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Sodium valproate/valproic acid (VPA), a histone deacetylase inhibitor, and 5-aza-2-deoxycytidine (5-aza-CdR), a DNA methyltransferase 1 (DNMT1) inhibitor, induce DNA demethylation in several cell types. In HeLa cells, although VPA leads to decreased DNA 5-methylcytosine (5mC) levels, the demethylation pathway involved in this effect is not fully understood. We investigated this process using flow cytometry, ELISA, immunocytochemistry, Western blotting and RT-qPCR in G1 phase-arrested and proliferative HeLa cells compared to the presumably passive demethylation promoted by 5-aza-CdR. The results revealed that VPA acts predominantly on active DNA demethylation because it induced TET2 gene and protein overexpression, decreased 5mC abundance, and increased 5-hydroxy-methylcytosine (5hmC) abundance, in both G1-arrested and proliferative cells. However, because VPA caused decreased DNMT1 gene expression levels, it may also act on the passive demethylation pathway. 5-aza-CdR attenuated DNMT1 gene expression levels but increased TET2 and 5hmC abundance in replicating cells, although it did not affect the gene expression of TETs at any stage of the cell cycle. Therefore, 5-aza-CdR may also function in the active pathway. Because VPA reduces DNA methylation levels in non-replicating HeLa cells, it could be tested as a candidate for the therapeutic reversal of DNA methylation in cells in which cell division is arrested.

The enterococci, which are among the leading causes of multidrug-resistant (MDR) hospital infection, are notable for their environmental ruggedness, which extends to intrinsic antibiotic resistance. To identify genes that confer this unique property, we used Tn-seq to comprehensively explore the genome of MDR Enterococcus faecalis strain MMH594 for genes important for growth in nutrient-containing medium and with low-level antibiotic challenge. As expected, a large core of genes for DNA replication, expression, and central metabolism, shared with other bacteria, are intolerant to transposon disruption. However, genes were identified that are important to E. faecalis that are either absent from or unimportant for Staphylococcus aureus and Streptococcus pneumoniae fitness when similarly tested. Further, 217 genes were identified that when challenged by sub-MIC antibiotic levels exhibited reduced tolerance to transposon disruption, including those previously shown to contribute to intrinsic resistance, and others not previously ascribed this role. E. faecalis is one of the few Gram-positive bacteria experimentally shown to possess a functional Entner-Doudoroff pathway for carbon metabolism, a pathway that contributes to stress tolerance in other microbes. Through functional genomics and network analysis we defined the unusual structure of this pathway in E. faecalis and assessed its importance. These approaches also identified toxin-antitoxin and related systems that are unique and active in E. faecalis. Finally, we identified genes that are absent in the closest nonenterococcal relatives, the vagococci, and that contribute importantly to fitness with and without antibiotic selection, advancing an understanding of the unique biology of enterococci. IMPORTANCE Enterococci are leading causes of antibiotic-resistant infection transmitted in hospitals. The intrinsic hardiness of these organisms allows them to survive disinfection practices and then proliferate in the gastrointestinal tracts of antibiotic-treated patients. The objective of this study was to identify the underlying genetic basis for its unusual hardiness. Using a functional genomic approach, we identified traits and pathways of general importance for enterococcal survival and growth that distinguish them from closely related pathogens as well as ancestrally related species. We further identified unique traits that enable them to survive antibiotic challenge, revealing a large set of genes that contribute to intrinsic antibiotic resistance and a smaller set of uniquely important genes that are rare outside enterococci.

Made available in DSpace on 2020-12-12T01:23:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Constitutive heterochromatin typically exhibits low gene density and is commonly found adjacent or close to the nuclear periphery, in contrast to transcriptionally active genes concentrated in the innermost nuclear region. In Triatoma infestans cells, conspicuous constitutive heterochromatin forms deeply stained structures named chromocenters. However, to the best of our knowledge, no information exists regarding whether these chromocenters acquire a precise topology in the cell nuclei or whether their 18S rDNA, which is important for ribosome function, faces the nuclear center preferentially. In this work, the spatial distribution of fluorescent Feulgen-stained chromocenters and the distribution of their 18S rDNA was analyzed in Malpighian tubule cells of T. infestans using confocal microscopy. The chromocenters were shown to be spatially positioned relatively close to the nuclear periphery, though not adjacent to it. The variable distance between the chromocenters and the nuclear periphery suggests mobility of these bodies within the cell nuclei. The distribution of 18S rDNA at the edge of the chromocenters was not found to face the nuclear interior exclusively. Because the genome regions containing 18S rDNA in the chromocenters also face the nuclear periphery, the proximity of the chromocenters to this nuclear region is not assumed to be associated with overall gene silencing. Department of Structural and Functional Biology Institute of Biology University of Campinas (Unicamp), Rua Monteiro Lobato 255 Department of Biology Institute of Biosciences State University of S o Paulo (Unesp), Avenida 24-A, 1515 Superintendence for Control of Endemic Diseases (SUCEN), Rua Afonso Pessini, 86 Department of Biology Institute of Biosciences State University of S o Paulo (Unesp), Avenida 24-A, 1515

The plant-based biopesticides have been proposed as insect pest control tools that seem to be safer for the environment and human health when compared to synthetic conventional molecules. However, such assumptions are generally made without considering the absence of detrimental effects on sublethally-exposed non-target organisms or showing the physiological basis of the selective action of such botanical products. Thus, by using in silico-based and in vivo toxicological approaches, the present investigation aimed to disentangle the ecotoxicological selectivity of clove, Syzygium aromaticum, essential oil against the aphid Rhopalosiphum maidis and the non-target ladybeetle, Coleomegilla maculata. We also investigated whether the sublethal exposure to clove essential oil would affect the locomotory and predatory abilities of C. maculata. We found that the clove essential oil concentration estimated to kill 95% (LC95: 0.17 μL/cm2) of the aphids was lethal to

Summary Chrysocyon brachyurus, the largest South American canid, is a native species of the Brazilian cerrado. The present study is aimed to report the occurrence of the trematode, Athesmia foxi, in the liver of a new host, C. brachyurus, and to describe its morphology and pathology. One C. brachyurus individual was necropsied and examined for the presence of parasites. Worms were collected from the bile ducts and based on morphological and morphometrical characteristics, such as a relatively large, slender, aspinose, elongated shape with vitellarium present on the upper left side of the body were identified as A. foxi. On the host, hepatic lesions limited to the bile ducts and periportal regions, were characterized as chronic-active cholangitis, biliary hyperplasia, and fibrosis. This is the first report of A. foxi parasitizing C. brachyurus, demonstrating that this parasite has no host specificity and can be widely distributed. A. foxi lesions noted in C. brachyurus are similar to those noted in various other mammalian hosts.

The larvae of the two distantly related nonsocial bees Ericrocis lata (Apidae) and Hesperapis (Carinapis) rhodocerata (Melittidae), which develop mostly under arid desert areas of North America, and that differ in that they either spin (E. lata) or do not spin (H. rhodocerata) protective cocoons before entering diapause, produce transparent films that cover the larval integument. To understand the nature of these films, their responses to topochemical tests and their characteristics when examined with fluorescence and high-performance polarization microscopy and microspectroscopy were studied. A positive staining by Sudan black B, birefringence of negative sign, and a Fourier transform-infrared (FT-IR) spectrum typical of lipids were detected for the integument covering of both species. The FT-IR signature, particularly, suggests a wax chemical composition for these lipid coverings, resembling the waxes that are used as construction materials in the honey cells produced by social bees. Considering the arid environmental conditions under which these larvae develop, we hypothesize that their covering films may have evolved as protection against water depletion. This hypothesis seems especially appropriate for H. rhodocerata larvae, which are capable of undergoing a long diapause period in the absence of a protective cocoon.

A previous study has not revealed the participation of a mucous component in the cocoon wall of the solitary bee, Lithurgus chrysurus, differing from the cocoon structure reported for many other bee species. However, uncertainty remains, because only the median and rear zones of this cocoon type have thus far been analyzed. Here, we studied the front zone of this cocoon, searching its components and their organization, to fill this knowledge gap. Topochemical assays, polarization microscopy and Fourier transform-infrared (FT-IR) microspectroscopy were used to study cross sections from L. chrysurus cocoon. Three main layers differing in structural organization were found to compose the cocoon wall. Silk fibroins were assumed to constitute the filamentous threads of the inner and outer layers and the laminar structure of the intermediate layer. Deduced from its topochemical properties and FT-IR spectral signature, a foamy material containing mucin glycoproteins and carboxylated acid glycosaminoglycans was found in the intermediate layer. FT-IR analysis using a Savitzky-Golay 2nd-derivative and absence of linear dichroism and birefringence phenomena suggest that a random-coil secondary structure predominates in the foam component. Co-existence of α-helical and β-sheet conformations is also hypothesized for the fibroin component of this cocoon. It is thus concluded that in addition to fibroin elements, a mucous component, likely contributed by a Malpighian tubule secretion, integrates the composition of the front zone of the cocoon wall of L. chrysurus. In addition, the FT-IR analysis of the inner layer silk of this cocoon suggests significant differences in comparison to the silk fibroins of the silkworm, and some minor spectral differences in comparison to published data on the honeybee silk, with respect to protein secondary structure.

Malpighian tubules of Triatoma infestans cultivated in vitro in presence of 0.05 mM sodium valproate (VPA) for 1-4 h had their nuclear phenotypes investigated regarding induction of chromatin remodeling. Under present conditions, although a few nuclei demonstrated heterochromatin decondensation, no overall differences resulted in comparison to tests in which live insects are injected with VPA.

Made available in DSpace on 2018-12-11T16:54:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Panstrongylus megistus, a potential vector of Chagas disease, currently occupies a wider geographic distribution in Brazil than Triatoma infestans, another member of the hemipteran Reduviidae family and a vector of the same disease. A small heterochromatic body (chromocenter) formed by the Y chromosome is evident in the somatic cells of P. megistus, differing in size and chromosome type contribution from the well-studied chromocenters present in T. infestans. While the overall distribution of histone epigenetic marks differ when comparing the heterochromatin and euchromatin territories in T. infestans, no similar data have been established for other hemipteran reduviids, including P. megistus. In the present work, histone acetylation and methylation marks were investigated in cells of Malpighian tubules of P. megistus 5th instar nymphs using immunocytochemical assays and compared to previously published data for T. infestans. Although similarities between these species were found regarding absence of acetylated H3K9, H4K8 and H4K16, and H3K9me and H3K9me2 in the chromocenter, presence of these marks in euchromatin, and presence of H3K9me3 in the chromocenter, no intimate association of acetylated H4K8 and 18S rDNA was revealed in the chromocenter of P. megistus. The elevated abundance of H3K9me2 marks at the nuclear periphery in P. megistus cells, differing from data for T. infestans, is suggested to reflect differences in the interaction of lamina-associated chromatin domains with the nuclear lamina, methyl-transferase modulation and/or association with the last DNA endoreplication step in 5th instar nymphs, which is a matter for further investigation. Department of Structural and Functional Biology Institute of Biology University of Campinas (Unicamp) Department of Biology Institute of Biosciences State University of São Paulo (Unesp) Superintendence for Control of Endemic Diseases SUCEN Department of Genetics “Luiz de Queiroz” College of Agriculture (ESALQ) University of São Paulo (USP) Department of Morphology Institute of Biomedical Sciences Federal University of Uberlândia Department of Biology Institute of Biosciences State University of São Paulo (Unesp) CNPq: 132341/2010-7 CNPq: 304668/2014-1

The cocoon of Lithurgus chrysurus Fonscolobe, a univoltine species belonging to the megachilid subfamily Lithurginae, was studied using polarization microscopy and topochemical methods. The aim of the study was to establish the composition and structure of the cocoon wall of this bee in comparison with reported data for other species of this subfamily. The cocoon was found to be composed by macromolecularly oriented, positively birefringent silk protein layers that acquire a complex and thicker multilayered distribution at its rear zone. Although the composition and distribution of the silk threads in this species' cocoon was considered similar to the previously described patterns for other lithurgine bees, the absence of a mucous layer, typically produced by Malpighian tubules, differed from reported data for other species of the same subfamily.

Potato virus Y (PVY) is a reemerging problem in potato production in North America. Although the “ordinary” strain, PVYO, is still the dominant isolate in U.S. seed potatoes, the recombinant strain of the virus PVYN-Wi (= PVYN:O) has become widespread. An increase in the prevalence of a PVY strain could be due to differences in the efficiency of transmission by aphid vectors. The transmission efficiency by a clone of Myzus persicae was determined for five isolates each of PVYO and PVYN-Wi. An aphid transmission assay was developed based on the use of potato seedlings from true potato seed, allowing for greater control of plant age and growth stage. No apparent differences in transmission by M. persicae were observed. Single isolates of PVYO and PVYN-Wi were tested for their ability to be transmitted from potato to potato by five aphid species: Aphis glycines, A. gossypii, A. nasturtii, M. persicae, and Rhopalosiphum padi. Both PVY isolates showed a similar transmission phenotype in being transmitted efficiently by M. persicae but very poorly or not at all by A. glycines, A. gossypii, and R. padi. The aphid A. nasturtii transmitted both isolates with an intermediate level of efficiency. The data do not support a model for a differential aphid transmissibility being responsible for the increase in the prevalence of PVYN-Wi.

Nuptial flight is a critical phase in the reproduction of most ant species. Here, we describe the nuptial flight and mating behaviour of Acromyrmex ameliae, a leaf-cutting ant social parasite. We observed a nuptial flight of this parasite in a parasitized colony of Acromyrmex subterraneus subterraneus – known as a host of the social parasite in nature – and we registered the initial and final times of copula as well as the time the male dispended attempting to mate. Mating flight occurred after the beginning of the light phase in the laboratory. Agitation of host workers preceded the nuptial flight, without aggression between host workers and sexuals. Alates were attracted to the light bulbs of the room and formed couples that dropped to the ground immediately after their formation. Copula lasted on the average 1.6 ± 0.6 min. During mating attempts, the male put his forelegs on the female's abdomen while she remained walking on the ground. Copula normally occurred when the female stopped or slowe...

RNA-silencing mechanisms control many aspects of gene regulation including the detection and degradation of viral RNA through the action of, among others, Dicer-like and Argonaute (AGO) proteins. However, the extent to which RNA silencing restricts virus host range has been difficult to separate from other factors that can affect virus-plant compatibility. Here we show that Potato virus X (PVX) can infect Arabidopsis (Arabidopsis thaliana), which is normally a nonhost for PVX, if coinfected with a second virus, Pepper ringspot virus. Here we show that the pepper ringspot virus 12K protein functions as a suppressor of silencing that appears to enable PVX to infect Arabidopsis. We also show that PVX is able to infect Arabidopsis Dicer-like mutants, indicating that RNA silencing is responsible for Arabidopsis nonhost resistance to PVX. Furthermore, we find that restriction of PVX on Arabidopsis also depends on AGO2, suggesting that this AGO protein has evolved to specialize in antiviral defenses.

This study aimed to contribute to our knowledge of the parasite-vector interaction associated with Trypanosoma cruzi (Chagas, 1909) infection in Panstrongylus megistus (Burmeister, 1835), an important vector of Chagas' disease in Brazil. The prevalence and intensity of T. cruzi infection, the incidence of metacyclogenesis and the frequency of nuclear phenotypes in Malpighian tubules were investigated in nymphs of P. megistus, reared at 28 °C and subjected to heat shock (40 °C, 1 hour) two days after infection with T. cruzi II (Y strain). Following the 45-day post-infection period, the frequency of epimastigotes was much higher than that of trypomastigotes in both heat-shocked and non-shocked insects, and the prevalence of infection was not altered by heat shock. Fewer epimastigotes and trypomastigotes were found in the infected insects subjected to the heat shock, indicating that the multiplication and metacyclogenesis of the parasites were affected by the stress. In infected specimens heat shock promoted an increased frequency of cell nuclei with heterochromatin decondensation, a cell survival response to stress, and did not affect insect survival. The effects of infection and heat shock, especially on the multiplication and metacyclogenesis of T. cruzi, and the observed resistance to heat shock developed by P. megistus nymphs are suggestive that they should be considered when adequate conditions for rearing these infected insects in the laboratory are pursued.

The impact of citrus stubborn disease, caused by Spiroplasma citri, on citrus production is associated with the symptom severity of infected trees but its association with bacterial levels and virulence are unknown. Fifty-eight S. citri isolates were cultivated from severely and mildly symptomatic trees and randomly amplified polymorphic DNA and short-sequence repeat fingerprinting differentiated four major S. citri genotypes among these isolates. Each genotype was present in both mildly and severely symptomatic trees, suggesting that readily detectable genetic differences in the S. citri populations did not account for differences in disease severity. No variation in the size of amplicons of the pathogenicity-related fructose operon was observed in isolates from trees having varying degrees of symptom expression. Quantitative polymerase chain reaction demonstrated that spiroplasma titer is over 6,000 times higher in fruit from severely symptomatic than from mildly symptomatic trees. The genotypic similarities among S. citri isolates from severely and mildly symptomatic trees, and the consistently higher bacterial titer in the former than in the latter, suggests that titer but not genotype is, at least in part, responsible for the greater symptom severity in some of the S. citri-affected trees in the orchard evaluated.

Cowpea chlorotic mottle virus (CCMV) is a bromovirus transmitted by species of chrysomelid beetles, including the spotted cucumber beetle, Diabrotica undecimpunctata howardii Barber. An experimental system was set up to identify the viral determinant(s) of the beetle transmission of CCMV. Nicotiana clevelandii was selected as an experimental plant host because it supports the replication and accumulation of both CCMV and a second member of the family Bromoviridae, cucumber mosaic virus (CMV). Using a reverse genetic system for CMV, a cDNA copy of the CCMV capsid protein (CP) gene was substituted for that of the CMV CP gene. The resulting 'CMV-hybrid' consisted of wild-type CMV RNA1, RNA2, and a chimeric CMV RNA3 expressing the CCMV structural protein. The CMV-hybrid replicated and formed virions in N. clevelandii; in electron micrographs the hybrid virus was indistinguishable from CCMV. In beetle feeding assays, both CCMV and the CMV-hybrid were transmitted by D. undecimpunctata, while beetle transmission of CMV was not observed. Conversely, only CMV was observed to be transmitted by the aphid Myzus persicae. Surprisingly, the CMV-hybrid was transmitted more efficiently than the parental CCMV, and a virus-induced alteration in beetle feeding behaviour is proposed to account for the difference. These results indicate that the CCMV CP is a viral determinant for beetle vector transmission.

Carrot purple leaf disease was first reported in 2006 in the state of Washington and was associated with Spiroplasma citri. The disease also was reported in California in 2008. The objectives of this work were to fulfill Koch's postulates and to determine 1) whether the beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), transmits carrot [Daucus carota L. subsp. Sativus (Hoffm.) Arcang] isolates of S. citri; and 2) whether carrot and citrus [Citrus sinensis (L.) Osb.]derived spiroplasmas are pathogenic to both plant species. C. tenellus adults received a 24-h acquisition access period to a diet containing carrot-derived S. citri. After 30 d, insects were transferred to healthy carrot seedlings (five per plant). Negative controls were carrot and periwinkle [Catharanthus roseus (L.) G. Don] plants exposed to diet-only-fed insects, and positive controls were periwinkle plants exposed to insects fed on spiroplasma-supplemented diet. Purple carrot leaves and small, chlorotic periwi...

Mello, L. G. S., and Rose, G. A. 2009. The acoustic dead zone: theoretical vs. empirical estimates, and its effect on density measurements of semi-demersal fish. – ICES Journal of Marine Science, 66: 1364–1369. The height of the acoustic dead zone, the region near the seabed where fish cannot be resolved acoustically, was calculated both theoretically (DZt) and empirically (DZe). The DZe was based on measurements of depth and trawl geometry from sensors (SCANMAR) mounted on a bottom trawl deployed during a survey off Newfoundland and Labrador in winter 2007. Acoustic data were acquired while trawling, using a 38-kHz echosounder (Simrad EK500) with a hull-mounted transducer. The DZe was calculated as the difference between the trawl-footrope depth and the corresponding acoustically sensed, seabed depth. EK500 and SCANMAR estimates of seabed depth were significantly different. The fish caught were mostly Atlantic cod (Gadus morhua). The estimates of DZe ranged between 2.0 and 3.5 m and were greater than DZt by 0.1–0.9 m in more than half the cases. Three values of acoustically derived cod densities were estimated for each tow, without dead-zone correction and with corrections for DZt and DZe. When compared with DZt corrections, DZe resulted in negative (6–12%) and positive (9–35%) corrections to cod density. A general linear model revealed that the seabed depth gradient, standard deviation of estimated fish density in the dead zone, and wind direction and force explained 85% of the difference between DZt and DZe estimates. These factors affected the detection of the seabed and biased acoustically derived indices of demersal-fish abundance.

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-α, interleukin-6, transforming growth factor-β, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright © 2008 John Wiley & Sons, Ltd.

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.

Yokomi, R. K., Mello, A. F. S., Saponari, M., and Fletcher, J. 2008. Polymerase chain reactionbased detection of Spiroplasma citri associated with citrus stubborn disease. Plant Dis. 92:253260. Polymerase chain reaction (PCR)-based detection of citrus stubborn disease was improved using primers based on sequences of the P89 putative adhesin gene and the P58 putative adhesin multigene of Spiroplasma citri. Real-time PCR also was developed with detection limits estimated to be between 10–4 and 10–4 ng by serial dilution of a recombinant S. citri plasmid into DNA extracts from healthy Madam Vinous sweet orange. PCR for the detection of S. citri by these new primers was validated by comparing culturing of the pathogen, the traditional method of diagnosis, with PCR assays from samples taken from two citrus plots in Kern County, CA. Fruit columella was collected from 384 and 377 individual trees in each of two fields, respectively; one portion was used for culturing and the other for DNA extraction and PCR. PCR results matched those of culturing 85 to 100% of the time depending on the primers used. More importantly, PCR detected S. citri from culture-negative trees in 5 to 15% of the cases, suggesting that PCR performed as well or better than culturing for detection of S. citri in field samples. Realtime PCR proved to be the best method for detection. Differential reaction of the samples to the P58 primer pairs suggested that two populations of S. citri occur in historical and present-day field isolates. Citrus stubborn disease incidence was estimated to be 58.3 and 3.7% in the two orchards. The results presented here support the use of PCR for reliable detection of S. citri in field trees. Additional keyword: epidemiology

Forms of the protozoan of the Hepatozoon genus are detected free in the circulation and also within some of the erythrocytes of infected snakes. In healthy snakes, DNA fragmentation and cell death usually affect a few circulating erythrocytes in agreement with the long life span expected for these cells. In the present study we investigated whether infection by Hepatozoon spp. affected the incidence of DNA fragmentation and cell death in erythrocytes from the rattlesnake, Crotalus durissus terrificus. Methods such as the kinetics of Feulgen-DNA hydrolysis, and the TUNEL and comet assays, previously used for the study of chromatin organization and DNA fragmentation in erythrocytes of healthy snakes, were used. The results indicated that Hepatozoon spp. increased the DNA fragmentation and chromatin condensation typical of cell death in circulating erythrocytes of C. d. terrificus, including cells that do not harbour the parasite. The Hepatozoon infection is thus suggested to accelerate destruction of erythrocytes in the rattlesnake, not only affecting cells harbouring the parasite, but also in those without it.

Antagonist plants have been used as an alternative to control plant-parasitic nematodes. One in vitro assay and two greenhouse experiments were carried out to evaluate the effects of Mexican-tea (Chenopodium ambrosioides) on the control of Pratylenchus brachyurus. In the in vitro test an extract of Mexican-tea was tested at four concentrations (20; 2; 0.2 and 0.02%), in suspensions containing 500 exemplars of P. brachyurus. After 48 h, living nematodes were collected and counted. A nematicidal action of Mexican-tea was observed by higher juvenile mortality in comparison with chemical control (aldicarb). In greenhouse experiments, 1 and 2, the Mexican-tea and soybean plants were inoculated with 1,500 and 5,000 P. brachyurus, respectively. Forty-five days later, plant shoots were incorporated in soil, as follows: shoots of Mexican-tea, were incorporated; shoots of soybean were incorporated; and plant shoots were not incorporated. One month later, each pot received one soybean plant, to act as a biological indicator of parasitism. At the end of experimental period (45 days later) the final nematode population was estimated by counting the nematodes extracted from soybean roots and soil, and data were collected of fresh root weight and shoot dry weight. Mexican-tea reduced the nematode population, although soybean plants had been affected by phytotoxicity.

The effect of fasting and of fasting followed by refeeding and heat shock was studied in Malpighian tu- bules of fourth instar nymphs of the blood-sucking hemipteran, Panstrongylus megistus (Burmeister). The aim was to detect different frequencies of nuclear changes (apoptosis, necrosis, heterochromatin decondensation) under conditions assumed to be stressful in blood-sucking hemipterans. The insects were fasted for up to 90 days at 28 o C and their survival was followed daily. Groups of nymphs were separated each month, with part of the group being refed and the other part kept fasting. Insects in each of these subgroups received either a heat shock at 40 o C for 1 h or were maintained at 28 o C (control for heat shock). The Malpighian tubules were removed one and seven days af- ter each assay and subjected to the Feulgen reaction for identification and counting of the various nuclear pheno- types. Insect survival was high (90%) even after 40 days of starvation but decreased thereafter. Necrosis rather than apoptosis, increased with fasting. Feeding after fasting increased the frequency of apoptosis but not of necrosis. The short heat shock as used here did not additionally affect the responses induced by fasting and refeeding. P. megistus nymphs could withstand relatively long periods of fasting although individual variation in the mean length of cell survival had been found especially after a three-month fasting. The results related to cell necrosis suggest that part of the Malpighian tubule cells may not have developed highly efficient mechanisms for dealing with fasting. For those cells resistant to fasting, feeding subsequent to fasting acted only as a mild stressing agent and heat shock was well tolerated. The ability of P. megistus nymphs to withstand and recover from periods of inadequate or poor nu- trition inclusive in association to a short heat shock as demonstrated here is certainly an important adaptation for the survival of the species.

Growth of 2659 Atlantic cod Gadus morhua aged 4 to 9 years examined in Placentia Bay, Newfoundland, peaked in most cases in June and was at a minimum in October or November. Water temperature, partial fullness index (J P ) and gonado-somatic index (I G ) explained between 31 and 52% of the monthly variability in growth. Temperature and I P of capelin Mallotus villosus had significant effects on growth of all age groups and explained most of the variance for ages 6-8 and 4-5 years, respectively. The I P of large invertebrates (ages 4 to 7 years), sandlance (Ammodytes sp. age 6 years) and demersal fishes (age 9 years) had age-specific effects in the model. Overall, amphipods, decapods and echinoderms dominated the Atlantic cod diet in most seasons, but fish consumption by Atlantic cod was high in June and July, particularly on capelin. The rapid increase in somatic mass during June and July occurred despite cold water temperatures (< 3° C at 50m) and moderate to high gonado-somatic index. The findings of this study suggest that when food was not a limiting factor, growth tended to increase even when Atlantic cod occupied colder waters, but when food was limiting, the opposite may have occured.

O mofo-branco, causado por Sclerotinia sclerotiorum, e uma doenca importante na cultura do tomateiro. O objetivo do trabalho foi avaliar o efeito de extratos de plantas, residuos animais e subprodutos industriais no desenvolvimento micelial e na producao de esclerodios do fungo "in vitro". Os tratamentos testados foram diferentes concentracoes de licor pirolenhoso, oleo de nim, glutamato monossodico, biossolido e composto orgânico [(borra de cafe (50%) cinza de carvao (10%), residuo de milho (25%), esterco de aves (12,5%) e farinha de aves (2,5%)], alem de duas testemunhas, sem adicao de produtos ao meio e com adicao do fungicida procimidone. O fungo submetido aos diferentes tratamentos foi incubado a temperatura de 22oC e luminosidade constante de 260 lux. Foram avaliadas o crescimento micelial, a producao de esclerodios e a viabilidade dos mesmos aos 7 e 17 dias apos a repicagem do fungo para meio de neon. O composto orgânico a 30 % mostrou-se eficiente na inibicao do crescimento micelial e na producao de esclerodios. Este tratamento, assim como o oleo de nim a 0,5%, foi analisado com relacao a sua influencia na microbiota no solo por meio de uma analise de respirometria, que indicou que ambos os tratamentos foram degradados rapidamente, nao causando assim maleficios a mesma.

Geostatistical methods were used to (i) quantify fish aggregation patterns over a range of scales (100 m to 67 km) using both simulated and acoustic density data of Atlantic cod (Gadus morhua) and (ii) examine how changes in aggregation patterns influenced the precision of geostatistical density indices. Variogram parameters (range, sill, and nugget) reflected changes in distribution patterns. Variograms of dispersed and low-density aggregations had large range and small sill and nugget values. In contrast, when fish were aggregated in a small portion of the study area, the range was low and the sill and nugget large. The precision of density indices (coefficient of variation) was below 20% in all cases but at a maximum during summer when cod were broadly distributed in small, moderate to dense aggregations. Geostatistical modeling allowed us to describe and quantify distribution patterns of fish density over different scales of observation, comparisons of spatiotemporal changes in density distribution, and estimations of the precision of density indices while accounting for the effects of heterogeneous distributions, outliers and the typically large number of zero and low-density observations. Geostatistical methods have particular applicability to fishes exhibiting gregarious behaviour and seasonally variable distributions, which include many temperate and high-latitude fish species.

The Malpighian tubules of the hemipteran, Triatoma infestans, grow through endopolyploidy. As a result, the number of tubule cells is constant throughout nymphal development and adult life, and nuclei with 32 C and 64 C Feulgen-DNA classes are maximally reached through DNA endoreplication. Feulgen-DNA classes > 64 C, with resulting giant nuclei, may occur only through nuclear and cellular fusions, elicited by stressing agents such as heat shock and fasting. Although apoptosis has been reported in cells of Malpighian tubule epithelia, especially under stress conditions, the association between cell death and the degree of polyploidy resulting from different phenomena (either development or stress) has not yet been investigated. In this study, image analysis was used to estimate the Feulgen-DNA values of apoptotic nuclei in the Malpighian tubule cells of 5th instar nymphs of T. infestans subjected to heat shock at 40°C for 1 h and fasted for 15 days before the shock. The susceptibility to apoptosis...

than in 2002 (41%), P= 0.027. ABPM recordings showed significantly fewer undiagnosed hypertensive individuals in 2012 (14%) that in 2002 (28%), P< 0.001. Significantly more hypertensive individuals were optimally treated in 2012, (44%) versus (19%), P < 0.001. Minimum and mean systolic BP were significantly lower in the 2012 cohort, but maximum systolic BP and diastolic BP did not differ. Use of ACE inhibitor, angiotensin receptor blockers and diuretics had significantly increased over the 10-year interval. Patient reported rates of falls, dizziness and syncope had not risen significantly. Ischaemic heart disease was significantly less common in the 2012 cohort, P< 0.05. Conclusion: Hypertension in older people is better recognised and more effectively managed now than 10 years ago. This has not been associated with a significant change in rates of falls, dizziness or syncope.

The nuclear phenotypes of Malpighian tubule cells in fifth instar nymphs of Triatoma infestans, one of the most important vectors of Chagas disease, were studied following sequential shocks at 0 degrees C, separated by intervals of 8 h and 24 h at 30 degrees C, under conditions of moderate fasting and full nourishment. The insects pertained to colonies reared in the laboratory and originated from domestic specimens collected in the Brazilian states of São Paulo (north) and Minas Gerais (south). Since nuclear phenotypes in this species are affected by single cold shocks, it was expected that these phenotypes could also be changed by sequential shocks. Nuclear phenotypes indicative of mechanisms of cell survival (nuclear fusion and heterochromatin decondensation) and cell death (apoptosis and necrosis) were observed concomitantly in all the conditions tested. Nuclear fusion and heterochromatin decondensation were not found relevant for the presumed acquisition of the cold-hardening response in T. infestans. The decreased frequency of apoptosis and necrosis following sequential cold shocks including under fasting conditions, indicated that tolerance to sequential cold shocks occurred in T. infestans of the mentioned origin.

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40 degrees C, 1 h) or cold (5 or 0 degrees C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0 degrees C, respectively, after 8, 18, 24 and 72 h at 28 degrees C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0 degrees C were more effective than the temperatures of 35 and 5 degrees C in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.

In this study, we examined cell survival and cell death in response to heat shock in an insect organ composed of highly polyploid cells no longer capable of cell division. For this, the frequency of nuclear phenotypes in Feulgen-stained Malpighian tubules of the blood-sucking insect, Triatoma infestans, was analyzed at various times after a short heat shock with or without subsequent moderate fasting. Cell death DNA fragmentation was studied immunocytochemically. Normal phenotypes and phenotypes indicative of cell survival (heterochromatin decondensation, nuclear fusion) and death (apoptosis, necrosis) were observed, especially in heat-shocked specimens. While the number of total and normal nuclei decreased following heat shock, the frequency of apoptosis increased during a short period (7 days) after heat shock. During a 30-day period following heat shock, the frequency of necrosis in fasted but not in fully nourished nymphs increased simultaneously with a decrease in the frequency of apoptosis. This finding suggests that the stress promoted by heat shock, but not that associated with heat shock plus fasting, can be dealt with by the apoptosis program. When considering the forms of cell survival, heterochromatin decondensation was more relevant in fully nourished nymphs, whereas nuclear and cell fusions were more important in fasted specimens. The forms of cell survival and cell death reported here may have protected the organ from damage by the stressing agents. In cells with no induction or accumulation of heat-shock proteins, cell death and the forms of cell survival observed here were the probable consequence.Key words: heat shock, fasting, apoptosis, necrosis, cell survival, Triatoma infestans.

The nuclear phenotypes of Malpighian tubule epithelial cells of male nymphs of the blood-sucking insect, Panstrongylus megistus, subjected to short- and long-duration heat shocks at 40ºC were analyzed immediately after the shock and 10 and 30 days later. Normal nuclei with a usual heterochromatic body as well as phenotypes indicative of survival (unravelled heterochromatin, giants) and death (apoptosis, necrosis) responses were observed in control and treated specimens. However, all nuclear phenotypes, except the normal ones, were more frequent in shocked specimens. Similarly altered phenotypes have also been reported in Triatoma infestans following heat shock, although at different frequencies. The frequency of the various nuclear phenotypes observed in this study suggests that the forms of cell survival observed were not sufficient or efficient enough to protect all of the Malpighian tubule cells from the deleterious effects of stress. In agreement with studies on P. megistus survival following heat shock, only long-duration shock produced strongly deleterious effects.

We report a case of mixed intranasal infection caused by Fusarium solani and a zygomycete, with probable sinus and brain involvement. The patient had chronic myelogenous leukaemia and was treated with chemotherapy. Diagnosis of the infection was established by direct examination of the intranasal eschar and histopathological study of an excisional biopsy. Only F. solani was isolated from both specimens. Difficulties in diagnosis and treatment of these infections are discussed.

The nuclear phenotypes and survival of the hemipteran, Triatoma infestans (Hemiptera, Reduviidae), were studied in specimens treated with copper sulfate and methylmercury. The objective was to determine whether changes in chromatin supraorganization and insect survival similar to those promoted by other stressing agents could also be induced by heavy metals. At the concentrations used, copper sulfate and methylmercury were toxic to the cells, mainly inducing nuclear degeneration in the Malpighian tubules and being lethal to a large part of the insect population. Although some individual resistance was found, especially in fasted specimens, heavy metals were found to be much more lethal than was, for instance, a thermal shock at 0 °C for 12 h. The nuclear phenotypes detected after heavy metal treatment were similar to those reported under other stressing conditions. However, the frequency at which nuclei exhibited aspects of heterochromatin unraveling was much higher than that found in fasted and thermal-shocked specimens, and was independent of the heavy metal type used. If this phenotype represents an attempt to improve opportunities for extensive cell and insect survival, it was not sufficiently effective. In 5th instar nymphs, the effect of CuSO4 on chromatin supraorganization was detected at early steps of spermatogenesis but not in the cells which were at late spermiogenesis when the metal was administered. This is probably due to changes in nuclear protein composition and to the tightly packed state of the DNA- protein complexes occurring at spermiogenesis, which may protect chromatin from damages. However, when CuSO4 was supplied to 4th instar nymphs, it slowed down the spermiogenesis process, possibly due to several factors including Cu2+ binding to DNA phosphates. Resume L'effet des metaux lourds sur l'organisation superieure de la chromatine, les phenotypes nucleaires et la survie de Triatoma infestans Klug (Hemiptera, Reduviidae) Les phenotypes nucleaires et la survie de l'hemiptere Triatoma infestans ont ete etudies sur des specimens traites avec du sulfate de cuivre et du methyle de mercure. L'objectif a ete de determiner si les changements provoques par d'autres agents stressants sur l'organisation superieure de la chromatine et la survie de l'insecte peuvent etre induit aussi par les metaux lourds. Il est apparu qu'aux concentrations utilisees, le sulfate de cuivre et le methyle de mercure sont toxiques pour les cellules, provoquent principalement une degeneration nucleaire dans des organes tels que les tubes de Malpighi, et sont lethaux pour une grande partie de la population. Bien qu'une certaine resistance individuelle ait ete trouvee particulierement chez les specimens a jeun, les metaux lourds se sont reveles beaucoup plus lethaux que, par example, un choc thermique de 12 h a 0 °C. Les phenotypes nucleaires observes apres traitement aux metaux lourds sont similaires a ceux decrits dans d'autres conditions stressantes. Toutefois, la frequence avec laquelle les noyaux ont montre des aspects deroules de l'heterochromatine a ete beaucoup elevee que chez les specimens soumis au jeun ou a un choc thermique, quelque soit le metal utilisee. Si ce phenotype correspond a une tentative d'amelioration de la survie de la cellule et de l'insecte, cela n'est pas suffisant. Chez les nymphes au 5e. etage, l'effet du sulfate de cuivre sur l'organisation superieure de la chromatine a ete observe des les premieres etages de la spermatogenese, mais pas sur les cellules qui se trouvaient en spermiogenese avancee lorsque le metal fut fourni. Cela est probablement du a des changements dans la composition proteinique du noyaux et a la structure dense des complexes ADN-proteines lors de la spermiogenese, qui protegerait la chromatine. Toutefois, ce meme metal, fourni a des nymphes au 4e etage, relentit la spermiogenese, probablement a cause de facteurs diverses, inclusivement son attachement avec les phosphates de l'ADN.

BACKGROUND: Valproic acid (VPA) is a potent anticonvulsant that inhibits histone deacetylases. Because of this inhibitory action, we investigated whether VPA would affect chromatin supraorganization, mitotic indices and the frequency of chromosome abnormalities and cell death in HeLa cells. METHODOLOGY/PRINCIPAL FINDINGS: Image analysis was performed by scanning microspectrophotometry for cells cultivated for 24 h, treated with 0.05, 0.5 or 1.0 mM VPA for 1-24 h, and subjected to the Feulgen reaction. TSA-treated cells were used as a predictable positive control. DNA fragmentation was investigated with the TUNEL assay. Chromatin decondensation was demonstrated under TSA and all VPA treatments, but no changes in chromosome abnormalities, mitotic indices or morphologically identified cell death were found with the VPA treatment conditions mentioned above, although decreased mitotic indices were detected under higher VPA concentration and longer exposure time. The frequency of DNA fragmentation identified with the TUNEL assay in HeLa cells increased after a 24-h VPA treatment, although this fragmentation occurred much earlier after treatment with TSA. CONCLUSIONS/SIGNIFICANCE: The inhibition of histone deacetylases by VPA induces chromatin remodeling in HeLa cells, which suggests an association to altered gene expression. Under VPA doses close to the therapeutic antiepileptic plasma range no changes in cell proliferation or chromosome abnormalities are elicited. The DNA fragmentation results indicate that a longer exposure to VPA or a higher VPA concentration is required for the induction of cell death.

The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A(3)/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus.

Mg(2+)-dependent ATPases were investigated in Malpighian tubules of the blood-sucking insect, Triatoma infestans, with cytochemical procedures for light and electron microscopy. The aim was to establish patterns of enzyme occurrence in the blood-sucking insect under control rearing conditions for further comparisons with animals subjected to the action of stress factors. Enzyme activity was found in laminated "concretions" present in distal cells, in edges of urate crystals at the lumen of the proximal region of tubules, in the basement membrane of proximal cells, and variously distributed in plasmalemma invaginations of both distal and proximal cells. Presence of ATPases in the "concretions" and urate crystals is presumed to be due to engulfment of other ATPase-containing components during formation of these structures. Cytochemical reactivity in the basement membrane and plasmalemma invaginations is assumed to be involved with active transport of waste molecules from and to hemolymph and differs as a function of the Malpighian tubule region. This paper provides a basic understanding of the enzyme occurrence in the blood sucking insects, and can be used as a pattern for comparative means of the staining patterns among Triatominae species.

Although the Allium cepa test has been widely used to identify potentially cytotoxic and genotoxic pollutants in aquatic environments, variable non-standardized choices have been made regarding the number of plant bulbs and roots analyzed. We propose numbers for bulbs and roots per bulb when comparing the frequencies of micronuclei, mitotic anomalies and mitotic index with this test. Roots that had been treated with aqueous solutions, such as water samples collected in August 2007 from the Paraiba do Sul River at the Brazilian cities of Tremembe and Aparecida; negative and positive controls were used for bioassays. The presence of pollutants in the river water had been presumed based on our previous cytological data and an official report by the Sao Paulo State Environmental Agency (Brazil) on presence of fecal contaminants (Tremembe and Aparecida) and elevated dissolved aluminium (Aparecida) in the water under study. The sampling of ten bulbs and five roots per bulb was found adequate for comparative studies to evaluate with the A. cepa test the potential damage inflicted by pollutants in aquatic environments. Furthermore, even one bulb and one root per bulb was sufficient in discerning this damage, thereby shortening the time required to attain a statistically confident comparative evaluation. However, to allow for the use of statistical programs based on the evaluation of average values, and to avoid criticism based on genetic variability, we propose that three bulbs and three roots per bulb be considered as standard sample sizes for the A. cepa test.

Citrus stubborn disease, caused by Spiroplasma citri, has occurred in California for over 90 yr; however, detection methods for estimating disease incidence have not been optimized. Two 8 ha commercial citrus plots were sampled in 2005 and 2006. Different tissues of sweet orange were tested as sources for spiroplasma cultivation and three sampling procedures for estimating disease incidence were compared using cultivation and PCR. Fruit receptacles and columellas yielded cultivable spiroplasmas more consistently than did leaves, midribs, petioles, or bark. Stat sampling, in which every fifth tree every fifth row was sampled, resulted in estimated incidences of 45.9% and 1.3% by cultivation in groves 1 and 2, respectively. Hierarchical sampling, in which every fourth quadrat was sampled, yielded non-transformed incidences of 71.4% and 3.6% in the same groves by culturing, and 73.3% and 3.6% by PCR. In every-tree sampling, all trees in six blocks of 64 trees in each grove, sampled individually, yielded incidences of 50% and 1.6% by culturing and 58.4% and 2.1% by PCR. Thus, stubborn incidence in grove 1 was confirmed as high and that of grove 2 low. In these tests, PCR was superior to culturing; it is relatively inexpensive, sensitive, and rapid, permitting analysis of a large number of samples. Index words: epidemiology, bacterial cultivation, PCR, pathogen detection

Although citrus stubborn disease (CSD), caused by the phloem resident mollicute Spiroplasma citri, is a significant threat to California citrus industry, our knowledge of its epidemiology is mostly anecdotal. We optimized multiple pathogen-detection protocols, measured disease incidence in two plots of commercial California groves, assessed pathogen impact on fruit quality and yield, and evaluated genetic diversity among S. citri isolates. Fruit columellas and receptacles were more suitable than leaves or bark for bacterial cultivation. Using cultivation and S. citri-specific PCR for detection, the incidence of CSD in two orchards, respectively, ranged from 46 to 85% and 1 to 4%, depending on the sampling technique. Yield and quality of fruits produced by trees that were mildly or severely CSD-symptomatic were compared to those of S. citri-free trees in one California orchard in 2006 and 2007. These infected trees had reduced fruit quality and up to 32% lower yield relative to S. citri-free trees. Using RAPD markers to compare 35 S. citri isolates collected 20 years ago from the United States and Mediterranean region with 34 isolates recently collected from California, significant genetic diversity was identified but was not correlated with the time or location of collection. Our findings suggest that CSD incidence in the commercial groves evaluated could be as high as 85% and its impact on yield and fruit quality are significant. Accepted for publication 1 March 2010. Published 26 May 2010.

Foram caracterizadas e identificadas as frações proteicas constituintes do capim-mombaça submetido a quatro doses de nitrogênio (0, 100, 300 e 500kg.ha-1) em duas alturas de corte no período chuvoso e no seco. Utilizou-se delineamento experimental em blocos casualizados, em esquema fatorial 4 x 2, com quatro repetições. Os teores de proteína bruta (PB) e das frações A, B1, B2, B3 e C da proteína do capim-mombaça, cortado a 0,20 e 0,40m da superfície do solo foram avaliados pelo Cornell Net Carbohydrate and Protein System. Em relação ao teor de PB, ocorreu diferença significativa (P

Made available in DSpace on 2019-09-12T16:25:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 Este trabalho é parte de uma investigação sobre o potencial citotóxico e genotóxico da água do Rio Paraíba do Sul (Brasil) utilizando raízes de Allium cepa. Foi analisado um parâmetro anátomo-morfológico (crescimento das raízes), bem como o índice mitótico e a frequência de micronúcleos. Aleatoriamente oito bulbos foram submetidos aos tratamentos de 24 a 120 horas com água do Rio proveniente dos pontos de coleta das cidades de Tremembé e Aparecida nos anos de 2005 e 2006. O comprimento das raízes de cada bulbo foi acompanhado diariamente ao longo do experimento. O índice mitótico (IM) e a frequência de micronúcleos (MN) foram determinados pela análise de 2.000 células por raiz, sendo utilizadas 3-5 raízes de outros bulbos (7-10). Somente as raízes tratadas com amostras de água coletadas em 2005 na cidade de Tremembé, apresentaram decréscimo no comprimento das raízes quando comparadas com o controle. Entretanto, foi observada redução do IM nas raízes tratadas com água de ambos os pontos de coleta no mesmo ano. Considerando os dados de crescimento de raiz e especialmente IM, um potencial citotóxico é sugerido para a água do Rio Paraíba do Sul em Tremembé e Aparecida, no ano de 2005. Por outro lado, para este mesmo ano, a frequência de micronúcleos não foi alterada; assim, a genotoxicidade não foi assumida para a água do Rio nos pontos mencionados. This work investigated the cytotoxic and genotoxic potential of water from the River Paraíba do Sul (Brazil) using Allium cepa roots. An anatomo-morphological parameter (root length), mitotic indices, and frequency of micronuclei were analysed. Eight bulbs were chosen at random for treatment for 24 to 120 hours with the River water collected in the years of 2005 and 2006 from sites in the cities of Tremembé and Aparecida (São Paulo state, Brazil). Daily measurements of the length of the roots grown from each bulb were carried out throughout the experiment. Mitotic index (MI) and frequency of micronuclei (MN) were determined for 2000 cells per root, using 3-5 root tips from other bulbs (7-10). Only in the roots treated with samples of the River water collected in 2005 in Tremembé city was there a decrease in the root length growth compared to the respective control. However, a reduction in MI values was verified for both sites analysed for that year. Considering the data involving root length growth and especially MI values, a cytotoxic potential is suggested for the water of the River Paraíba do Sul at Tremembé and Aparecida, in the year of 2005. On the other hand, since in this year the MN frequency was not affected with the river water treatments, genotoxicity is not assumed for the river water sampled at the aforementioned places. [Barbério, A.; Voltolini, JC.] Universidade de Taubaté, Brazil Barbério, A.; Barros, L.; Mello, MLS.] Universidade Estadual de Campinas, Brazil

A sobrevivência e a ocorrência de mudas em espécimes de Triatoma infestans foram estudados num período de 30 dias após choques de temperatura. Foi demonstrado que choques hipertérmico e hipotérmico interferem nesses processos, na dependência da temperatura do choque, tempo de sua duração, fase de desenvolvimento e sexo dos espécimes. Dentre as situações experimentais utilizadas, o choque a 0°C por 12 h pareceu produzir a ação mais deletéria, porém choques a 40°C e 0°C mesmo por uma hora são admitidos como afetando o desenvolvimento hormonal que controla a muda. Casos de aumento de sobrevivência pós-choque são sugeridos como tendo sido favorecidos por ação de proteínas de choque térmico.

A variable effect of Cu2+ and Hg2+ ions on the intensity and localization of the SDH activity was cytochemically detected in the Malpighian tubule cells of the blood-sucking insect, Triatoma infestans. Unaffected and decreased enzyme activities as well as a change in the spatial distribution of mitochondria were detected on the basis of the cytochemical localization of the enzyme. It is assumed that these metal ions could affect not only the mitochondrial performance but also other cell functions which require a specific mitochondrial distribution.

Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD). Losses due to CSD in California orchards have grown over the past decade. To investigate the possibility of introduction or emergence of a new strain, a study of genetic diversity among S. citri strains from various locations was conducted using random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR) of 35 strains cultured from 1980 to 1993, and of 35 strains cultured from 2005 to 2006. Analysis using 20 primer pairs revealed considerable diversity among strains. However, no unique genetic signatures were associated with recently collected strains compared with those collected 15 to 28 years ago, and no geographically associated pattern was distinguishable. S. citri strains from carrot and daikon radish contain some unique DNA fragments, suggesting some host plant influence. Multiple strains from single trees also showed genetic diversity. Sequencing of five RAPD bands that differed among strains showed that diversity-related gene sequences include virus fragments, and fragments potentially encoding a membrane lipoprotein, a DNA modification enzyme, and a mobilization element. No differences in colony morphology were observed among the strains. The lack of correlation between PCR patterns and isolation date or collection site is inconsistent with the hypothesis that recent infections are due to the introduction or emergence of novel pathogen strains.

Feulgen-DNA values and nuclear areas were evaluated microspectrophotometrically for epithelial cells of the ovarian follicle during oogenesis in Chrysomya putoria. The aim was to investigate whether polyploidization occurred in the cells surrounding the nurse chamber and/or in those around the oocyte as well as whether different DNA amounts were found regarding the cell types considered. Four DNA endoreduplicative cycles could be demonstrated for the epithelial cells regardless of their localization on the follicle, during oogenesis. A small percentage of epithelial cells reaches a 32C degree. The nuclear area, however, did not increase at the same rate in cells covering the oocyte as in those covering the nurse chamber, in some of the oogenesis stages. The meaning of endopolyploidy for these cells is discussed, considering reports on relevance of secretory activities and their maintenance in polyploid cell systems.

Background: The stingless bee genus Melipona has been divided into two groups, based on their heterochromatin content. Melipona quadrifasciata and Melipona rufiventris have low and high levels of heterochromatin, respectively. Since condensed chromatin may be rich in methylated DNA sequences, M. quadrifasciata and M. rufiventris nuclei may contain different amounts of methylated CpG. These differences could be assessed by comparing Feulgen-DNA values obtained by image analysis of cells treated with the restriction enzymes Msp I and Hpa II that distinguish between methylated and unmethylated DNA. Msp I and Hpa II cleave the sequence -CCGG-, but there is no cleavage by Hpa II if the cytosine of the central CG dinucleotide is methylated. Methods: Malpighian tubules of M. quadrifasciata and M. rufiventris were treated with Msp I and Hpa II prior to the Feulgen reaction, and analyzed by automatic scanning microspectrophotometry. Results: The Feulgen-DNA values for the heterochromatin of M. rufiventris and for the small heterochromatin and some euchromatin domains of M. quadrifasciata mostly decreased after treatment with Msp I, but were unchanged after treatment with Hpa II. Conclusion: CpG methylation, although detected in diverse chromatin compartments in different bee species, may induce silencing effects required for the same cell physiology. © 2006 International Society for Analytical Cytology

The pigmentation of black (wild) and red (mutant) eyes of Triatoma infestans was studied spectrophotometrically and compared with red-eyed (wild) and white-eyed (mutant) forms of Drosophila melanogaster. The spectral absorption profiles of the black and red eye pigments of T. infestans were similar to each other and to that of the wild-type eyes of D. melanogaster. The similarity to the wild form of D. melanogaster indicated that both eye forms of T. infestans contained ommochromes of the xanthommatin type, a finding confirmed by ascending paper chromatography. Pteridines, melanins, and ommins were not detected as eye pigments in T. infestans. The eye color difference in T. infestans was assumed to be a function of the xanthommatin concentration, with a smaller content of ommochrome in red eyes, although this probably did not affect the insect's visual acuity. These data support other findings regarding the similarities between black- and red-eyed specimens of T. infestans for other characteristics.