Gilberto Grandi

Transcriptome sequencing and de novo annotation of the critically endangered Adriatic sturgeon

BackgroundSturgeons are a group of Condrostean fish with very high evolutionary, economical and conservation interest. The eggs of these living fossils represent one of the most high prized foods of animal origin. The intense fishing pressure on wild stocks to harvest caviar has caused in the last decades a dramatic decline of their distribution and abundance leading the International Union for Conservation of Nature to list them as the more endangered group of species. As a direct consequence, world-wide efforts have been made to develop sturgeon aquaculture programmes for caviar production. In this context, the characterization of the genes involved in sex determination could provide relevant information for the selective farming of the more profitable females.ResultsThe 454 sequencing of two cDNA libraries from the gonads and brain of one male and one female full-sib A. naccarii, yielded 182,066 and 167,776 reads respectively, which, after strict quality control, were iterative assembled into more than 55,000 high quality ESTs. The average per-base coverage reached by assembling the two libraries was 4X. The multi-step annotation process resulted in 16% successfully annotated sequences with GO terms. We screened the transcriptome for 32 sex-related genes and highlighted 7 genes that are potentially specifically expressed, 5 in male and 2 in females, at the first life stage at which sex is histologically identifiable. In addition we identified 21,791 putative EST-linked SNPs and 5,295 SSRs.ConclusionsThis study represents the first large massive release of sturgeon transcriptome information that we organized into the public database AnaccariiBase, which is freely available at http://compgen.bio.unipd.it/anaccariibase/. This transcriptomic data represents an important source of information for further studies on sturgeon species. The hundreds of putative EST-linked molecular makers discovered in this study will be invaluable for sturgeon reintroduction and breeding programs.

Histological and ultrastructural investigation of early gonad development and sex differentiation in Adriatic sturgeon (Acipenser naccarii, Acipenseriformes, Chondrostei)

Gonad development and sex differentiation from embryos to 594‐day‐old individuals were investigated in farmed Acipenser naccarii using light and transmission electron microscopy. The migrating primordial germ cells first appear along the dorsal wall of the body cavity in embryos 1.5 days before hatching. The gonadal ridge, containing a few primary primordial germ cells (PGC‐1) surrounded by enveloping cells, appears in 16‐day‐old larvae. At 60 days, the undifferentiated gonad is lamellar and PGC‐1 multiply, producing PGC‐2. In 105‐day‐old juveniles, a distinct germinal area with advanced PGC‐2 appears on the lateral side near the mesogonium and the first blood vessels are visible. At 180 days, putative ovaries with a notched gonadal epithelium and putative testes with a smooth one appear, together with adipose tissue on the distal side. In 210‐day‐old juveniles, active proliferation of germ cells begins in the putative ovaries, whereas putative testes still contain only a few germ cells. The onset of meiosis and reorganization of stromal tissue occurs in ovaries of 292‐day‐old individuals. Ovaries with developed lamellae enclosing early oocyte clusters and follicles with perinucleolar oocytes occur at 594 days. Meiotic stages are never found, even in anastomozing tubular testes of 594‐day‐old individuals. Steroid producing cells are detected in the undifferentiated gonad and in the differentiated ones of both sexes. Anatomical differentiation of the gonad precedes cytological differentiation and female differentiation largely precedes that of the male. Gonad development and differentiation are also associated with structural changes of connective tissue, viz. collagen‐rich areas are massive in developing testes and reduced in ovaries. J. Morphol., 2008.

Testis cytological structure, plasma sex steroids, and gonad cytosol free steroid receptors of heterologous gonadotropin (hCG)-stimulated silver eel, Anguilla anguilla L

Complete testicular maturation was induced in silver eels, kept at 24 degrees in fresh water, by a single injection of 1000 I.U. of heterologous gonadotropin (hCG). Each week, for 4 weeks, some eels were examined for testis structural pattern, plasma sex steroids, and gonad cytosol steroid receptors. The first effect of the hCG was on the tubular organization of the testis, followed by spermatogenesis. Plasma androgens were not detectable in the untreated eels, whereas a peak was detected a week after in those treated with the injection and afterward a decline. Plasma progesterone and estradiol showed a peak 2 weeks after treatment. Untreated eel gonads showed a high content of cytosolic free estradiol receptors which disappeared in the hCG-treated ones, a peak of free progesterone receptors was found 1 week after injection. The results are discussed in relation to the differentiation and maturation of eels testes.

Immunocytochemical studies on the pituitary gland of Anguilla anguilla L., in relation to early growth stages and diet-induced sex differentiation

Mammalian and teleost antisera against pituitary hormones were used to identify and localize pituitary cell types in the European eel (Anguilla anguilla L.). The investigation was conducted on unpigmented glass eels of 5.6-6.2 of total body length (L(T)) caught in river mouths, then on yellow eels reared from the pigmented glass eel (or elver) stage up to 12-14 cm of L(T), in an eel farm in warm freshwater. Treated elvers were fed with commercial paste food supplemented with mature carp ovaries, containing oestradiol, that induced an early ovarian differentiation and a higher growth rate. The antisera detected seven types of immunoreactive (ir) cells, six of which were already found in glass eel adenohypophysis, suggesting differentiation of these cell types during the leptocephalus stage. In 12-14 cm treated yellow eels with small ovaries, a seventh type (ir-GtH) was detected in the proximal pars distalis; in the same animals the ir-TSH cells increased in number and size. From unpigmented glass eels to 12-14 cm yellow eels, the whole pituitary volume of controls increased nearly four times, while that of treated ones increased nearly six times. The larger volume of pituitary in treated eels was mainly due to volume increase of proximal pars distalis and rostral pars distalis. The %GH, that is the potential index of GH production, was significantly higher in treated yellow eels with gonads differentiating into ovaries than in controls; no difference was detected in %PRL between treated and control eels. The above results strongly suggest that in eels the feminizing effects of oestrogen is first exerted on the pituitary, probably through the hypothalamus, and later on the gonads.

Contamination, parasitism and condition of Anguilla anguilla in three Italian stocks

In conjunction with habitat loss and overfishing, pollution and parasitism are believed to be relevant causes of collapse of Anguilla, as these can affect eel swimming ability and the development of gonads and embryos. The present study investigated Persistent Organic Pollutant (POP) concentrations, infection levels of Anguillicoloides crassus, lipid content and gonad abnormalities in eels sampled in 2007–2008 in three Italian water bodies (Caprolace Lake, Lesina Lagoon and Tevere River) that vary in salinity, trophic condition, contamination level and fishing pressure. Our analysis revealed that low-to-moderate levels of contamination and parasitism were not associated with gonad abnormalities in Caprolace Lake and Lesina Lagoon. On the contrary, POP concentrations and abundances of swim bladder nematodes were remarkably high in eels from the heavily urbanized Tevere River and were associated with significant gonad and swim bladder alterations. Contamination and infestation levels were so high to potentially impair spawner successful migration and reproduction. POP concentrations in Tevere eels also exceeded levels considered safe for food consumption. Though marginally contaminated, eels from the oligotrophic Caprolace Lake were in critical health condition: their lipid reserve was so low as to be considered insufficient to sustain the energetic costs of the transoceanic migration. Lesina eel stock was the only one displaying relatively good quality but here spawner abundance is likely limited by overfishing. Our results suggest that multiple stressors may potentially affect eel reproductive success. More definitive studies are needed to assess whether health effects caused by these multiple stressors are additive, compensatory or synergistic.

Oogenesis in Kalotermes flavicollis (Fabr.) (Isoptera: Kalotermitidae). I. Differentiation and maturation of oocytes in female supplementary reproductives

Abstract The ovary of female supplementary reproductives is constituted by panoistic‐type ovarioles containing a linear array of germ cells in successive stages of differentiation. In the distal region of the germarium, germ cells cannot be morphologically and ultrastructu‐rally discriminated from somatic cells. In the middle and proximal region of the germarium, oogonia at two developmental stages have been identified. The oocytes at the beginning of meiosis are characterized by nuclear vacuoles and by a polar localization of cell organelles, in addition to synaptonemal complexes. The post‐pachytcne oocytes are partially isolated by prefollicular cell extensions and show annulate lamellae and Balbiani bodies. During previtellogenesis, the oolemma forms microvilli, with coated pits and vesicles, about 60 nm in diameter, at their bases. At the onset of vitellogenesis, coated pits and vesicles about 120 nm in diameter and early yolk globules appear in the cortical ooplasm. At a later stage, wide spaces fill...

Probing behaviour of Cacopsylla pyri on a resistant pear selection

European pear psylla Cacopsylla pyri L. (Hemiptera Psyllidae) is one of the worst pests of pear (Pyrus communis L.) in Europe. We investigated probing behaviour in adults and nymphs of C. pyri by full EPG on a psylla‐resistant pear selection, NY 10353. Concerning stylet probing behaviour on the plant surface, the results showed no significant differences between the resistant selection and the susceptible cultivar Bartlett, and no differences were also detected for epidermis and mesophyll resistance in the same conditions. For mesophyll/phloem, no differences were found in adults. However, in nymphs, weak resistance factors (longer stylet penetration and mesophyll salivation) were detected in the resistant selection. In phloem, EPG data indicate strong resistance factors in NY 10353, especially for nymphs and summer‐form adults (longer time before the first phloem ingestion and a lower duration of each phloem ingestion event). No prolonged (>10 min) phloem ingestion was performed by nymphs and adults in the resistant selection. The results support the hypothesis that NY 10353 resistance factors are located in the phloem sap and cause high C. pyri nymph mortality: this could be useful as a basis for further investigations of resistance mechanisms at the metabolic, chemical and genetic levels.

Ultrastructural study of testis development and spermatogenesis in Kalotermes flavicollis (Fabr.) (Isoptera, Kalotermitidae)

Abstract Testes of larvae, nymphs, young winged imagoes and primary reproductives of Kalotermes flavicollis (Fabr.) were examined by light and electron microscopy. They are composed of 7–8 lobes enveloped by a tri‐layered wall made up of: i) an outer monolayered cellular sheath above an acellular lamina; ii) a lacuna in which plasmatocytes and granulocytes are found; iii) a testis tunica propria. In the 3rd and 4th larval instar it is not yet possible to discriminate the somatic cells from the germ ones. In the 5th‐ instar larvae, lobes exhibit the typical organization of insect testis follicles with four, linearly arranged regions. In the germarium a group of apical cells, in close connection to predefinitive primary spermatogonia, is present. In 5th instar larvae, spermatogenesis is completed and, in the testis terminal chamber, sperms are always found. In nymphs, young winged imagoes, and primary reproductives, all regions of testis follicles appear progressively larger than 5th instar ones and cysts o...

Oogenesis in Kalotermes flavicollis (Fabr.) (Isoptera, Kalotermitidae) III. Choriogenesis and corpus luteum formation in female supplementary reproductives

Abstract Follicular cell ultrastructure during chorion and corpus luteum formation was studied in supplementary reproductives of the termite Kalotermes flavicollis. During choriogenesis the follicular epithelium is formed by large flattened cells joined together by specialized junctions. Follicular cells have an extended rough endoplasmic reticulum (RER) with cisternae parallel to the cell long axis and many Golgi complexes surrounded by secretion vesicles moving towards the egg surface. At the end of choriogenesis the follicular cells move away from the just completed chorion and show few RER cisternae and scarce Golgi apparati. When the egg is ejected from the ovariole, the empty ovarian follicle collapses; the irregularly shaped follicular cells, now exhibiting in their cytoplasm small autophagic vacuoles and polymembranous structures, form the initial corpus luteum. This undergoes a further shrinking, becoming an ovoidal mass (the advanced corpus luteum) formed by irregularly shaped follicular cells c...

Oogenesis in Kalotermes flavicollis (Fabr.) (Isoptera: Kalotermitidae). II‐prefollicular and follicular cell ultrastructure during oogenesis in female supplementary reproductives

Abstract Ovariole somatic cells were examined by light and electron microscopy. Prefollicular, follicular and interfollicular cells were identified. Prefollicular cells envelop oogonia and oocytes at the beginning of meiosis; they have a scarcely differentiated cytoplasm with protrusions penetrating between germ cells and between these and the tunica propria. Follicular cells form a flattened epithelium around initial previtellogenic oocytes, and during previtellogenic growth they proliferate and change from flattened to cuboidal and then columnar shape. They adhere tightly to each other and apically form microvilli which extensively interlock with oocyte microvilli. During vitellogenesis, the follicular cells show a globose shape; wide intercellular spaces appear between adjacent cells and between follicular cells and the oocyte. Their cytoplasm becomes enriched in rough endoplasmic reticulum cisternae, Golgi complexes, mitochondria and microtubules, particularly near the basal area. At the end of vitell...