A male-specific nuclease-resistant chromatin fraction in the mealybug Planococcus lilacinus

In mealybugs, chromatin condensation is related to both genomic imprinting and sex determination. The paternal chromosomal complement is condensed and genetically inactive in sons but not in daughters. During a study of chromatin organization in Planococcus lilacinus, digestion with micrococcal nuclease showed that 3% to 5% of the male genome is resistant to the enzyme. This Nuclease Resistant Chromatin (NRC) apparently has a nucleosomal organization. Southern hybridization of genomic DNA suggests that NRC sequences are present in both sexes and occur throughout the genome. Cloned NRC DNA is A+T-rich with stretches of adenines similar to those present in mouse alpha-satellite sequences. NRC DNA also contains sequence motifs that are typically associated with the nuclear matrix. Salt-fractionation experiments showed that NRC sequences are matrix associated. These observations are discussed in relation to the unusual cytological features of mealybug chromosomes, including the possible existence of multiple centres of inactivation.

The evolution of genomic imprinting

We explore three possible pathways for the evolution of genomic imprinting. (1) Imprinting may be advantageous in itself when imprinted and unimprinted alleles of a locus confer different phenotypes. If a segment of DNA is imprinted in the gametes of one sex but not in those of the other, it might lead to effects correlated with sexual dimorphism. More fundamentally, in certain organisms, sex determination might have evolved because of imprinting. When imprinting leads to chromosome elimination or inactivation and occurs in some embryos but not in others, two classes of embryos, differing in the number of functional gene copies, would result. A model for sex determination based on inequality in the actual or effective copy-number of particular noncoding, regulatory sequences of DNA has been proposed (Chandra, Proc. natn. Acad. Sci. U.S.A. 82. 1165–1169 and 6947–6949, 1985). Maternal control of offspring sex is another possible consequence of imprinting; this would indicate a potential role for imprinting in sex ratio evolution. (2) Genes responsible for imprinting may have pleiotropic effects and they may have been selected for reasons other than their imprinting ability. Lack of evidence precludes further consideration of this possibility. (3) Imprinting could have co-evolved with other traits. For instance, gamete-specific imprinting could lead to a lowered fitness of androgenetic or gynogenetic diploids relative to the fitness of ‘normal’ diploids. This in turn would reinforce the evolution of anisogamy. The reversibility of imprinting raises the possibility of occasional incomplete or improper erasure. If the site of imprinting is the egg – as appears to be the case with the human X (Chandra and Brown, Nature 253. 165–168, 1975) – either improper imprinting or improper erasure could lead to unusual patterns of inheritance (as in the fragile-X syndrome) or fitness effects skipping generations.

Differential expression of estrogen receptor alpha in the embryonic adrenal-kidney-gonadal complex of the oviparous lizard, Calotes versicolor (Daud.)

Estrogen signalling is critical for ovarian differentiation in reptiles with temperature-dependent sex determination (TSD). To elucidate the involvement of estrogen in this process, adrenal-kidney-gonadal (AKG) expression of estrogen receptor (ER alpha) was studied at female-producing temperature (FPT) in the developing embryos of the lizard, Calotes versicolor which exhibits a distinct pattern of TSD. The eggs of this lizard were incubated at 31.5 +/- 0.5 degrees C (100% FPT). The torso of embryos containing adrenal-kidney-gonadal complex (AKG) was collected during different stages of development and subjected to Western blotting and immunohistochemistry analysis. The ER alpha, antibody recognized two protein bands with apparent molecular weight similar to 55 and similar to 45 kDa in the total protein extracts of embryonic AKG complex of C. versicolor. The observed results suggest the occurrence of isoforms of ER alpha. The differential expression of two different protein isoforms may reveal their distinct role in cell proliferation during gonadal differentiation. This is the first report to reveal two isoforms of the ER alpha in a reptile during development. Immunohistochemical studies reveal a weak, but specific, cytoplasmic ER alpha immunostaining exclusively in the AKG during late thermo-sensitive period suggesting the responsiveness of AKG to estrogens before gonadal differentiation at FPT. Further, cytoplasmic as well as nuclear expression of ER alpha in the medulla and in oogonia of the cortex (faint activity) at gonadal differentiation stage suggests that the onset of gonadal estrogen activity coincides with sexual differentiation of gonad. Intensity and pattern of the immunoreactions of ER alpha in the medullary region at FPT suggest endogenous production of estrogen which may act in a paracrine fashion to induce neighboring cells into ovarian differentiation pathway. (C) 2014 Elsevier Inc. All rights reserved.

The effects of size-selective fisheries on the stock dynamics of and sperm limitation in sex-changing fish

Fisheries models have traditionally focused on patterns of growth, fecundity, and survival of fish. However, reproductive rates are the outcome of a variety of interconnected factors such as life-history strategies, mating patterns, population sex ratio, social interactions, and individual fecundity and fertility. Behaviorally appropriate models are necessary to understand stock dynamics and predict the success of management strategies. Protogynous sex-changing fish present a challenge for management because size-selective fisheries can drastically reduce reproductive rates. We present a general framework using an individual-based simulation model to determine the effect of life-history pattern, sperm production, mating system, and management strategy on stock dynamics. We apply this general approach to the specific question of how size-selective fisheries that remove mainly males will impact the stock dynamics of a protogynous population with fixed sex change compared to an otherwise identical dioecious population. In this dioecious population, we kept all aspects of the stock constant except for the pattern of sex determination (i.e. whether the species changes sex or is dioecious). Protogynous stocks with fixed sex change are predicted to be very sensitive to the size-selective fishing pattern. If all male size classes are fished, protogynous populations are predicted to crash even at relatively low fishing mortality. When some male size classes escape fishing, we predict that the mean population size of sex-changing stocks will decrease proportionally less than the mean population size of dioecious species experiencing the same fishing mortality. For protogynous species, spawning-per-recruit measures that ignore fertilization rates are not good indicators of the impact of fishing on the population. Decreased mating aggregation size is predicted to lead to an increased effect of sperm limitation at constant fishing mortality and effort. Marine protected areas have the potential to mitigate some effects of fishing on sperm limitation in sex-changing populations.

Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers

DMRT1 has been suggested to play different roles in sex determination and gonad differentiation, because different expression patterns have been reported among different vertebrates. The groupers, since their gonads first develop as ovary and then reverse into testis, have been thought as good models to study sex differentiation and determination. In this study, we cloned the full-length cDNAs of DMRT] gene from orange-spotted grouper (Epinephelus coioides), and prepared corresponding anti-EcDMRT1] antiserum to study the relationship of DMRT] to sex reversal. One important finding is that the grouper DMRT] is not only differentially expressed in different stage gonads, but also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, we confirmed that EcSox3 is expressed not only in oogonia and different stage oocytes, but also in Sertoli cells and spermatogonia, and EcSox9 is expressed only in Sertoli cells. The data suggested that grouper DMRT1 might be a more specific sex differentiation gene for spermatogenesis, and play its role at the specific stages from spermatogonia to spermatocytes. In addition, no introns were found in the grouper DMRT1, and no duplicated DMRT1, genes were detected. The finding implicates that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad. (c) 2006 Published by Elsevier Ireland Ltd.

Sex-linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta)

P>In our microsatellite analysis of three male and three female gametophytes of Undaria pinnatifida (Harv.) Suringar, a microsatellite marker (part of the locus Up-AC-2A8, GenBank accession no. AY738602.1) was only polymerase chain reaction-amplified in three female gametophytes. This putative female-specific marker was further tested by the use of 32 male and 21 female gametophytes maintained in the Marine Biological Culture Collection Centre, China. In addition, three sporophytes were included for confirmation. Results showed that the marker was present in all of the female gametophytes and sporophyte cultures, but absent in all of the male gametophytes. To our knowledge, this is the first sex-related marker ever reported in U. pinnatifida. The discovery of this marker will accelerate gender identification and shed light on our understanding of the mechanisms of sex determination at a molecular level in this commercially important seaweed.

An investigation into mechanisms of visceral pain in rodents

Visceral pain is a debilitating symptom of irritable bowel syndrome (IBS), a disorder affecting up to 30% of adults. A better understanding of the mechanisms underlying visceral hypersensitivity may facilitate development of more targeted therapies, improving the quality of life of these individuals. The studies performed in this thesis were designed to investigate important factors of visceral pain, including early-life manipulations, genetic predisposition and sex hormones. Maternal separation (MS) consistently reproduces visceral hypersensitivity and altered anxiety-like behaviours in rats, symptoms associated with IBS. It has been found that 5-HT2B receptor antagonism blocks visceral pain but no difference in relative 5-HT2B receptor mRNA expression was found in hippocampus, amygdala and colon. The neuronal activation patterns of prefrontal cortex and amygdala of MS rats were then investigated. MS animals are characterised by differential activation of the prefrontal cortex (anterior cingulate cortex (ACC), infralibic cortex, prelimbic cortex) as well as the central nucleus of the amygdala (CeA). Genetic factors also contribute to pain syndromes such as IBS. We utilised the Wistar Kyoto (WKY) rat, a stress-sensitive strain, as an animal model of brain-gut axis dysfunction. WKY rats have a lower expression of the glutamate transporter EAAT2 and mGlu4 receptor in the ACC. Another early-life factor that can increase susceptibility to functional gastrointestinal symptoms later life is disruption of the gut microbiota, thus early-life antibiotic treatment was used to assess this effect. Antibiotic treatment induced visceral hypersensitivity in adulthood and may be related to observed reductions in spinal cord alpha-2A adrenoreceptor (adra2A) mRNA. Lastly, we investigated sex differences in visceral sensitivity. EAAT1 & 2 mRNA levels are lower in females, potentially increasing glutamatergic concentration at the symaptic level. Moreover, NR1 and NR2B subunits mRNA of NMDA receptor were increased in caudal ACC of females. These findings may account for sex differences in visceral sensitivity.

Conservation genetics of Neotropical pollinators revisited: microsatellite analysis suggests that diploid males are rare in orchid bees

Allozyme analyses have suggested that Neotropical orchid bee (Euglossini) pollinators are vulnerable because of putative high frequencies of diploid males, a result of loss of sex allele diversity in small hymenopteran populations with single locus complementary sex determination. Our analysis of 1010 males from 27 species of euglossine bees sampled across the Neotropics at 2-11 polymorphic microsatellite loci revealed only 5 diploid males at an overall frequency of 0.005 (95% CIs 0.002-0.010); errors through genetic non-detection of diploid males were likely small. In contrast to allozyme-based studies, we detected very weak or insignificant population genetic structure, even for a pair of populations >500 km apart, possibly accounting for low diploid male frequencies. Technical flaws in previous allozyme-based analyses have probably led to considerable overestimation of diploid male production in orchid bees. Other factors may have a more immediate impact on population persistence than the genetic load imposed by diploid males on these important Neotropical pollinators.

Shift of spawning season and effects of climate warming on developmental stages of a grayling (Salmonidae)

River-dwelling fish, such as European graylings (Thymallus thymallus), are susceptible to changes in climate because they can often not avoid suboptimal temperatures, especially during early developmental stages. We analyzed data collected in a 62-year-long (1948-2009) population monitoring program. Male and female graylings were sampled about three times/week during the yearly spawning season in order to follow the development of the population. The occurrence of females bearing ripe eggs was used to approximate the timing of each spawning season. In the last years of the study, spawning season was more than 3 weeks earlier than in the first years. This shift was linked to increasing water temperatures as recorded over the last 39 years with a temperature logger at the spawning site. In early spring water temperatures rose more slowly than in later spring. Thus, embryos and larvae were exposed to increasingly colder water at a stage that is critical for sex determination and pathogen resistance in other salmonids. In summer, however, fry were exposed to increasingly warmer temperatures. The changes in water temperatures that we found embryos, larvae, and fry were exposed to could be contributing to the decline in abundance that has occurred over the last 30-40 years.

Aspectos da biologia e morfologia da broca do pedúnculo floral do coqueiro, Homalinotus coriaceus (Gyllenhal) (Coleoptera: Curculionidae)

A broca do pedúnculo floral do coqueiro, Homalinotus coriaceus (Gyllenhal), é uma praga limitante à produção de coco no Brasil, provocando a queda das flores e dos frutos imaturos. Sua biologia é pouco conhecida o que tem dificultado a seu manejo adequado no campo. Dessa forma, foi realizado o estudo da biologia desse inseto no Laboratório de Entomologia da Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Tabuleiros Costeiros - CPATC, em Aracaju, SE, à temperatura de 25 ± 2°C, umidade relativa de 70% e fotofase de 12h. Para alimentação e oviposição dos adultos coletados no campo foram utilizados toletes de cana-de-açúcar como substrato, nos quais as fêmeas colocaram ovos individualizados, cujo período de incubação variou de seis a 14 dias. As larvas foram criadas no mesocarpo de coco, desenvolvendo-se em 144 dias em média, passando por cinco a sete ínstares. O período pupal médio foi de 31 dias. A longevidade do adulto variou de 303 a 695 dias para fêmeas e de 246 a 635 dias para machos, sendo o ciclo de ovo a adulto de 181,9 dias para fêmeas e 188,5 para machos. Foram observadas diferenças morfológicas nas pupas e nos adultos, que podem ser utilizadas para separação dos sexos. Ao contrário dos machos, as fêmeas apresentam, na fase de pupa, duas estruturas arredondadas e elevadas situadas na face ventral e final do abdome e, na fase adulta, o penúltimo tergito abdominal projeta-se sobre o último.

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sex chromosome evolution in fish: the formation of the neo-Y chromosome in Eigenmannia (Gymnotiformes)

Chromosomes of a species of Eigenmannia presenting a X1X1X2X2:X1X2Y sex chromosome system, resulting from a Y-autosome Robertsonian translocation, were analyzed using the C-banding technique, chromomycin A(3) (CMA(3)) and mithramycin (MM) staining and in situ digestion by the restriction endonuclease AluI. A comparison of the metacentric Y chromosome of males with the corresponding acrocentrics in females indicated that a C-band-positive, CMA(3)/MM-fluorescent and AluI digestion-resistant region had been lost during the process of translocation, resulting in a diminution of heterochromatin in the males. It is hypothesized that the presence of a smaller amount of G+C-rich heterochromatin in the sex chromosomes of the heteromorphic sex when compared with the homomorphic sex may be associated with the sex determination mechanism in this species and may be a more widely occurring phenomenon in fish with differentiated sex chromosomes than was initially thought.

Genetics of neotropical fish: from chromosomes to populations

The Neotropical freshwater fish fauna is very rich-according to the most recent catalogue 71 families and 4,475 species have been described. However, only a small amount of general information is available on the composition of Neotropical marine fishes. In Brazil, 1,298 marine species have been recorded. General analysis of available cytogenetic and population genetic data clearly indicates research has been mainly concentrated on freshwater fishes. Thus, today, cytogenetic information is available for 475 species of Characiformes, 318 species of Siluriformes, 48 species of Gymnotiformes, 199 freshwater species that do not belong to the superorder Ostariophysi, and only 109 species of marine fishes. For the species studied, only about 6% have sex chromosomes and about 5% have supernumerary or B chromosomes. A review of the cytogenetic studies shows that these data have provided valuable information about the relationships between fish groups, the occurrence of cryptic species and species complexes, the mechanism of sex determination and sex chromosome evolution, the distribution of nucleolus organizer regions, the existence supernumerary chromosomes, and the relationship between polyploidy and evolution. In relation to populations in Neotropical marine waters, the studies have shown the presence of cryptic species, which has important implications for fishery management. Different levels of genetic structuring can be found among Neotropical freshwater migratory fish species. This raises important implications for fish population genetic diversity and consequently its sustainable utilization in inland fisheries and aquaculture, specifically for conservation of ichthyo-diversity and survival.

Reproductive cycle and recruitment period of Ocypode quadrata (Decapoda, Ocypodidae) at a sandy beach in southeastern Brazil

The reproductive cycle and recruitment period of a ghost crab population from Ubatuba, São Paulo, Brazil were investigated by means of examining the developmental stages of gonads of breeding crabs and the ingress of young recruits to the studied population. Monthly collections over a one-year period were carried out during nocturnal low-tide periods at Vermelha beach. The morphology of the abdomen and pleopods was used for sex determination. All captured crabs were measured for carapace width and dissected for the determination of the development stage of the gonads. A total of 582 specimens was captured: 271 males, 241 females, and 70 juveniles. Size ranged from 8.5 to 37.5 mm for males, from 9.5 to 39.2 mm for females, and from 5.8 to 12 mm for early juveniles. Median size of males and females did not differ statistically. The frequency of ovigerous females was markedly low. The onset of sexual maturity in females is achieved at around 23 mm of carapace width. Mature females with advanced gonad stages were not recorded from May to September. Recruitment of young was highest during summer, but the presence of early and late juvenile specimens throughout the year indicates that continuous recruitment is taking place in the studied population.

Development of eleven polymorphic microsatellite markers for the Chaco Treefrog, Hypsiboas raniceps

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)