Marine Ranching: Proceedings of the Eighteenth U.S.-Japan Meeting on Aquaculture Port Ludlow, Washington, 18-19 September 1989

Female reproduction in penaeid shrimp is carefully regulated by several different endocrine factors. Their precise modes of action have not yet been fully elucidated. Three endocrine factors, each representing a different chemical class of hormones, have been investigated in the penaeid shrimp Sicyonia ingentis in our laboratory: ecdysteroids, vitellogenesis-inhibiting hormone (VIH) , and methyl farnesoate (MF). Ecdysteroids (the steroid molting hormones of arthropods; predominantly 20-hydroxyecdysone), are initially present in low levels (<10 ng/mg) in shrimp embryos. As development of the embryos nears time of hatch, the ecdysteroid levels increase to approximately 150 ng/ mg, indicating that they may be of embryonic origin and involved in embryonic development. An assay was developed for shrimp VIH, which presumably is a protein. Delay of onset of the next reproductive cycle was observed following injection of sinus gland extracts into shrimp that had previously had their eyestalks removed. A photoaffinity analog was synthesized for the putative shrimp reproductive hormone MF-a terpenoid. This analog, farnesyl diazomethyl ketone (FDK) , was used to demonstrate the presence of specific binding proteins for MF in shrimp hemolymph. (PDF file contains 136 pages.)

Endocrine disruption in the terrestrial isopod porcellio scaber

Nas ��ltimas d��cadas tem-se assistido a uma preocupa����o crescente relativamente ��s poss��veis consequ��ncias da exposi����o a compostosxen��bioticos capazes de modular ou causar disrup����o do sistema end��crino, os denominados Compostos Disruptores End��crinos (CDEs). A maioria dos estudos efectuados tem-se centrado principalmente nos efeitos dos CDEs em vertebrados, enquanto que os seus efeitos em invertebrados t��msido negligenciados, embora este grupo represente mais de 95% de todas asesp��cies animais. Is��podes como o Porcellio scaber, combinam caracter��sticas associadas ��s mudas e aos processos reprodutivos mediados por mecanismos end��crinosconhecidos com um modo de vida terrestre, tornando-os potenciais esp��cies sentinela para estudos de disrup����o end��crina (DE) em ambientes terrestres. Neste estudo, is��podes machos adultos, machos e f��meas juvenis e casais foram expostos a concentra����es crescentes dedois CDEs, vinclozolina (Vz) e bisfenol A (BPA). Testou-se a hip��tese nula que a Vz e o BPA n��o interferem com o desenvolvimento e reprodu����o deste is��pode terrestre. Foi investigadaa poss��vel liga����o entre os efeitos causados pelos compostos propostos e DE assim como a liga����o a outros potenciais mecanismos de toxicidade.Par��metros como concentra����o de 20-hidroxiecdisona (20E), muda, crescimento, r��cios sexuais ediversos par��metros reprodutivos foram estudados. Adicionalmente, de modo a estudar os alvos moleculares destes t��xicos, analisou-se a express��o proteica do intestino, hepatop��ncreas e test��culos do is��pode ap��s exposi����o aos qu��micos. Os resultados demonstram que a Vz e o BPA estimulam o aumento dos n��veis de 20E de um modo dependente da dose. Excep����o feita para a concentra����o mais baixa de BPA testada (10 mg/kg solo), para a qual concentra����es significativamente mais altas de 20E foram determinadas, sugerindo a ocorr��ncia dos ���efeitos de baixas doses t��picos de DE��� j�� demonstrados por outros autores. O BPA tamb��m distorceu o r��cio sexual favorecendo asf��meas na concentra����o mais baixa. A mortalidade devido �� ecdise incompleta foi relacionada com o hiper-ecdisonismo nas concentra����es mais elevadas de Vz. Mais ainda, a Vz tende a atrasar a muda e o BPA a induzi-la. N��o obstante, ambos os compostos provocam toxicidade no desenvolvimento,uma vez que foi encontrada uma diminui����o generalizada nos par��metros de crescimento. Os juvenis mostraram ser mais sens��veis �� exposi����o aos t��xicos que os adultos. Estes compostos provocaram ainda toxicidade reprodutiva, com um decr��scimo generalizado do ���output��� reprodutivo. A toxicidadecausada pelos ecdister��ides e o seu papel na s��ntese de vitelogenina s��o alguns dos factores chave que poder��o influenciar negativamente a reprodu����o.A Vz e o BPA afectaram a express��o de prote��nas envolvidas nometabolismo energ��tico e induziram v��rias respostas de stress. Interferiram ainda com prote��nas intimamente ligadas com o sucesso reprodutivo. Conclui-se assim,que ambos os CDEs propostos provocam toxicidade nodesenvolvimento e na reprodu����o de P. scaber, tendo sido evidenciada umaliga����o a DE. Alvos moleculares de natureza n��o-end��crina foram tamb��m revelados, atrav��s da express��o diferencial de algumas prote��naspreviamente descritas para invertebrados aqu��ticos e mesmo alguns vertebrados.

Germination requirements of Plantago algarbiensis seeds, an endangered species endemic to the Algarve region (South of Portugal)

The ecdysteroid, 20-hydroxyecdysone or beta-ecdysone, is a steroid hormone which plays a crucial role in molting, metamor- phosis and reproduction of arthropods. This ecdysteroid and its analogues have high potential to be used as insecticides. Previous studies in our laboratory have demonstrated that Vitex glabrata R.Br. (commonly known as Khai-Nao), an indigenous herbaceous plant of Thailand, synthesized and accumulated high quantity of 20-hydroxyecdysone. Therefore, the aim of this study was to investigate the effect of precursor and elicitors feeding on cell growth and 20-hydroxyecdysone production of V. glabrata suspension cultures. Plant cells were cultured in half strength MS medium containing 30 g/l glucose and supplemented with 2.0 mg/l 6- benzylaminopurine (BAP) and 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Cells were incubated on a rotary shaker at 120 rpm under continuous light of 2000 lux at 25 ��C. Sterilized cholesterol (5 and 10 mg/l) as precursor was added to the cell cultures on the day of inoculation, while chitosan (50, 100 and 200 mg/l) and methyl jusmonate (100 and 200 mM) as elicitors were added to the cell cultures on day 6 after cultivation.

Efeito da adi����o de colesterol e ecdisona na produ����o in vitro do baculov��rus spodoptera frugiperda MNPV

Among the pests that attack corn crop in Brazil, there is Spodoptera frugiperda (JE Smith, 1797) (Lepidoptera: Noctuidae), known as fall armyworm, which is the major corn pest. Due to genetic instability during serial passage of baculoviruses in insect cell culture, the viral bioinseticides in vitro production development is the greatest challenge for mass production of this bioproduct. Successive passages of virus using extracellular viruses (BVs), necessary during viral bioinseticides production scaling up, leads to the appearance of aberrant forms of virus, a process so called as "passage effect ". The main consequence of passage effect is the production of occlusion bodies (OB) decrease, preventing its production using in vitro process. In this study, it was carried out a serial passage of baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus, isolate 18, using Sf21 cells. A decrease in the production of occlusion bodies from 170 to 92 in the third to fourth passage was observed. A factorial experimental design (22) was employed to verify the influence of two input variables, concentration of the hormone 20 - hydroxyecdysone (CH) and cholesterol (CC) on the values of response variables (volumetric and the specific OB production) of the process, seeking to define the optimum operating ranges trying to reverse or minimize the passage effect. The result indicated a negative influence of the cholesterol addition and positive effect in the hormone supplementation which the optimum range found for the concentrations studied were 8 to 10&#956;g/mL and 5 to 6.5 mg / mL, for cholesterol and hormone concentrations respectively. New experiments were performed with addition of hormone and cholesterol in order to check the influence of these additives on the OB production independently. While the best result obtained from the factorial experiment was 9.4 x 107 OB/mL and 128.4 specific OB/cell, with the addition of only 6&#956;g/mL 20-hydroxyecdysone these concentrations increased to 1.9 x 108 OB/mL and 182.9 OB/cell for volumetric and specific OB production, respectively. This result confirms that the addition of the hormone 20-hydroxyecdysone enhances the SfMNPV in vitro production process performance using Sf21 cells

Selectivity of diacylhydrazine insecticides to the predatory bug Orius laevigatus: In vivo and modeling/docking experiments

BACKGROUND: Knowledge of pesticide selectivity to natural enemies is necessary for a successful implementation of biological and chemical control methods in integrated pest management (IPM) programs. Diacylhydrazine (DAH)-based ecdysone agonists also known as molting-accelerating compounds (MACs) are considered a selective group of insecticides, and their compatibility with predatory Heteroptera, which are used as biological control agents, is known. However, their molecular mode of action has not been explored in beneficial insects such as Orius laevigatus (Fieber) (Hemiptera: Anthocoridae). RESULTS: In this project in vivo toxicity assays demonstrated that the DAH-based RH-5849, tebufenozide and methoxyfenozide have no toxic effect against O. laevigatus. The ligand-binding domain (LBD) of the ecdysone receptor (EcR) of O. laevigatus was sequenced and a homology protein model was constructed which confirmed a cavity structure with 12 ?-helixes, harboring the natural insect molting hormone 20-hydroxyecdysone. However, docking studies showed that a steric clash occurred for the DAH-based insecticides due to a restricted extent of the ligand-binding cavity of the EcR of O. laevigatus. CONCLUSIONS: The insect toxicity assays demonstrated that MACs are selective for O. laevigatus. The modeling/docking experiments are indications that these pesticides do not bind with the LBD-EcR of O. laevigatus and support that they show no biological effects in the predatory bug. These data help in explaining the compatible use of MACs together with predatory bugs in IPM programs. Keywords: Orius laevigatus, selectivity, diacylhydrazine insecticides, ecdysone receptor, homology modelling, docking studies.

Nonsteroidal moulting hormone agonists: effects on protein synthesis and cuticle formation in colorado potato beetle larvae.

Larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say), that were orally treated with RH-0345 at 0.1 mg l?1, RH-5849 at 10 and 50 mg l?1, tebufenozide at 2 g l?1, and 20-hydroxyecdysone at 2 g l?1, showed symptoms of prematuremoulting, followed by inhibition of ecdysis. In addition, fresh weight gain and total protein content were blocked. The effects on haemolymphal and cuticular polypeptides after PAGE were linked with premature, new epicuticle deposition as was observed under the electron microscope. These observations support the concept that the ecdysteroid-mimicking action of the three nonsteroidal molecules is specific

Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5��� flanking sequence (���855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5��� flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the Aedes aegypti ecdysone receptor/Ultraspiracle nuclear receptor complex, and, seemingly, the endogenous Anopheles gambiae nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an A. gambiae cell line.

Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members.

We have designed a rapid cloning and screening strategy to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, we isolated three Drosophila genes, designated DHR38, DHR78, and DHR96. All three genes are expressed throughout third-instar larval and prepupal development. DHR38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. DHR78 and DHR96 are orphan receptor genes. DHR78 is induced by 20-hydroxyecdysone (20E) in cultured larval organs, and its encoded protein binds to two AGGTCA half-sites arranged as either direct or palindromic repeats. DHR96 is also 20E-inducible, and its encoded protein binds selectively to the hsp27 20E response element. The 20E receptor can bind to each of the sequences recognized by DHR78 and DHR96, indicating that these proteins may compete with the receptor for binding to a common set of target sequences.

���Just try harder and you will shine��� : a Study of 20 lazy children

Attributions of laziness, reflected in teacher comments such as ���just try harder and you will shine��� may mask specific cognitive, learning, attentional or emotional problems that could explain low motivation in some children. This paper reports findings from an investigation of 20 children, aged 7 to 10 years, who were regarded as lazy by their parents and teachers. Questionnaire measures provided evidence of low levels of motivation and classroom engagement. Psychometric assessments revealed the presence of a range of difficulties including phonologically-based learning disabilities and significant problems with attention in 17 of the 20 children. The paper concludes that the special needs of an unknown number of children may be overlooked because they are simply presumed to be lazy.

A psychometric comparison of the Toronto Alexithymia Scale (TAS-20) and the Observer Alexithymia Scale (OAS) in an alcohol-dependent sample

Alexithymia is characterised by deficits in emotional insight and self reflection, that impact on the efficacy of psychological treatments. Given the high prevalence of alexithymia in Alcohol Use Disorders, valid assessment tools are critical. The majority of research on the relationship between alexithymia and alcohol-dependence has employed the self-administered Toronto Alexithymia Scale (TAS-20). The Observer Alexithymia Scale (OAS) has also been recommended. The aim of the present study was to assess the validity and reliability of the OAS and the TAS-20 in an alcohol-dependent sample. Two hundred and ten alcohol-dependent participants in an outpatient Cognitive Behavioral Treatment program were administered the TAS-20 at assessment and upon treatment completion at 12 weeks. Clinical psychologists provided observer assessment data for a subsample of 159 patients. The findings confirmed acceptable internal consistency, test-retest reliability and scale homogeneity for both the OAS and TAS-20, except for the low internal consistency of the TAS-20 EOT scale. The TAS-20 was more strongly associated with alcohol problems than the OAS.

A confirmatory factor analysis of the Toronto Alexithymia Scale (TAS-20) in an alcohol-dependent sample

Confirmatory factor analyses were conducted to evaluate the factorial validity of the Toronto Alexithymia Scale in an alcohol-dependent sample. Several factor models were examined, but all models were rejected given their poor fit. A revision of the TAS-20 in alcohol-dependent populations may be needed.

The effect of metamictization on the Raman spectroscopy of the uranyl titanate mineral davidite (La,Ce)(Y,U,Fe2+)(Ti,Fe3+)20(O,OH)38

Raman spectra of the uranyl titanate mineral davidite-(La) (La,Ce)(Y,U,Fe2+)(Ti,Fe3+)20(O,OH)38 were analysed and related to the mineral structure. Observed bands are attributed to the TiO and (UO2)2+ stretching and bending vibrations, U-OH bending vibrations, H2O and (OH)- stretching, bending and libration modes. U-O bond lengths in uranyls and O-H���O bond lengths are calculated from the wavenumbers assigned to the stretching vibrations. Raman bands of davidite-(La) are in harmony with those of the uranyl oxyhydroxides. The mineral davidite-(La) is metamict as is evidenced by the intensity of the UO stretching and bending modes being of lower intensity than expected and with bands that are significantly broader.

meso-Porphyrinylphosphine Oxides: Mono- and Bidentate Ligands for Supramolecular Chemistry and the Crystal Structures of Monomeric [10,20-Diphenylporphyrinatonickel(II)-5,15-diyl]-bis-[P(O)Ph2] and Polymeric Self-Coordinated ([10,20-Diphenylporphyrinatozinc(II)-5,15-diyl]-bis-[P(O)Ph2])

A series of porphyrins substituted in one or two meso-positions by diphenylphosphine oxide groups has been prepared by the palladium catalysed reaction of diphenylphosphine or its oxide with the corresponding bromoporphyrins. Compounds {MDPP-[P(O)Ph2]n} (M = H2, Ni, Zn; H2DPP = 5,15-diphenylporphyrin; n = 1, 2) were isolated in yields of 60-95%. The reaction is believed to proceed via the conventional oxidative addition, phosphination and reductive elimination steps, as the stoichiometric reaction of ��1-palladio(II) porphyrin [PdBr(H2DPP)(dppe)] (H2DPP = 5,15-diphenylporphyrin; dppe = 1,2-bis(diphenylphosphino)ethane) with diphenylphosphine oxide also results in the desired mono-porphyrinylphosphine oxide [H2DPP-P(O)Ph2]. Attempts to isolate the tertiary phosphines failed due to their extreme air-sensitivity. Variable temperature 1H NMR studies of [H2DPP-P(O)Ph2] revealed an intrinsic lack of symmetry, while fluorescence spectroscopy showed that the phosphine oxide group does not behave as a "heavy atom" quencher. The electron withdrawing effect of the phosphine oxide group was confirmed by voltammetry. The ligands were characterised by multinuclear NMR and UV-visible spectroscopy as well as mass spectrometry. Single crystal X-ray crystallography showed that the bis(phosphine oxide) nickel(II) complex {[NiDPP-[P(O)Ph2]2} is monomeric in the solid state, with a ruffled porphyrin core and the two P=O fragments on the same side of the average plane of the molecule. On the other hand, the corresponding zinc(II) complex formed infinite chains through coordination of one Ph2PO substituent to the neighbouring zinc porphyrin through an almost linear P=O---Zn unit, leaving the other Ph2PO group facing into a parallel channel filled with disordered water molecules. These new phosphine oxides are attractive ligands for supramolecular porphyrin chemistry.

Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats

BACKGROUND: There has been some difficulty getting standard laboratory rats to voluntarily consume large amounts of ethanol without the use of initiation procedures. It has previously been shown that standard laboratory rats will voluntarily consume high levels of ethanol if given intermittent-access to 20% ethanol in a 2-bottle-choice setting [Wise, Psychopharmacologia 29 (1973), 203]. In this study, we have further characterized this drinking model. METHODS: Ethanol-na��ve Long-Evans rats were given intermittent-access to 20% ethanol (three 24-hour sessions per week). No sucrose fading was needed and water was always available ad libitum. Ethanol consumption, preference, and long-term drinking behaviors were investigated. Furthermore, to pharmacologically validate the intermittent-access 20% ethanol drinking paradigm, the efficacy of acamprosate and naltrexone in decreasing ethanol consumption were compared with those of groups given continuous-access to 10 or 20% ethanol, respectively. Additionally, ethanol consumption was investigated in Wistar and out-bred alcohol preferring (P) rats following intermittent-access to 20% ethanol. RESULTS: The intermittent-access 20% ethanol 2-bottle-choice drinking paradigm led standard laboratory rats to escalate their ethanol intake over the first 5 to 6 drinking sessions, reaching stable baseline consumption of high amounts of ethanol (Long-Evans: 5.1 +/- 0.6; Wistar: 5.8 +/- 0.8 g/kg/24 h, respectively). Furthermore, the cycles of excessive drinking and abstinence led to an increase in ethanol preference and increased efficacy of both acamprosate and naltrexone in Long-Evans rats. P-rats initiate drinking at a higher level than both Long-Evans and Wistar rats using the intermittent-access 20% ethanol paradigm and showed a trend toward a further escalation in ethanol intake over time (mean ethanol intake: 6.3 +/- 0.8 g/kg/24 h). CONCLUSION: Standard laboratory rats will voluntarily consume ethanol using the intermittent-access 20% ethanol drinking paradigm without the use of any initiation procedures. This model promises to be a valuable tool in the alcohol research field.