Isolation of antibacterial response genes from the dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) immunized with Escherichia coli

Dot blot hybridization of total RNA from normal and bacteria-injected larvae of the dung beetle Copris tripartitus identi- fied 13 cDNA clones that seem to be associated with the immune response. A cDNA encoding a dung beetle homolog of the heat shock protein HSP70 (CTHSP70) was characterized by full-length sequencing and expression pattern analysis. CTHSP70 was upregulated 4 h after bacterial injection, reached maximum level after 8 h, and declined gradually after 16 h. Our data suggest that CTHSP70 and another 12 genes are involved in the anti-microbial defense that is particularly important for dung beetle larvae living in pathogen-rich conditions.


INTRODUCTION
To defend themselves against infection by a variety of bacteria, fungi and parasites, insects have evolved several efficient defense mechanisms, including the production of antimicrobial compounds.So far, more than 200 antibacterial peptides have been identified in insects.These peptides are known to play important roles in the humoral defense reaction (Hultmark, 2003;Lemaitre, 2004;Hoffmann, 2003;Hoffman et al., 1999;Engstrom, 1999;Garcia-Orozco, 2005).Humoral reactions include synthesis of antimicrobial proteins, which are secreted into the haemolymph, and proteolytic cascades leading to localized coagulation (Hoffman, 1995).The insect antibacterial peptides are classified into three groups, which are linear alpha-helical peptides devoid of cysteine residues, peptides in which proline and/or glycine residues are over-represented, and cysteine-rich peptides with cysteine established -motif (Bulet et al., 1999).Copris tripartitus spends a lot of time in dung, which contains an abundance of pathogens.The larvae feed on the fungi, decaying organic matter, dung and other organic materials in dung balls.Thus, it is likely that C. tripartitus defends itself against invading pathogens by means of antimicrobial compounds.Therefore, the purpose of the present study was to isolate and identify immune related genes in the dung beetle, C. tripartitus.In this study, we cloned immunerelated genes differentially expressed in control and immunized larvae, and detected the time course of expression using Northern blot.

Dot blot anlalysis and DNA sequencing
A total of 1862 random cDNA clones were arrayed onto a Hybond-N membrane (Amersham Biosciences, Uppsala, Sweden) using a 96-well format dot blotter (Bio-Rad).Two cDNA probes labelled with [ -32 P]dCTP were generated by reverse transcription of 0.5-1.0µgpoly(A) + RNA samples prepared from the normal and the immunized larvae.Probes were mixed with hybridization solution containing 50% formamide, 5 × SSC, 10 × Denhardt's solution (0.2% each of bovine serum albumin, ficoll and polyvinylpyrrolidone), 25 µg/ml sonicated salmon sperm DNA, and 50 mM sodium phosphate (pH 7.0).Hybridization was for 3 h at 65°C.After hybridization the membranes were washed in buffers of increasing stringency, from 2 × SSC containing 0.1% SDS to 0.1 × SSC containing 0.1% SDS, 30 min in each and then exposed to X-ray film (Agfa, Wiesbaden, Germany) for 1 or 3 days at -70°C.cDNA clones selected by the dot blot analysis were sequenced using the ABI 310 Automated Sequencer (Applied Biosystems, Foster City, CA, USA).The sequences were analyzed with the BLASTX program and protein database (http://www.ncbi.nlm.nih.gov/BLAST/) of the National Center for Biotechnology Information (NCBI).

RACE (rapid amplification of cDNA ends)-PCR
The 5'-end and 3'-end RACE-PCR were performed using Marathon cDNA Amplification Kit (Clontech, Mauntain View, CA, USA) according to the manufacture's protocol.Briefly, gene-specific primers were designed from the partial cDNA sequence of the CTHSP70.These primers were used in conjunction with the anchor primer API (Clontech) to amplify the 5' and 3' ends of the DDC gene from cDNA.PCR was performed for 30 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C, followed by a 5 min final extension step at 72°C.PCR products were isolated and subcloned into the pGEM T Easy Vector (Promega) and sequenced by the dideoxy-mediated chain termination method using an ABI 310 Automated Sequencer.

Northern Blot Analysis
Total RNA was extracted with a Trizol reagent (Life technologies, Inc., Gaithersburg, MD, USA) from the whole larvae 0, 4, 8, 16, and 24 h after E. coli injection and quantified by ultraviolet spectroscopy.Aliquots of 10 µg total RNA were fractionated on 1% agarose / 6.7% formaldehyde gels and blotted onto nylon membranes (Schleicher & Schwell BioScience, Sanford, ME, USA) using 20 × SSC buffer.Membranes were hybridized with cDNA probes labelled with [ -32 P]dCTP using a random primer labelling kit (Stratagene, La Jolla, CA, USA).Hybridization was performed for 3 h at 65°C in the Express-edHyb hybridization solution, according to the manufacturer's instruction (Clontech).Membranes were subsequently exposed to Kodak BIOMAX film (Eastman Kodak Co., Rochester, NY, USA) at 70°C.As an internal marker, 28S rRNA was visualized by ethidium bromide staining.

RESULTS AND DISCUSSION
As an initial step in the search for antibacterial peptides in Copris tripartitus, a full-length cDNA library was constructed from whole larvae of C. tripartitus immunized with E. coli.The cDNA library was plated out on LB-agarose plates covering an estimated 100,000 clones expressed in the library, and then 1,862 randomly selected cDNA clones were selected randomly from the cDNA library.Amplified PCR products were prepared and blotted onto duplicate nylon membranes.Screening was performed by differential hybridization with two types of [ -32 P]dCTP-labeled total cDNA probes synthesized from the normal and the immunized larvae.
Differential expression profiles of 1,862 clones (Fig. 1) revealed 13 cDNAs that were expressed preferentially or exclusively in the immunized larvae.Sequences of these cDNAs were determined by single-run partial sequencing and compared with the data in public databases.Seven cDNAs represented well known genes of the immune response: defensin, heat shock protein 70, peptidoglycan recognition protein, actin superfamily AT14584P, calreticulin, catalase, and transferrin.Each of these differentially expressed genes exhibited a significant protein sequence identity to the corresponding genes known from insects (Table 1).The remaining 6 cDNAs were functionally unidentified.
Insect defensins are generally cationic peptides composed of 34-46 residues with molecular mass ranging from 2~6 kDa.Initially discovered in Diptera (Matsuyama & Natori, 1988), defensins have since been described in various insects of the Neoptera cohort.Most of them act against a broad spectrum of Gram-positive bacteria and have little effect on Gram-negative bacteria and fungi (Dimarcq et al., 1994).HSP70 can be induced by various chemicals and biological stresses such as heat shock, oxidative stress, heavy metals, and amino acid ana-  logues, inhibitors of energy for energy metabolism, UV and irradiation, viral and bacterial infections, parasitism, alcohol, crowding, and inflammation (Lindquist & Craig, 1988;Hartl, 1996;Morimoto et al., 1990Morimoto et al., , 1992;;Abe et al., 1995;Fukamachi et al., 1998;Wagner et al., 1999;Sierra-River et al., 1993;Hatayama et al., 1993;Goodman & Blank, 1998;Gething & Sambrook, 1992;Sørensen & Loeschcke, 2001).Peptidoglycan recognition proteins are evolutionarily conserved molecules that are structurally related to bacterial amidases.Peptidodoglycans occur in the cell walls of almost all bacteria and in many insects induce strong antibacterial responses that include activation of phenoloxidase leading to melanin formation in infected wounds.Recently, peptidoglycan recognition proteins, SC1 and SC2, were reported as downregulators in the Drosophila immune response (Bischoff et al., 2006).In insects, calreticulin is an early stage capsulation protein involved in non-self recognition in the cellular defense reaction (Zhang et al., 2006).Catalase is one of the regulatory enzymes and facilitates the degradation of hydrogen peroxide to oxygen and water (Collins et al., 2004).Insect transferrin is considered to be an infection-inducible gene (Yoshiga et al., 1997, Thompson et al., 2003) and has been implicated in innate immunity to microbial infection by means sequestering iron.
According to the aforementioned reports, HSP70 is known to be induced in response to various biological stresses including viral and bacterial infections.CTHSP70, a dung beetle homologue of HSP70, was selected for further studies because of the infection-inducible genes identified in C. tripartitus HSP70 is the most likely to be a potential antimicrobial.We determined the nucleotide sequence of the CTHSP70 cDNA encoding the heat shock protein homolog of C. tripartitus.
In order to obtain the full cDNA sequence of CTHSP70, we performed 5'-and 3'-RACE-PCR.The full cDNA sequence of 2,354 bases contains initiation codon ATG in position 103 and the stop codon TAA at 2059 and encodes a protein of 652 amino acids (Fig. 2).Blast analysis of CTHSP70 revealed close matches with other known HSP70.The deduced amino acid sequence of CTHSP70 was highly similar to HSP70 in invertebrate and vertebrates, plants and yeasts (data not shown).The similarity was more than 70% in all matches identified from vertebrates and invertebrates.
Northern blot analysis was performed to verify differential expression of the Cthsp70 gene in the E. coli-immunized larvae (Fig. 3).The expression was upregulated 4 h after E. coli injection, reached a maximum after 8 h and declined gradually after 16 h.These data suggest that HSP70 is involved in the immune response, which is consistent with a previous report that HSP70 expression accompanies immune response in a scallop (Song et al., 2006).Highly conserved heat shock proteins such as HSP70 are known to protect cells against stress by facilitating proper folding of nascent peptides.HSPs have been classified into several families according to their apparent mass, such as HSP90 (85-90 kDa), HSP70 (68-73 kDa), HSP60, HSP47, and low molecular mass HSPs (16-24 kDa) (Tanaka et al., 1988;Morimoto et al., 1990).Heat shock proteins are expressed in response to a wide range of abiotic stressors, but also to biological stressors such as infectious pathogens.Recent data indicate that these stress proteins have the ability to modulate the cellular immune response and play a role in protecting organisms from pathogenic stress (Encomio & Chu, 2004;Forsyth et al., 1997;Brown et al., 1993: Moseley, 2000;Iwama et al., 1998).
In conclusion, we used dot-blot hybridization to identify some pathogen-induced genes in dung beetle larvae immunized with E. coli.A full-length sequence of a dung beetle homolog of hsp70 was determined and found to be up-regulated in larvae in response to infection with the pathogen E. coli.Copris tripartitus larvae were injected with 50 µl of E. coli JM109 (5 × 10 5 cells) suspended in physiological saline (150 mM NaCl/5 mM KCl).Larvae were kept for 0, 4, 8, 16, and 24 h. 10 µg aliquots of total RNA were resolved on formaldehyde containing agarose gels and blotted onto nitrocellulose membranes.The probe was labelled with [ -32 P]dCTP.As internal marker, 28S rRNA was stained with ethidium bromide.

Fig. 1 .
Fig. 1.Randomly selected 1,862 genes from cDNA library of Copris tripartitus larvae, spotted onto nylon membranes and hybridized with [ -32 P]dCTP-labelled RNA samples from normal and E. coli immunized larvae.Arrows indicate differently expressed clones in E. coli immunized larvae.Among the total of 1,862 EST clones, 13 cDNA transcripts were expressed differentially in E. coli immunized larvae.

Fig. 2 .
Fig. 2. Nucleotide and deduced amino acid sequences of the Cthsp70 gene isolated from Copris tripartitus.The predicted amino acid sequence (single-letter abbreviation) is shown below the nucleotide within the open reading frame.Codons for initiation, termination, polyadenylation, and poly(A) tail are in bold.The asterisk denotes a stop codon.

TABLE 1 .
Differentially expressed genes in E. coli immunized larvae.