In mammals, the relationship between the immune system and behavior is widely studied. In fish, however, the knowledge concerning the brain immune response and behavioral changes during brain viral infection is very limited. To further investigate this subject, we used the model of tilapia lake virus (TiLV) infection of zebrafish (Danio rerio), which was previously developed in our laboratory. We demonstrated that TiLV persists in the brain of adult zebrafish for at least 90 days, even when the virus is not detectable in other peripheral organs. The virions were found in the whole brain. During TiLV infection, zebrafish displayed a clear sickness behavior: decreased locomotor activity, reduced food intake, and primarily localizes near the bottom zone of aquaria. Moreover, during swimming, individual fish exhibited also unusual spiral movement patterns. Gene expression study revealed that TiLV induces in the brain of adult fish strong antiviral and inflammatory response and upregulates expression of genes encoding microglia/macrophage markers. Finally, using zebrafish larvae, we showed that TiLV infection induces histopathological abnormalities in the brain and causes activation of the microglia which is manifested by changes in cell shape from a resting ramified state in mock-infected to a highly ameboid active state in TiLV-infected larvae. This is the first study presenting a comprehensive analysis of the brain immune response associated with microglia activation and subsequent sickness behavior during systemic viral infection in zebrafish.

This article presents the scanning and transmission electron microscopy of the spermatozoa and sperm packets of three inseminating species of the glandulocaudine tribe Xenurobryconini. All three species, Scopaeocharax rhinodus, Tyttocharax cochui, and T. tambopatensis produce unencapsulated sperm packets (= spermatozeugmata) of similar morphology. The external anterior surface of each spermatozeugma is comprised of elongate sperm heads arranged in parallel, and the posterior part is made up of tightly packed flagella. The interior of the anterior portion consists of alternating layers of sperm heads and flagella. The remarkable integrity of each packet appears to be maintained through an electron-dense secretion seen among all parts of the cells. Spermatozeugma formation takes place within the spermatocysts at the end of spermiogenesis and at spermiation fully formed packets are released. Morphology of the mature spermatozoa was similar in all three species. Each nucleus is elongate, flattened along most of its length, and tapers at either end. The two centrioles are nearly parallel to one another and are located just anterior to the nucleus. Elongate mitochondria are located along the nucleus. The single flagellum, which lacks axonemal fins, is initially contained within a short cytoplasmic collar. Accessory microtubules run parallel to the long axis of the nucleus just beneath the plasma membrane. During spermiogenesis, no nuclear rotation occurs and the cytoplasmic canal containing the flagellum elongates along with the nucleus. However, prior to spermiation all but the anterior portion of the collar degenerates. The sperm modifications observed in these species are discussed as adaptations to the unique reproductive habit of insemination.

The aim of this study was to investigate consecutive stages of gonadal development of the yellow-bellied toad (Bombina variegata) with particular emphasis on the origin of somatic and germ cell lineages as well as the timing of gonial cell migration. Changes in gonadal basal lamina distribution helped to explain the exceptional mode of gonadal differentiation in this species. Atypical and rapid differentiation of the male gonad in B. variegata is the result of the ability of gonial cells to migrate into the center of the gonad relatively early. Thus, the testis medulla contains germ cells from the onset of gonadal differentiation into cortex and medulla, whereas in other anurans a sterile medulla is characteristic of both future testes and ovaries; germ cells translocate into the medulla during the subsequent stage of testis development. This atypical testiculogenesis is probably the result of an acceleration of the sex determination period, indicating a contribution of sex determination heterochrony to the course of gonadogenesis. The results also suggest that medullar cells are derived from proliferating coelomic epithelial cells. Moreover, Sertoli cells constitute an integral part of the germinal epithelium in B. variegata, as in other vertebrates. Spermatids do not contact Sertoli cells just before spermiation and do not form bundles.

The vitamin A (retinol) and its metabolites such as retinoic acid (RA) affect vertebrate gametogenesis. The level of RA in cells relies on the balance between its synthesis and degradation. The sex-dependent equilibrium is reached in different ways in various species. It is known that RA induces meiosis in developing gonads in mouse, chicken and urodel amphibians, but its role in anuran amphibians has not been studied. Here we show in six anuran species (Xenopus laevis, Bombina bombina, Hyla arborea, Bufo viridis, Rana arvalis and Rana temporaria) that cultured undifferentiated gonads were insensitive to RA treatment, but the RA induced ectopic meiosis in cultured larval testes. In larval testes of all studied species, the exogenous RA induced leptotene phase of I meiotic prophase in gonia, but only in H. arborea and B. viridis gonia progressed to zygotene phase. In the cultured developing ovaries, exogenous RA led to increase in the number of oocytes as compared to the control. Inhibition of either RA synthesis or RA-receptors prevented meiotic entry in larval gonads of all species. Exogenous RA rescued this inhibitory effect demonstrating that the balance in RA homeostasis plays a key role in meiotic entry in anuran gonads. The localization of two enzymes, Raldh2 and Cyp26b1, which antagonistically control RA levels and whose abundance suggests the sites of RA synthesis and degradation respectively, showed two distinct expression patterns specific for (i) X. laevis, H. arborea, R. arvalis, R. temporaria and (ii) B. bombina, B. viridis. Thus, RA, in correlation with specific expression patterns of Raldh2 and Cyp26b, induces meiosis during gonad development in anurans. In addition, in B. viridis, RA signalling seems important for development of the Bidder's organ containing oocytes both in males and females.

Additional Supporting Information may be found in the online version of this article: Figure S1. Peptide-blocking of anti-OT antibody staining. Figure S2. Structure of prepro-OT mRNA. Figure S3. Quantitative PCR analysis of OT mRNA level in keratinocytes. University, ul. Gronostajowa 7, Poland, Abstract: Pheomelanin is supposed to be the first type of melanin found in vertebrates, in contrast to the main typeeumelanin. Our study aimed at detecting pheomelanin in the skin of Hymenochirus boettgerii. We employed electron paramagnetic resonance (EPR) spectroscopy, and transmission electron microscopy (TEM), supplemented with standard histology and immunochemistry. We identified pheomelanin in the dorsal skin of adult frogs (not only in the dermis, but also in the epidermis) and in the dorsal tadpole. Our work identifies Hymenochirus boettgerii as a model in the basic study on the mechanism, evolution and role of melanogenesis in animals, including human.