Immune transcriptome analysis in predatory beetles reveals two cecropin genes overexpressed in mandibles.

The great complexity and variety of the innate immune system and the production of antimicrobial peptides in insects is correlated with their evolutionary success and adaptation to different environments. Tiger beetles are an example of non-pest species with a cosmopolitan distribution, but the immune system is barely known and its study could provide useful information about the humoral immunity of predatory insects. Suppression subtractive hybridization (SSH) was performed in Calomera littoralis beetles to obtain a screening of those genes that were overexpressed after an injection with Escherichia coli lipopolysaccharide (LPS). Several genes were identified to be related to immune defense. Among those genes, two members of the cecropin antimicrobial peptides were characterized and identified as CliCec-A and CliCec-B2. Both protein sequences showed cecropin characteristics including 37 and 38 residue mature peptides, composed by two α-helices structures with amphipathic and hydrophobic nature, as shown in their predicted three-dimensional structure. Chemically synthesized CliCec-B2 confirmed cecropin antimicrobial activity against some Gram (+) and Gram (-) bacteria, but not against yeast. Expression of both cecropin genes was assessed by qPCR and showed increases after a LPS injection and highlighted their overexpression in adult beetle mandibles, which could be related to their alimentary habits.

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.

Validation of reference genes for quantitative real-time PCR in tiger beetles across sexes, body parts, sexual maturity and immune challenge.

Reference genes are frequently used as normalizers for expression studies despite not being previously verified to present suitable stabilities. Considering the interest that tiger beetles have generated in the past years, resulting in a variety of studies, it is crucial to dispose of a validated reference gene panel for expression studies. Nine candidate genes were tested in Cicindela campestris and Calomera littoralis across several conditions and their transcription levels were assessed with geNorm, NormFinder, BestKeeper and ΔCT method algorithms. Results showed high stabilities across sexes, immune challenge and gonad developmental stages for all genes tested, while body parts comparison presented less constant expression values. Only two genes are sufficient to perform a proper normalization for most of the conditions tested, except for the body parts comparison in C. littoralis, which requires the use of at least three reference genes. On the whole, no universal gene is found to be suitable for all situations, but according to the acceptable range of values, NADH, B-t, Vatpase and ArgKin seem to present the most constant expression stability, indicating their suitability as reference genes in most of the conditions. This is the first report evaluating the stability of housekeeping genes in adephagan beetles.

Real-time cell analysis and heat shock protein gene expression in the TcA Tribolium castaneum cell line in response to environmental stress conditions.

The rust red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environmental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real-Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL-TcA-CLG1 (TcA) of T. castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV-A light with the aim of measuring the expression levels of Hsp27, Hsp68a, and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up-regulation of all studied Hsp genes is observed after 1 h of exposure to 40 °C and UV light. All 3 genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real-time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management.

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.

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.