Characterisation and expression analysis of UBC9 and UBS27 genes in developing gonads of cicindelids (Coleoptera: Cicindelidae).

Ubiquitin and small ubiquitin-like modifiers (SUMO) are post-translational modifiers essential in a variety of cellular processes, including gametogenesis. SUMO-conjugating enzyme (UBC9) and the ubiquitin ribosomal fusion protein UBS27 have been characterised in several model species. However, their expression in coleopteran remains unstudied. In this study, UBC9 and UBS27 genes have been characterised in the tiger beetle Cicindela campestris for the first time. Bioinformatic analysis showed that the Cc-UBC9 gene encoded a 159 amino acid protein with a predicted molecular weight of 18.18kDa, and the Cc-UBS27 gene encoded a 156 amino acid protein with a predicted molecular weight of 17.71kDa. Selection analyses carried out in several cicindelid species revealed that both genes were affected by purifying selection. Real time quantitative PCR analysis demonstrated that Cc-UBC9 and Cc-UBS27 were expressed in different tissues. The highest expression on both genes was found in the ovary and testis, and there were differential expression levels between immature and mature stages of testis development. The expression patterns of Cc-UBC9 and Cc-UBS27 suggest that these genes play important roles in gametogenesis in C. campestris. This information is relevant to better understand the reproductive process in cicindelids and the function of ubiquitin and small ubiquitin-related modifier genes in the Coleoptera.

Identification and Characterization of Pathogen-Response Genes (repat) in Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm (Spodoptera frugiperda, Noctuidae, Lepidoptera) is one of the most important crop pests in the Americas, causing significant damage to maize, rice and sorghum. The mechanisms that determine its defences against pathogens are particularly relevant for the development of management and control strategies. We used an in silico approach to identify and characterize pathogen response genes (repat) present in different tissue libraries of S. fugiperda. The analyses revealed complete cDNA for nine repat genes; of these, repat15 and repat39 were found in libraries from a specific tissue--the midgut of larvae fed with xenobiotic substances. High expression levels of some genes were found in different libraries: 39 hits in repat30 in challenged hemocytes, 16 hits in repat31 in fat body, 10 hits in repat32 in fat body and 10 in challenged hemocytes, and 10 hits in repat38 in midgut of non-treated larvae and midgut of larvae fed with natural and xenobiotic substances. The genes corresponded to two ontology categories, stress response and immune response, and their phylogenetic relationships, nucleotide similarity, number of amino acid residues and molecular weights agree with what has been described for repat genes. It is noteworthy that proteins encoded by the repat genes of S. frugiperda have important defence functions in other tissues beyond midgut and that their functional categories are likely diverse, as they are related to cell envelope structure, energy metabolism, transport and binding.

Identification, structural characterisation and expression analysis of a defensin gene from the tiger beetle Calomera littoralis (Coleoptera: Cicindelidae).

In this study, a defensin gene (Clit-Def) has been characterised in the tiger beetle Calomera littoralis for the first time. Bioinformatic analysis showed that the gene has an open reading frame of 246bp that contains a 46 amino acid mature peptide. The phylogenetic analysis showed a high variability in the coleopteran defensins analysed. The Clit-Def mature peptide has the features to be involved in the antimicrobial function: a predicted cationic isoelectric point of 8.94, six cysteine residues that form three disulfide bonds, and the typical cysteine-stabilized α-helix ß-sheet (CSαß) structural fold. Real time quantitative PCR analysis showed that Clit-Def was upregulated in the different body parts analysed after infection with lipopolysaccharides of Escherichia coli, and also indicated that has an expression peak at 12h post infection. The expression patterns of Clit-Def suggest that this gene plays important roles in the humoral system in the adephagan beetle Calomera littoralis.

Diversity of culturable Gram-negative bacteria isolated from irrigation water of two rice crop regions in Southern Brazil.

In this study, we assessed the diversity of Gram-negative bacteria found in water used for irrigation of rice crops in two growing areas of southern Brazil. Samples were collected from the main irrigation channel and field drain area. Twenty-two bacterial species were found in Cachoeirinha and 28 in Camaquã. In both areas, the most frequent bacterial families were Enterobacteriaceae and Aeromonadaceae. Differences in microbial diversity were observed in both study areas. Thirty-five Gram-negative species were identified; however, only 15 were common in both locations. In addition, there were found pathogenic and drug-resistant species, such as Acinetobacter sp., Brucella spp., and Chryseobacterium meningosepticum. This study demonstrates the existence of a number of pathogenic species in aquatic ecosystems analyzed in three consecutive crop years, especially water used for rice production.

Microbial, physical and chemical properties of irrigation water in rice fields of Southern Brazil.

This paper presents the results of the statistical analysis of microbiological, physical and chemical parameters related to the quality of the water used in rice fields in Southern Brazil. Data were collected during three consecutive crop years, within structure of a comprehensive monitoring program. The indicators used were: potential hydrogen, electrical conductivity, turbidity, nitrogen, phosphorus, potassium, calcium, total and fecal coliforms. Principal Component and Discriminant Analysis showed consistent differences between the water irrigation and drainage, as the temporal variation demonstrated a clear reduction in the concentration of most of the variables analyzed. The pattern of this reduction is not the same in the two regions - that is, the importance of each of the different variables in the observed differentiation is modified in two locations. These results suggested that the variations in the water quality utilized for rice irrigation was influenced by certain specific aspects of each rice region in South Brazilian - such as anthropic action or soil/climate conditions in each hydrographic basin.

Identification and characterisation of putative seminal fluid proteins from male reproductive tissue EST libraries in tiger beetles.

BACKGROUND: The study of proteins transferred through semen can provide important information for biological questions such as adaptive evolution, the origin of new species and species richness. The objective of this study was to identify seminal fluid proteins (SFPs) that may contribute to the study of the reproductive system of tiger beetles (cicindelids), a group of more than 2,500 species distributed worldwide that occupy a great diversity of habitats. RESULTS: Two cDNA libraries were constructed from the male gonads of Calomera littoralis and Cephalota litorea. Expressed sequence tags (ESTs) were analysed by bioinformatics approaches and 14 unigenes were selected as candidate SFPs, which were submitted to Reverse Transcription Polymerase Chain Reaction (RT-PCR) to identify patterns of tissue-specific expression. We have identified four novel putative SFPs of cicindelids, of which similarity searches did not show homologues with known function. However, two of the protein classes (immune response and hormone) predicted by Protfun are similar to SFPs reported in other insects. Searches for homology in other cicindelids showed one lineage specific SFPs (rapidly evolving proteins), only present in the closely related species C. littoralis and Lophyra flexuosa and two conserved SFP present in other tiger beetles species tested. CONCLUSIONS: This work represents the first characterisation of putative SFPs in Adephagan species of the order Coleoptera. The results will serve as a foundation for further studies aimed to understand gene (and protein) functions and their evolutionary implications in this group of ecologically relevant beetles.

RNA interference: a new strategy in the evolutionary arms race between human control strategies and insect pests.

The relationship between humans and the insect pests of cultivated plants may be considered to be an indirect coevolutionary process, i.e., an arms race. Over time, humans have developed several strategies to minimize the negative impacts of insects on agricultural production. However, insects have made adaptive responses via the evolution of resistance to insecticides, and more recently against Bacillus thuriengiensis. Thus, we need to continuously invest resources in the development of new strategies for crop protection. Recent advances in genomics have demonstrated the possibility of a new weapon or strategy in this war, i.e., gene silencing, which involves blocking the expression of specific genes via mRNA inactivation. In the last decade, several studies have demonstrated the effectiveness of this strategy in the control of different species of insects. However, several technical difficulties need to be overcome to transform this potential into reality, such as the selection of target genes, the concentration of dsRNA, the nucleotide sequence of the dsRNA, the length of dsRNA, persistence in the insect body, and the life stage of the target species where gene silencing is most efficient. This study analyzes several aspects related to the use of gene silencing in pest control and it includes an overview of the inactivation process, as well as the problems that need to be resolved to transform gene silencing into an effective pest control method.

Microbiological and physical-chemical water quality of the rice fields in Sinos River's basin, Southern Brazil.

In this study, we evaluate spatial and temporal variations of the water's quality used in rice fields. Every 15 days during the different phases of cultivation of the rice-vegetative, reproductive, and maturity-samples were collected from the main irrigation channel, from the surface water in the field, and from the excess-water drainage channel. The differences in the values of 13 variables were analyzed by the analysis of variance system and by the Principal Component Analysis (PCA) technique. The results demonstrate that the values observed for the presence of total coliforms, heat-tolerant coliforms, and pH were higher in the irrigation water and that the calcium and magnesium components were greater in the drainage water. The PCA results indicate that three groups of variance exist and that these three account for 77 % of the observed variances. The first principal component, (39 % of the variances), includes the variables pH, phosphorus, potassium, carbon, and turbidity; the second (28.1 %), calcium, magnesium, and conductivity; while the third accounts for only 9.9 % of the variation and incorporates the variable biological thermotolerant coliforms. The spatial pattern resulting from the distribution of the sampling locations as regards the first two principal components indicates a difference between the irrigation and drainage waters. The variables of the first and third items (except for the turbidity in the second component) reach higher values in the irrigation water, while the variables associated with the second component have higher values in the drainage water. The information provided is important for the analysis of the influence exercised by plantation management decisions on the microbiological, physical, and chemical quality of the water. The results confirm the ability of paddy rice field to filter out some of the chemicals and coliforms from the irrigation water as it passes through the agro ecosystem.

Phylogenetic relationships and the evolution of mimicry in the Chauliognathus yellow-black species complex (Coleoptera: Cantharidae) inferred from mitochondrial COI sequences

The phylogenetic relationships of twelve species of Chauliognathus were investigated by studying the mitochondrial cytochrome oxidase I gene. A 678 bp fragment of the COI gene was sequenced to test the hypothesis that the Müllerian mimicry species of the "yellow-black" complex make up a monophyletic clade, separated from species with other colour patterns. The data set was analysed by neighbour-joining, maximum parsimony and maximum likelihood procedures. The results support a single origin of the yellow-black colour pattern during the evolution of the genus, with one main clade formed by Chauliognathus lineatus, C. tetrapunctatus, C. riograndensis, C. flavipes, C. octomaculatus, C. fallax, and another one formed by two species, C. expansus and C sp 1, plus an orange-black-coloured species. The nucleotide divergences found between C. sp 3 (black) and the other species studied fall within the level expected for species from different genera. The similarity of colour patterns of the yellow-black species has been considered an example of Müllerian mimicry by conservation of the ancestral state with some minor modifications.

Elytra colour polymorphism and randomness of matings in Chauliognathus fallax Germar 1824 from southern Brazil (Coleoptera, Cantharidae)

Beetles of the species Chauliognathus fallax Germar 1824 are polymorphic for elytron colouration with six morphs distinguished on the basis of black pigmentation on a yellow background. We investigated samples of C. fallax taken in eight consecutive weeks aiming to determine the frequency of the morphs which were grouped, for statistical analysis, into three classes according to elytra pigmentation as well as the possible occurrence of assortative mating. Our results showed a variation in the frequency of the classes throughout the season, both in males and females, with the maximum frequency of each class at the fourth and fifth week. The three classes (A, B, C) showed the same pattern of variation, and class B was always the more frequent. To test randomness of matings two approaches were taken: in the first, we compared the frequency of each class in copulating and non-copulating insects. In the second, the frequency of each class in the whole sample was taken as the probability of occurrence of the respective class; then, using the criterion of the probability of independent events we calculated the expected proportion of copulating insects for each pair of events. Both methods gave non-significant differences, suggesting that the matings were random.