Diversity and phylogenetic analysis of endosymbiotic bacteria of the date palm root borer Oryctes agamemnon (Coleoptera: Scarabaeidae)
© El-Sayed and Ibrahim; licensee BioMed Central. 2015
Received: 14 November 2014
Accepted: 8 April 2015
Published: 22 April 2015
The date palm root borer Oryctes agamemnon (Coleoptera: Scarabaeidae) is one of the major pests of palms. In Saudi Arabia, both larvae and adults of Oryctes are particularly troublesome, especially during the establishment of young date palm orchards. Endosymbiotic bacteria are known to have a key role in food digestion and insecticide resistance mechanisms, and therefore are essential to their host insect. Identification of these bacteria in their insect host can lead to development of new insect pest control strategies.
Metagenomic DNA from larval midgut of the date palm root borer, O. agamemnon, was analyzed for endosymbiotic bacterial communities using denatured gradient gel electrophoresis (DGGE) utilizing 16S rRNA genes. The DGGE fingerprints with metagenomic DNA showed predominance of eleven major operational taxonomic units (OTUs) identified as members of Photobacterium, Vibrio, Allomonas, Shewanella, Cellulomonas, and Citrobacter, as well as uncultured bacteria, including some uncultured Vibrio members. DGGE profiles also showed shifts in the dominant bacterial populations of the original soil compared with those that existed in the larval midguts. The endosymbiotic bacterial community was dominated by members of the family Vibrionaceae (54.5%), followed by uncultured bacteria (18.2%), Enterobacteriaceae (9.1%), Shewanellaceae (9.1%), and Cellulomonadaceae (9.1%). Phylogenetic studies confirmed the affiliation of the dominant OTUs into specified families revealed by clustering of each phylotype to its corresponding clade. Relative frequency of each phylotype in larval midguts revealed predominance of Vibrio furnisii and Vibrio navarrensis, followed by uncultured bacterial spp., then Cellulomonas hominis, Shewanella algae, and Citrobacter freundii.
Analysis of metagenomic DNA for endosymbiotic bacterial communities from the midgut of Oryctes larvae showed strong selection of specific bacterial populations that may have a key role in digestion, as well as other benefits to the larvae of O. agamemnon. Determination of the distinct endosymbiotic community structure and its possible biological functions within the insect could provide us with basic information for future pest control research.
Several insect pests attack date palm (Phoenix dactylifera L.) orchards, causing serious damage and economic losses. In many Arabian countries, three species of rhinoceros beetles, Oryctes (Coleoptera: Scarabaeidae), O. elegans, O. agamemnon and O. rhinoceros, are known to infest date palm orchards . The most widespread is O. agamemnon, which is a root borer in its larval stage and a frond borer in the adult stage. The other two species, O. rhinoceros and O. elegans, are fruit stalk borers and can also act as root borers . Oryctes spp. have a wide host range, attacking and causing serious damage and crop loss on many hosts, including date palm, coconut palm, betel nut, sago palm and oil palm . Recently, Oryctes spp. have emerged as major pests of different date palm cultivars. In Saudi Arabia, both larvae and adults of Oryctes are particularly troublesome, especially during the establishment of young date palm orchards. The development time of the larval stage is long and may extend for several years in some species. The larvae feed on roots and rotten wood whereas the adults feed on nectar, plant sap and fruit [4-7].
Certain mutualists may influence host plant range and enable insect pests to modify plant physiology for their own benefit. There is increasing evidence for the role of microbial mutualistic symbioses in insect–plant interactions . The horizontal transmission of mutualists among their host insects can be achieved through a route involving its host plant. Where this transmission occurs, the insect mutualist might either become a plant pathogen and damage the plant or change the way the plant interacts with its natural enemies and host competitors .
Insect intestinal tracts harbor rich communities of nonpathogenic microorganisms . A single gut can harbor 105–109 prokaryotic cells  that have been affiliated to twenty-six phyla, at least for the insects studied to date. It is increasingly evident that insect microbiota are essential for normal growth and development . It has been shown that about 65% of insects possess symbiotic bacteria. Wolbachia spp. is the most commonly reported genus [13-15]. The symbiotic relationship between bacteria and insects varies from being mutualistic and commensal to pathogenic [16,17]. Based on their role, intracellular symbionts in insects are classified as primary or secondary endosymbionts. Primary (obligate) symbionts are essential for the insect due to their role in nutrient supplementation, whereas secondary symbionts have a useful but not essential role for insect survival [18,19]. Insect endosymbionts are detected in specific organs referred to as bacteriomes or mycetomes, usually resulting in a strict vertical transmission from mother to offspring.
Understanding relationships between endosymbiotic bacteria and their insect hosts is not only relevant from an evolutionary view, but can also lead to the identification of new targets for insect pest control . Since many of the relevant endosymbionts cannot be cultured, their functional characterization and/or identification has been difficult. Certain symbionts have been developed as biological control agents and were found to be effective against Chagas disease vectored by Rhodnius prolixus. In this example, the endosymbiotic organism, Rhodococcus rhodnii was genetically transformed to express an anti-trypanosomal output in the insect gut .
The date palm root borers of the genus Oryctes are regarded as devastating and invasive pests in a wide variety of palms worldwide. Little is known about the presence of endosymbionts in the genus Oryctes. Exploring bacteria-insect associations in this regard would be useful for potential insect pest control. For example, if obligate endosymbionts exist in Oryctes, then eliminating them using baits could be a potential control strategy. Investigation of endosymbiosis in this genus may help to understand the host-symbiont interactions and the evolution of different reproductive strategies in these beetles, and ultimately provide a future basis for development of novel pest management strategies. Therefore, the objective of this study was to analyze the diversity of the larval midgut microbiota of the date palm root borer, O. agamemnon.
Results and discussion
Endosymbionts of Oryctes agamemnon larvae
Endosymbiotic bacterial community structure
Bacterial species identified in the midgut of O. agamemnon larvae
Photobacterium ganghwense FR1311
Photobacterium sp. RSBAUOCAS0005B
Vibrio fluvialis MBTD-CMFRI-Vf05
Vibrio sp. BTOK10
Vibrio vulnificus MP-4
Photobacterium ganghwense FR1311
Vibrio fortis H083
Uncultured bacterium clone SWH04_PR
Uncultured bacterium clone BT12G08
Uncultured bacterium clone SWG11_MS
Uncultured bacterium clone nbw223h08c1
Allomonas enterica JC102, D09-37
Uncultured Vibrio sp. clone D004025F04
Uncultured bacterium clone LGH02-B-135
Vibrio navarrensis AM37820
Vibrio navarrensis 2544-86
Vibrio navarrensis 1397-6T
Vibrio sp. U15
Vibrio furnisii (ATCC 35016T)
Uncultured Vibrio sp. clone KR-SUC-9-A10
Shewanella algae H5
Shewanella haliotis NIOT-CS16
Shewanella sp. MPTDBS
Cellulomonas hominis PuiC5.18
Cellulosimicrobium cellulans S17
Cellulomonas aerilata JCM 16376
Citrobacter freundii C09
Citrobacter youngae GTC 01314
Citrobacter murliniae M-T-MRS_22
Uncultured bacterium clone SWH04_PR
Uncultured bacterium clone BT12G08
Uncultured bacterium clone SWG11_MS
Gut bacteria have been reported to exert many useful functions, such as preventing disease, degrading insecticides, and directly or indirectly contributing to food digestion . Food materials may be important in regulating the dynamics of the bacterial community within the insect gut. For example, S. marcescens is a facultative anaerobe that aids in consuming oxygen at the periphery of the Formosan termite’s stomach, thereby maintaining a habitable gut for the strict anaerobes that digest cellulose . In addition to aiding digestion, Citrobacter detected in our study is believed to have the same role in establishing anaerobic conditions for the succession of Shewanella spp. involved in anaerobic fermentation of ingested materials.
Analysis of larval midgut bacterial populations in O. agamemnon revealed a predominance of members belonging to the genus Vibrio. Dominance of certain bacterial taxa as endosymbionts in some insects has been reported. Using sequence-based bacterial typing, Hirsch et al.  identified bacterial endosymbionts in four species of Otiorhynchus. More than 90% of all sequence reads belonged to the genus Rickettsia. Tagliavia et al.  analyzed the gut microbiota of larvae of the red palm weevil. High abundance of Enterobacteriaceae was detected. Fujiwara et al.  surveyed symbiotic bacteria from Bemisia tabaci species and reported the dominance of Rickettsia in all examined whitefly species.
In contrast to our results with larvae, in a study of gut microbiota of adult Oryctes monoceros by Desai and Bhamre , a completely different microbial population, except for Citrobacter, was reported, and included Dienococcus proteolyticus, Micrococcus varians, Micrococcus kristinae, Micrococcus roseus, Micrococcus lylae, Citrobacter amalonacticus, Corynebacterium xerosis and Bacillus fermentas.
Cellulolytic bacteria are important for digestion of cellulosic materials. In our study, Cellulomonas sp. has been detected as a member of the O. agamemnon midgut bacterial population indicating its involvement in the digestion process. Huang et al.  isolated strains of aerobic and facultatively anaerobic cellulolytic bacteria from the gut of Holotrichia parallela (Coleoptera: Scarabaeidae) larvae. The cellulolytic bacterial community was dominated by Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes (1.45%). However, Cellulomonas sp. in particular, was not detected among this community.
Diversity of Oryctes agamemnon endosymbionts
The versatility and diversity of insect-bacteria interactions leads to an enormous potential regarding the mechanisms for the modulation and control of insect pests with both medical and agricultural implications . Through TFLP analyses of bacterial rRNA extracted from the guts of Harpalus pensylvanicus and Anisodactylus sanctaecrucis (Coleoptera: Carabidae), Lundgren et al.  revealed that gut-associated bacterial communities were of low diversity. The bacterial community in these beetles comprised Serratia sp., Burkholderia fungorum, H. alvei, Phenylbacterium sp., Caedibacter sp., Spiroplasma sp., Enterobacter strain B-14, and Weissella viridescens. Some of these organisms, but not all have been previously associated with insects. However, none of them has been detected in O. agamemnon, suggesting that their larvae have a unique bacterial community. In comparison to previously reported insect microbiota, our study revealed low diversity and a highly unique pattern for O. agamemnon microbiota.
The 16S rRNA genes are used for phylogenetic affiliation of Eubacteria and Archaea. Partial sequences of 16S rRNA gene of bacterial microbiota from the larval midgut of O. agamemnon have been analyzed. Sequences were compared with their closest matches with BLAST search tool to obtain the nearest phylogenetic neighbors. About 72.7% of the bacterial community was assigned to Gammaproteobacteria. The remainder of the bacterial community was assigned to Actinobacteria (9.1%) and uncultured bacterial members (18.2%). Bacteria belonging to Gammaproteobacteria were classified as members of three families; Vibrionaceae, Enterobacteriaceae, and Shewanellaceae, with predominance of the former. Actinobacteria comprised only one family, Cellulomonadaceae enclosing Cellulomonas sp.
Tagliavia et al.  analyzed the gut microbiota of larvae of the red palm weevil. They assigned 98% of the total population to only three phyla: Proteobacteria, Bacteroidetes, and Firmicutes, and three main families (Enterobacteriaceae, Porphyromonadaceae and Streptococcaceae). Bacterial members have been identified as Dysgonomonas, Lactococcus, Salmonella, Enterobacter, Budvicia, Entomoplasma, Bacteroides and Comamonas. The major phylogenetic microbiota of the hindgut of P. ephippiata were identified through a 16S rRNA gene clone library and revealed that Clostridia, Betaproteobacteria, and Bacteroidetes, followed by Bacillales and Deltaproteobacteria, were dominant.
In conclusion, endosymbiotic bacteria are known to be involved in protecting their host insect against natural antagonists, contributing to insecticide resistance mechanisms, and aiding in food digestion and are, therefore, essential for normal growth and development of their host insect. In this regard, endosymbiotic bacteria could be manipulated, potentially offering new approaches for insect control. Therefore, identification of endosymbiotic bacteria of O. agamemnon is an important step in this process. Metagenomic DNA from midguts of Oryctes larvae was analyzed for endosymbiotic bacterial communities. Except for the Enterobacteriacaea group, Oryctes larvae were found to harbor unique endosymbiotic bacteria when compared with previously reported microbiota. Such distinct microbial community structure and its possible biological function within the insect will provide us with basic information for development of pest control strategies utilizing intrinsic endosymbiotic bacteria. Finally, there is an ultimate question we have to answer, what would be resulted in the absence (either intentional or accidental) of each single symbiont or a specific symbiotic group? If this question is correctly answered, this means a successful control strategy for this insect pest is achieved. Therefore, further studies are now required to clarify the biological function of these endosymbiotic bacteria in Oryctes larvae and their potential as novel targets for beetle control.
Oryctes agamemnon larvae were field-collected from a date palm orchard about 80 km north Almadinah Almunawarah region, of Saudi Arabia at longitude (39°11ʹ6ʺ) and latitude (24°47ʹ6ʺ). The 3rd larval instar was dominant in sampling. Samples of larvae were collected in sterilized plastic containers. The larvae were kept in the laboratory for one week prior to dissection to avoid possible infestations from the field and to reduce any potential insecticide residual effects. All stages were kept in plastic containers half-filled with soil and date palm pieces. The larvae were dissected in dissection trays containing 0.65% saline and the midguts were aseptically removed . The midguts were homogenized in a sterile glass homogenizer containing 0.85% saline. The supernatant suspension was used for bacterial enrichment and DNA extraction. Each sample consisted of the content of three pooled midguts taken from three larvae of the same instar. Metagenomic DNA was extracted from soil infested with larvae for comparative purposes.
DNA extraction and PCR amplification of 16S rRNA genes
Total community DNA was extracted with the Ultra Clean Soil DNA purification kit (Mo Bio Laboratories, Solana Beach, Calif.). Harvested cells were transferred to bead beating tubes and vortexed horizontally for 1 min at room temperature. Supernatant was collected and DNA was precipitated and purified according to the instruction manual. Amplification of 16S rRNA genes for DGGE analysis was performed using GC-clamp primers (EUB341F-GC: 5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAGCAG-3′ and EUB517R: 5′-ATTACCGCGGCTGCTGG-3′) that correspond to positions 341 and 517 in Escherichia coli . Amplification were performed in 25 μl reaction vessel containing: 2.5 μl of 10 × Taq buffer (100 mM Tris–HCl, pH 8), 1.25 mM MgCl2, 100 μM dNTPs (Invitrogen, USA), 1.2 μM forward primer and reverse primer set (Invitrogen, USA), 0.5U Taq DNA polymerase (Invitrogen, USA), and about 5 ng of template DNA. PCR was performed in Thermal Cycler (Applied Biosystems 2720, USA). A touchdown PCR program was implemented as follows: initial denaturation step at 95°C for 5 min; 5 cycles of 94°C for 40 sec, annealing at 65°C for 40 sec, and extension at 72°C for 40 sec; 5 cycles of 94°C for 40 sec, annealing at 60°C for 40 sec, and extension at 72°C for 40 sec; 10 cycles of 94°C for 40 sec, annealing at 55°C for 40 sec, and extension at 72°C for 40 sec; 10 cycles of 94°C for 40 sec, annealing at 50°C for 40 sec, and extension at 72°C for 40 sec were performed, followed by a final hold at 72°C for 7 min. Amplicons were analyzed by electrophoresis on 1% agarose gels with the size markers (1 kb DNA ladder, Invitrogen, USA) and visualized using ethidium bromide.
DGGE was performed using Dcode Mutation Detection System (Bio-Rad Laboratories Ltd., Hertfordshire, UK). PCR products were electrophoresed with 0.5 × TAE buffer (1 × TAE buffer is 0.04 M Tris base, 0.02 M sodium acetate, and 10 mM EDTA [pH 7.4]) on 8% acrylamide gel containing 25 to 50% denaturating gradient of formamide and urea. DGGE was conducted at 60°C for 5 h at voltage of 200 V. The gel was stained with SYBR Green I Nucleic acid gel stain (Cambrex Bio Science Rockland, USA), photographed and analyzed for DGGE band profile with a UV gel documentation system (Bio-Rad Laboratories Inc., CA, USA).
Numerical analysis of the DGGE fingerprints
where P i is a relative intensity of DNA band in the fingerprint, n i is densitometrically measured intensity of individual DNA band, and N i is the total amount of DNA in the fingerprint. The relative intensity of each band (Pi) was used to express the relative frequency of each phylotype .
Sequencing of DGGE bands
Dominant DGGE bands were cut off with a sterile scalpel and eluted by incubation in 100 μl of TE buffer at 100°C for 5 min. Supernatant was used as template for PCR amplification. Reamplification of 16Sr RNA genes from excised DNA fragments was performed using bacterial primers EUB314F without GC clamp and EUB517R. Amplification was verified by electrophoresis on 1% agarose gel. PCR products were directly sequenced using a BigDye terminator cycle sequencing  at GenoScreen sequencing facility (Genoscreen, Lille, France).
The sequences obtained from the 16S rRNA genes were analyzed by Genetyx-Win MFC application software version 4.0. The reference 16S rRNA gene sequences were retrieved from the GenBank database (National Center for Biotechnology Information, National Library of Medicine, USA) . Sequences were compared with their closest matches in GenBank with nucleotide-nucleotide BLAST to obtain the nearest phylogenetic neighbors (www.ncbi.nlm.nih.gov/BLAST/). Sequence alignments were performed by Clustal W1.83 XP  and phylogenetic trees were constructed using neighbor-joining method  using MEGA6 software .
Accession numbers and data deposition
The 16S rDNA sequences identified in this study have been deposited in the GenBank database under the accession numbers LC009469 to LC009479. The data of the phylogenetic analysis are available from the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.59h51.
The authors acknowledge Deanship of Scientific Research (DSR) at Taibah University, KSA for their financial support of this work. Authors extend their appreciation to Biology Department, Faculty of Science, Taibah University for providing research facilities. We are also grateful to Fares Zayadi for his help in collecting larvae of the date palm root borer.
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