The genetics of behaviour and other adaptive traits in nine-spined sticklebacks (Pungitius pungitius)

One of the main goals in current evolutionary biology research is to identify genes behind adaptive phenotypic variations. The advances in genomic technologies have made it possible to identify genetic loci behind these variations, also concerning non-model species. This thesis investigates the genetics of the behaviour and other adaptive traits of the nine-spined stickleback (Pungitius pungitius) through the application of different genetic approaches. Fennoscandian nine-spined stickleback populations express large phenotypical differences especially in behaviour, life –history traits and morphology. However the underlying genetic bases for these phenotypical differences have not been studied in detail. The results of the project will lay the foundation for further genetics studies and provide valuable information for our understanding of the genetics of the adaptive divergence of the nine-spined stickleback. A candidate gene approach was used to develop microsatellite markers situating close to candidate genes for behaviour in the nine-spined stickleback. Altogether 13 markers were developed and these markers were used in the subsequent studies with the anonymous random markers and physiologically important gene markers which are already currently available for nine-spined sticklebacks. It was shown that heterozygosity correlated with behaviour in one of the marine nine-spined stickleback populations but with contrasting effects: correlations with behaviour were negative when using physiological gene markers and positive with random markers. No correlation was found between behavioural markers and behaviour. From the physiological gene markers, a strong correlation was found between osmoregulation-related gene markers and behaviour. These results indicate that both local (physiological) and general (random) effects are important in the shaping of behaviour and that heterozygosity– behaviour correlations are population dependent. In this thesis a second linkage map for nine-spined sticklebacks was constructed. Compared to the earlier nine-spined stickleback linkage map, genomic rearrangements were observed between autosomal (LG7) and sex-determing (LG12) linkage groups. This newly constructed map was used in QTL mapping studies in order to locate genomic regions associated with pelvic structures, behaviour and body size/growth. One major QTL was found for pelvic structures and Pitx1 gene was related to these traits as was predicted from three-spined stickleback studies, but this was in contrast to earlier nine-spined stickleback study. The QTL studies also revealed that behaviour and body size/growth were genetically more complex by having more QTL than pelvic traits. However, in many cases, pelvic structure, body size/growth and behaviour were linked to similar map locations indicating possible pleiotropic effects of genes locating in these QTL regions. Many of the gene related markers resided in the QTL area. In the future, studying these possible candidate genes in depth might reveal the underlying mechanism behind the measured traits.

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity were studied. The morphological evaluation of the ovaries was performed by histological paraffin embedded and stained with HE. The immunohistochemical expressions of CDC47, VEGF, Flk-1, angiopoietin, Tie-2 and thyroid receptor (TRα) were performed by the technique of streptavidein-biotin-peroxidase. Apoptosis was assessed using the TUNEL kit. The relative expression of thyroid hormone receptors (TRα and TRβ) was assessed by RT-PCR real time. The nuclear expression of CDC47 increased with the stage of maturation of the oocyte and was observed in the follicle cells. Apoptotic bodies were observed in the follicular cells of atretic follicles and postovulatory follicles from the ovaries of 150g and 350g fish. Expression of VEGF and its receptor Flk-1 was also observed in the follicular cells, and the expression of both increased with the maturity of the oocyte, with a higher intensity observed in the full-grown follicle. The expression of angiopoietin and of its receptor (Tie 2) was discrete and moderate respectively. TRα expression was independent of follicular development. However, the 350 g tilapia exhibited higher expression of TRβ compared with the 50 g tilapia. We conclude that the proliferative activity and the expression of VEGF and its receptor increase with follicular maturation and that the TRs expression increases with ovarian maturity in tilapia (Oreochromis niloticus).

Mating system in Myracrodruon urundeuva (Anarcadiaceae): implications for conservation genetics

The aims of this study were to investigate the mating system of a fragmented population of the dioecious tropical tree Myracrodruon urundeuva Allemão, using five microsatellite loci and the mixed mating and correlated mating models. The study was conducted in the Estação Ecológica de Paulo de Farias (436 ha), where the population occupies about 142 ha. The mating system was estimated using 514 open-pollinated offspring, collected from 30 seed-trees. Estimates of the multilocus outcrossing rate confirm that the species is dioecious (t m = 1.0). Low levels of mating among relatives were detected in the population (1 - t s = 0.020). The estimate of paternity correlation (r p(m)) indicated that offsprings were composed of mixtures of half-sibs and full-sibs, with the latter occurring at a low frequency (average of 0.148). The estimated coancestry coefficient within families (Θ = 0.147) was larger and the effective population size (Ne(v)) was lower (Ne(v) = 2.98) than expected in progenies from panmictic populations (Θ = 0.125, Ne(v) = 4, respectively). In terms of conservation, the results indicate that to retain an effective population size of 150, is necessary to collect seeds from at least 50 seed-trees.

Genetics of resistance to the fungus Helminthosporium sativum in wheat: use of culture filtrates in tissue culture

Six wheat genotypes and their F1 and F2 generations were exposed to the action of Helminthosporium sativum culture filtrates to examine the genetics of hexaploid wheat resistance. The objective was to improve the efficiency of breeding programs by identifying the action and number of genes involved in the resistance. The varied response of the tested genotypes to the culture filtrates allowed division of the genotypes into four groups: resistant, moderately resistant, moderately susceptible and susceptible. This variability was detected in the progeny, suggesting that the parents have distinct genetic constitutions. Additive gene action predominated and genetic gain was shown to be possible through selection. The genetic control of the resistance trait seems to be complex because of the presence of gene interaction and the difficulty of eliminating the environmental effects. The inheritance seems to be oligogenic

Thyroid function in post-weaning rats whose dams were fed a low-protein diet during suckling

This study was designed to evaluate the thyroid and pituitary hormone levels in post-weaning rats whose dams were fed a low-protein diet during suckling (21 days). The dams and pups were divided into 2 groups: a control group fed a diet containing 22% protein that supplies the necessary amount of protein for the rat and is the usual content of protein in most commercial rat chow, and a diet group fed a low-protein (8%) diet in which the protein was substituted by an isocaloric amount of starch. After weaning all dams and pups received the 22% protein diet. Two hours before sacrifice of pups aged 21, 30 and 60 days, a tracer dose (0.6 µCi) of 125I was injected (ip) into each animal. Blood and thyroid glands of pups were collected for the determination of serum T4, T3 and TSH and radioiodine uptake. Low protein diet caused a slight decrease in radioiodine uptake at 21 days, and a significant decrease in T3 levels (128 ± 14 vs 74 ± 9 ng/dl, P<0.05), while T4 levels did not change and TSH was increased slightly. At 30 days, T3 and TSH did not change while there was a significant increase in both T4 levels (4.8 ± 0.3 vs 6.1 ± 0.2 µg/dl, P<0.05) and in radioiodine uptake levels (0.34 ± 0.02 vs 0.50 ± 0.03%/mg thyroid, P<0.05). At 60 days serum T3, T4 and TSH levels were normal, but radioiodine uptake was still significantly increased (0.33 ± 0.02 vs 0.41 ± 0.03%/mg thyroid, P<0.05). Thus, it seems that protein malnutrition of the dams during suckling causes hypothyroidism in the pups at 21 days that has a compensatory mechanism increasing thyroid function after refeeding with a 22% protein diet. The radioiodine uptake still remained altered at 60 days, when all the hormonal serum levels returned to the normal values, suggesting a permanent change in the thyroid function

Pituitary-thyroid axis in short- and long-term experimental diabetes mellitus

Short-term experimental diabetes mellitus (DM) produces a significant decrease in serum thyroid hormones, a decreased or normal serum thyroid-stimulating hormone (TSH) and a reduction in hepatic and renal T4-5'-deiodination. However, little is known about the effects of chronic diabetes mellitus on the pituitary-thyroid axis function. We evaluated the changes induced by very short-term (6 days), short-term (15 days) and chronic (6 months) streptozotocin-induced diabetes mellitus in 3-month old female Dutch-Miranda rat serum T4, serum TSH and T4-5'-deiodinase activity in the thyroid and pituitary glands. Serum hormones were determined by specific radioimmunoassays. Iodothyronine-5'-deiodinase activities were assayed in the thyroid and pituitary microsomal fractions using 2 µM T4 as substrate. Mean serum T4 was significantly decreased from 3.3 to 2.0 µg/dl 6 days after diabetes mellitus induction, and from 2.2 to 1.5 µg/dl after 15 days of DM, with no significant changes in serum TSH, indicating a decreased pituitary TSH responsiveness to the diminished suppression by T4, even though pituitary T4-5'-deiodinase activity was unchanged. Thyroid T4-5'-deiodinase was unchanged after 6 days of diabetes mellitus, but was significantly increased from 20.6 to 37.0 pmol T3/mg protein after 15 days. Six months after diabetes mellitus induction, both serum T4 and thyroid T4-5'-deiodinase returned to normal ranges and serum TSH was unchanged, although pituitary T4-5'-deiodinase was now significantly decreased from 2.7 to 1.7 pmol T3/mg protein. These findings indicate that some kind of adaptation to chronic insulinopenia may occur at the thyroid level, but this does not seem to be true for the pituitary

Effects of estradiol benzoate on 5'-iodothyronine deiodinase activities in female rat anterior pituitary gland, liver and thyroid gland

There is little information on the possible effects of estrogen on the activity of 5'-deiodinase (5'-ID), an enzyme responsible for the generation of T3, the biologically active thyroid hormone. In the present study, anterior pituitary sonicates or hepatic and thyroid microsomes from ovariectomized (OVX) rats treated or not with estradiol benzoate (EB, 0.7 or 14 µg/100 g body weight, sc, for 10 days) were assayed for type I 5'-ID (5'-ID-I) and type II 5'-ID (5'-ID-II, only in pituitary) activities. The 5'-ID activity was evaluated by the release of 125I from deiodinated 125I rT3, using specific assay conditions for type I or type II. Serum TSH and free T3 and free T4 were measured by radioimmunoassay. OVX alone induced a reduction in pituitary 5'-ID-I (control = 723.7 ± 67.9 vs OVX = 413.9 ± 26.9; P<0.05), while the EB-treated OVX group showed activity similar to that of the normal group. Thyroid 5'-ID-I showed the same pattern of changes, but these changes were not statistically significant. Pituitary and hepatic 5'-ID-II did not show major alterations. The treatment with the higher EB dose (14 µg), contrary to the results obtained with the lower dose, had no effect on the reduced pituitary 5'-ID-I of OVX rats. However, it induced an important increment of 5'-ID-I in the thyroid gland (0.8 times higher than that of the normal group: control = 131.9 ± 23.7 vs ovx + EB 14 µg = 248.0 ± 31.2; P<0.05), which is associated with increased serum TSH (0.6-fold vs OVX, P<0.05) but normal serum free T3 and free T4. The data suggest that estrogen is a physiological stimulator of anterior pituitary 5'-ID-I and a potent stimulator of the thyroid enzyme when employed at high doses

Autoradiographic thyroid evaluation in short-term experimental diabetes mellitus

Previous studies have shown that in vitro thyroid peroxidase (TPO) iodide oxidation activity is decreased and thyroid T4-5'-deiodinase activity is increased 15 days after induction of experimental diabetes mellitus (DM). In the present study we used thyroid histoautoradiography, an indirect assay of in vivo TPO activity, to determine the possible parallelism between the in vitro and in vivo changes induced by experimental DM. DM was induced in male Wistar rats (about 250 g body weight) by a single ip streptozotocin injection (45 mg/kg), while control (C) animals received a single injection of the vehicle. Seven and 30 days after diabetes induction, each diabetic and control animal was given ip a tracer dose of 125I (2 µCi), 2.5 h before thyroid excision. The glands were counted, weighed, fixed in Bouin's solution, embedded in paraffin and cut. The sections were stained with HE and exposed to NTB-2 emulsion (Kodak). The autohistograms were developed and the quantitative distribution of silver grains was evaluated with a computerized image analyzer system. Thyroid radioiodine uptake was significantly decreased only after 30 days of DM (C: 0.38 ± 0.05 vs DM: 0.20 ± 0.04%/mg thyroid, P<0.05) while in vivo TPO activity was significantly decreased 7 and 30 days after DM induction (C: 5.3 and 4.5 grains/100 µm2 vs DM: 2.9 and 1.6 grains/100 µm2, respectively, P<0.05 ). These data suggest that insulin deficiency first reduces in vivo TPO activity during short-term experimental diabetes mellitus

Thyroid hormone regulates protein expression in C6 glioma cells

Thyroid hormone (T3) is essential to normal brain development. Previously, we have shown that T3 induces cerebellar astrocyte proliferation. This effect is accompanied by alteration in glial fibrillary acidic protein (GFAP) and fibronectin organization. In the present study, we report that the C6 glioma cell line, which expresses GFAP and is classified as an undifferentiated astrocytic cell type, is a target for T3 action. The C6 monolayers were treated with 50 nM T3 for 3 days, after which the cells were maintained for 2 days without medium changes. In C6 cells, T3 induced the expression of proteins of 107, 73 and 62 kDa. The hormone also up-regulated protein bands of 100 (+50%), 37 (+50%) and 25.5 kDa (+50%) and down-regulated proteins of 94 (-100%), 86.5 (-100%), 68 (-100%), 60 (-100%), 54 (-33%), 51 (-33%) and 43.5 kDa (-33%). We suggest, on the basis of molecular mass, that the 54-, 51- and 43.5-kDa proteins could be the cytoskeletal proteins vimentin, GFAP and actin, respectively. The down-regulation of these proteins may be involved in the effects of thyroid hormone on C6 differentiation.

Reactivity of anti-thyroid antibodies to thyroglobulin tryptic fragments: comparison of autoimmune and non-autoimmune thyroid diseases

Studies concerning the antigenicity of thyroglobulin fragments allow the characterization of the epitopes but do not consider the role of heavier antigenic fragments that could result in vivo from the action of endoproteases. Here we assess the relative importance of the fragments obtained from thyroglobulin by limited proteolysis with trypsin and compare by immunoblotting their reactivity to serum from patients with autoimmune (Graves' disease and Hashimoto's thyroiditis) and non-autoimmune (subacute thyroiditis) disease. The results showed no difference in frequency of recognition of any peptide by sera from patients with autoimmune thyroiditis. In contrast, sera from patients with subacute thyroiditis reacted more frequently with a peptide of 80 kDa. These results suggest the presence of antibody subpopulations directed at fragments produced in vivo by enzymatic cleavage of thyroglobulin. This fragment and antibodies to it may represent markers for subacute thyroiditis.

Thyroid peroxidase activity is inhibited by amino acids

Normal in vitro thyroid peroxidase (TPO) iodide oxidation activity was completely inhibited by a hydrolyzed TPO preparation (0.15 mg/ml) or hydrolyzed bovine serum albumin (BSA, 0.2 mg/ml). A pancreatic hydrolysate of casein (trypticase peptone, 0.1 mg/ml) and some amino acids (cysteine, tryptophan and methionine, 50 µM each) also inhibited the TPO iodide oxidation reaction completely, whereas casamino acids (0.1 mg/ml), and tyrosine, phenylalanine and histidine (50 µM each) inhibited the TPO reaction by 54% or less. A pancreatic digest of gelatin (0.1 mg/ml) or any other amino acid (50 µM) tested did not significantly decrease TPO activity. The amino acids that impair iodide oxidation also inhibit the TPO albumin iodination activity. The inhibitory amino acids contain side chains with either sulfur atoms (cysteine and methionine) or aromatic rings (tyrosine, tryptophan, histidine and phenylalanine). Among the amino acids tested, only cysteine affected the TPO guaiacol oxidation reaction, producing a transient inhibition at 25 or 50 µM. The iodide oxidation inhibitory activity of cysteine, methionine and tryptophan was reversed by increasing iodide concentrations from 12 to 18 mM, while no such effect was observed when the cofactor (H2O2) concentration was increased. The inhibitory substances might interfere with the enzyme activity by competing with its normal substrates for their binding sites, binding to the free substrates or reducing their oxidized form.

Estradiol decreases iodide uptake by rat thyroid follicular FRTL-5 cells

Estradiol has well-known indirect effects on the thyroid. A direct effect of estradiol on thyroid follicular cells, increasing cell growth and reducing the expression of the sodium-iodide symporter gene, has been recently reported. The aim of the present investigation was to study the effect of estradiol on iodide uptake by thyroid follicular cells, using FRTL-5 cells as a model. Estradiol decreased basal iodide uptake by FRTL-5 cells from control levels of 2.490 ± 0.370 to 2.085 ± 0.364 pmol I-/µg DNA at 1 ng/ml (P<0.02), to 1.970 ± 0.302 pmol I-/µg DNA at 10 ng/ml (P<0.003), and to 2.038 ± 0.389 pmol I-/µg DNA at 100 ng/ml (P<0.02). In addition, 4 ng/ml estradiol decreased iodide uptake induced by 0.02 mIU/ml thyrotropin from 8.678 ± 0.408 to 7.312 ± 0.506 pmol I-/µg DNA (P<0.02). A decrease in iodide uptake by thyroid cells caused by estradiol has not been described previously and may have a role in goiter pathogenesis.