pieces of skin were fixed at various stages for immunocytochemical examinations, and the electrical properties were investigated on parallel pieces. Distinct cellular changes take place in the skin during metamorphosis, and lectin (PNA, WGA and ConA) binding indicates profound changes in glycoprotein composition of cell membranes, following metamorphosis. Band 3 and carbonic anhydrase I (CA I) were confined to mitochondria-rich (MR)-like cells, and were detected only in the larval stage. CA II on the other hand, was detected both in MR-like and in MR cells following metamorphosis. The electrical studies show that the skin becomes more tight (transepithelial resistance increases) upon metamorphosis, followed by manifestation of amiloride-sensitive short-circuit current (I(SC)) indicating that functional Na+ uptake has been acquired. The skin of metamorphosed adults had no finite transepithelial Cl- conductance, and band 3 was not detected in its MR cells. The functional properties of MR-like and MR cells remain to be established.