COVER PAGE
I. Manuscript Title: Exploration of Symbiotic Bacteria of Oryctes rhinoceros Larvae from Oil Palm Empty Fruit Bunches.
Marheni1*), Edhi Martono2) Octanina Sari Sijabat3)
1,3Faculty of Agriculture, University of Sumatera Utara, Padang Bulan, Medan 20155, Indonesia
2Faculty of Agriculture, University of Gadjah Mada, Yogyakarta Email :[email protected]
I. First author:
1. Name : Marheni
2. Afiliation : Faculty of Agriculture, University of Sumatera Utara, Medan, Indonesia 3. E-mail : [email protected]
4. Orcid ID :
5. Contribution to this Manuscript: First Author II. Second author:
1. Name : Edhi Martono
2. Afiliation : 2Faculty of Agriculture, University of Gadjah Mada, Yogyakarta 3. E-mail : [email protected]
4. Orcid ID :
5. Contribution to this Manuscript: Co- Author III.Third author:
1. Name : Octanina Sari Sijabat
2. Afiliation : Faculty of Agriculture, University of Sumatera Utara, Medan, Indonesia 3. E-mail : [email protected]
4. Orcid ID :
5. Contribution to this Manuscript: Co- Author
IV. Acknowledgement
The authors acknowledge the financial support for the Directorate General of Higher Education, Ministry of Research, Technology, Higher Education of Indonesia. Through the University of Sumatera Utara’s Research DRPM. The authors also convey their gratitude the Faculty of Agriculture, Univeristy of Sumatera Utara for giving permission and facilities to conduct research.
V. Reviewer Candidates
Requirements for the candidates:
1. The candidates should have speciality in authors’ research topic
2. The candidates should come from different institutions with authors (especially from different countries)
3. The candidates should not join the authors’ research project
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Exploration of Symbiotic Bacteria of Oryctes rhinoceros Larvae from Oil Palm Empty Fruit Bunches
Marheni1*Edhi Martono2, Octanina Sari Sijabat1
1Faculty of Agriculture, University of Sumatera Utara, Padang Bulan, Medan 20155, Indonesia
2Faculty of Agriculture, University of Gadjah Mada, Yogyakarta Email :[email protected]
*Corresponding author : Marheni, Faculty of Agriculture, Universitas Sumatera Utara Padang Bulan Medan 20155, Indonesia, Phone:
Email :[email protected]
Running title: Exploration of Symbiotic Bacteria of Oryctes rhinoceros Larvae
Significance statement: This study has found bacteria in the hindgut of the third instar larvae of Oryctes rhinoceros. Six different species of bacteria were found, indicate that there were bacterial interactions with the larvae in synthesizing organic material consumed by larvae.
Abstract
Background and Objective: Oil palm empty fruit bunches (OPEFB) were utilized as the source of organic fertilizer, but become a medium of Oryctes rhinoceros proliferation in oil palm plantations. O.
rhinoceros uses cellulose, hemicellulose and lignin in the OPEFB as energy sources for larvae growth and development. In the process of breaking and combining complex compounds into simple molecules, they are establishing symbiotic relationship with bacteria residing in the digestive tract of O. rhinoceros larvae. Isolation of the symbiotic bacteria of the O. rhinoceros larvae was carried out with the aim of identifying bacteria in the digestive tract of the third instar found in the OPEFB. Materials and Methods:
This research was done by using a descriptive method. Exploration of symbiotic bacteria of O.
rhinoceros larvae was conducted through two stages. The first step was attained by isolating, growing and multiplying the bacteria and conducting biochemical testing. The second phase was accomplished by molecular testing for bacterial identification in IPBCC Bogor. The order of 16S rDNA compared to the 16S rDNA sequence available in the Bank Gen database employed the BLAST program at the National Center for Biotechnology Information http: //www.ncbi.nlm.nih.gov/. Results: The results showed that bacterial isolates were found and identified molecularly for species analysis after applying sequencing of 16S rDNA species of bacteria, namely Bacillus stratosphericus, B. siamensis, B. cereus, Haemophilus parainfluenzae, Achromobacter xylosoxidans, and Alcaligenes faecalis. Conclusion: isolation and identification of symbiotic bacteria O. rhinoceros larvae in the OPEFB produced six species
Key words: Exploration, Larvae Oryctes rhinoceros, Symbiotic bacteria
INTRODUCTION
Oryctes rhinoceros is one of the important pests in oil palm plantations. O. rhinoceros which causes damage to oil palm plants and other types of palm plants is imago, both male and female. The O. rhinoceros attack can reduce crop yields, turn off young seedlings in the nursery and old plants, and damage new plant areas (replanting). In oil palm plants with mild attacks, O. rhinoceros causes leaf symptoms such as shear and decreased yield power, even at the beginning of generative growth production can be delayed. Adult beetles enter the growing point area and eat soft parts. When the attack reaches a growing point, the plant will die. These pests breed using empty palm bunches and palm oil waste which is in the plantation area ( Bintang et al., 2015).
Insects can be harbor gut microbial communities that range from simple to complex. Most studies to date have focused on dening community membership as a First step toward understanding the roles of community members in the insectology biology and in the functioning of the community itself. In a few systems that have been studied in more detail, important contributions to the host insect physiology and life history have been attributed to the gut-associated microbes. Little is known about the diversity, physiology, and ecology of microorganisms associated with the guts of diverse groups of beetles that develop within the wood and bark of trees (Baharuddin et al., 2014).
Fat body is composed of three cells including mycetocytes, which are cells that contain a number of microorganisms in the intestine and symbiosis with insects. Microorganisms synthesize various nutrients needed by insects. Herbivorous insects can utilize lignocellulosa as an energy source, this ability is supported by the presence of microbes in the intestine that produce hydrolytic enzymes (Sari et al., 2016).
In the digestive tract of insects there are nonpathogenic microorganisms, in the intestine which can store 105-109 prokaryotic cells that have been affiliated with twenty-six phyla. Insect microbiota is very important for normal growth and development because about 65% of insects have symbiotic bacteria. Symbiotic relationships between bacteria and insects vary from mutualistic and commensal.
Based on the role of the symbionic bacteria, intracellular symbionts in insects are classified as primary or secondary endosymbionts. Palm stem borer from the genus Oryctes is considered a very detrimental pest. The presence of endosymbionts in the genus Oryctes, which is an insect-bacterial association in this case will be useful to know (Elsayed et al., 2015).
The ability of microbes in the degradation process is because microbes have cellulase enzymes.
Microbes in insect gut are symbionic microbes that are interconnected with other organisms in the process of lignocellulose degradation (Aini, 2015).
In insects that digest wood components as nutrients often assisted by microorganisms in various ways including the first acquisition of microbial-generated enzymes on digestible substrates, pre-digestion of the substrate by microorganisms before digestion, then enrichment of nutrients in the form of microbial
cells and metabolites. Furthermore, the expenditure and detoxification of wood extractive substances, microbes living in the body produce and release enzymes and microorganisms that work as decomposers which release the main carbon source for assimilation of insects (Muin et al., 2010).
Identification of bacteria can be done using conventional and molecular methods. The conventional method can be done based on the typical phenotype, namely Gram staining, morphology and biochemical reactions. However, this conventional method of determining bacteria has several disadvantages. This method cannot be used for unidentified religions. Furthermore ,. Physical and biochemical characters are characters that do not change and change due to pressure and change, so this also affects the outcome of the meeting. From the findings of Polymerase Chain Reaction and DNA sequencing, determining the gene sequence of bacterial species displaying the 16S rDNA gene is very conserved in one species and between species with the same genus, and can be used as a new technique for bacteria at the species level (Setiawan et al., 2017)
There is Bacillus stratospherichus in isolation of Oryctes rhinoceros larvae from empty oil palm bunches while in larvae that live in rotten palm trees there is Bacillus siamensis ( Sijabat et al., 2017).
Bacteria have a variety of biochemical activities (growth and propagation) using raw material (nutrients) obtained from the surrounding environment. Biochemical transformations can arise inside and outside of bacteria regulated by biological catalysts known as enzymes. The original bacteria of the digestive tract have a mutual relationship with their host, which is using the host as their place of life. This study aims to determine the presence of symbionic bacteria in the digestive tract of O. rhinoceros larvae that develop in empty oil palm bunches.
MATERIAL AND METHODS
Location and Description of Experimental Layout
Experiments were conducted in laboratory Faculty of Agriculture, University of Sumatera Utara (USU), Medan, Indonesia from 15 Mei 2018 to Oktober 2018. The materials used in this study were O. rhinoceros L instar 3 larvae taken from the emptyfruit bunches collected from the oil palm plantation area. Media of CMC (Carboxy methyl cellulosa), SCA (Simmon's Citrate Agar), TSIA (Triple Sugar Iron Agar), SIM medium, Gelatin, NB, ingredients for gram coloring test (including crystal violet, lugol’siodine solution, safranin, 95% ethyl alcohol, and distilled water).Tools applied for isolation and identification encompass incubator, autoclave, pipette, Erlenmeyer flask, Bunsen burner, Petri dish, 0.1 g balance sheet, measuring cup, cotton, binocular microscope, glass object, inoculating needle, scalpel, thermometer, PCR Thermal block, electrophoresis machine, UV illuminator, laminar air flow, hot plate stirrer, freezer, bucket.
This research was determined using a descriptive method after field collections of larvae.
Exploration of symbiotic bacteria of O. rhinoceros larvae was conducted through two stages. The first step was attainedby isolating, growing and multiplying bacteria and testing biochemistry.
Isolation of bacteria from the back intestine of the O. rhinoceros larvae
The third instar larvae from the OPEFB were cleaned externally with 95% ethanol. They were subsequently dissected and mashed up, then a series of dilutions were carried out. The diluting processes were conducted by taking 1 g of sample which was put it in a test tube containing 9 mL of distilled water so that 10-1dilution was obtained. To acquire 10-2 dilution, taking 1 mL of 10-1 dilution which was entered into a test tube containing 9 ml of distilled water was carried out. The dilution series were completely continued up to 10-5. Afterward, from 10-4 and 10-5 dilutions 1 mL was taken and put it into a petri dish containing NA, flattened evenly, and then incubated with an inverted position for 24-48 hours at a temperature of 37ºC. After 48 hours of incubation, bacterial isolation was performed by following several steps of quadrant method to obtain one pure isolate.
Gram stain makes it easy to microscopic examination to observe bacterial cell shape so that it is easy to identify by knowing the reaction of bacterial cell walls through a series of staining. Gram staining reactions are based on differences in the chemical composition of bacterial cell walls. Gram positive cells have a thick peptidoglycan, while the peptidoglycan layer in gram negative cells in much thinner (James et al., 2014; Setiawan, 2016).
Biochemical Test
(a) Gram staining. - The object glass was cleaned with alcohol and passed several times on theBunsen burner’s flame, then the bacterial isolates weretaken by using aseptic needle and applied to the glass object. Subsequently, the isolates were dropped with purple violet and left for 1 minute, then washed with running water and aerated to dry. The bacterial isolates were diluted again and left for 1 minute, then washed with running water and allowed to dry. Furthermore, 95% of the bacterial isolates were tested for 30 seconds, then were watered and ultimately aerated to dry. The bacterial isolate were droppedwith safranin for 30 seconds and were washed with running water, dried with suction paper and aerated to dry, and then observed using a microscope. Gram-positive bacteria were marked in purple showing that the bacteria were able to bind crystalline violets, while gram-negative bacteria were marked with pink indicating that the bacteria could not bind violet crystal colors and were only colored by safranin
(b) Biochemical Tests. –The bacteria which had been grown and propagated were employed as the biochemical testing materials in determining the biochemical properties and helping the process of identifying bacteria.
Identification of Bacteria
Bacterial isolates were grown in NB medium at 37°C for 24 hours. The culture was centrifuged at 10,000 × g for 1 minute and the supernatant was removed. DNA extraction was carried out using Presto
™ Mini gDNA Bacteria Kit (Genaid, Taiwan) in accordance with manufacturer's instructions. Universal bacteria 63F (5'-CAGGCCTAACACATGCAAGTC-3') and 1387R (5'- CCCGGGAACGTATTCACCGC-3') were used to strengthen 16S rDNA from genomic DNA.
Polymerase chain reaction (PCR) was completed in Thermo Cycler (ApliedBiosystem).
Amplification was fulfilled as follows: initial denaturation for 5 minutes at 94°C, 35 cycles of each denaturation for 15s at 94°C, annealing for 15s at 55°C, and elongation for 15s at 72°C, and final extension for 7 minutes at 72°C. PCR purification of products and sequencing. The order of 16S rDNAwas compared to the16S rDNAorder available in the Bank Gen database by applying the BLAST program at the National Center for Biotechnology Information http: //www.ncbi.nlm.nih.gov/. Base sequences obtained from sequencing results were subsequently combined and analyzed with the BLAST program.
Phylogenetic Analysis
Phylogenetic analysis was carried out using MEGA 6.0 (Tamura et al., 2013). To evaluate branching statistics, bootstrap analysis was performed by replicating data 1000 times.
Result and Discussion
The study showed that six bacterial isolates were obtained from the hind part of the intestine of O. rhinoceros larvae. The bacterial staining test results demonstrated that three isolates of bluish purple bacteria were gained from positive-gram and 3 red isolates were from negative-gram bacteria. The color differences in gram-positive and gram-negative bacteria indicated that there were differences in cell wall structures between the two types of bacteria. Gram-positive bacteria have cell wall structures with thick peptidoglycan content while gram-negative bacteria are attributed to cell wallstructures with high lipid content (Lenni dan Yekki, 2011). Based on the color testing, purple was formed on the bacterial cell wallsbecause the bacteria hadlower lipid content so that the bacterial cell walls would be hydrated more easily due to treatment using alcohol.
Morphological observations on 6 isolates indicatedthat the bacteria possess quite large, round, and white colonies, convex surfaces, flat edges and spreading growths. The bacteria which are gram- positive and rod-shaped are considered as bacillus group.
In addition to the morphological observations, physiological observations also was included to the biochemical tests (Appendix 6). The simple biochemical tests incorporated into TSIA tests (Triple Sugar Iron), SCA (Simmons Citrate Agar), SA, Gelatin, and Catalase. The biochemical test results of 6 isolates are presented in table 1.
The result of the starch hydrolysis test was obtained by giving a few drops of iodine solution. The solution color dripped on the bacteria changed the color to blue. The color change indicated the presence of starch. If the starch on the medium can not be changed into simple molecules by the bacteria, the color willturn purple or blackish blue because Iodine leads to the bending of the straight structure of the starch which is visible as blue.
Positive gelatin test was indicated by gelatin medium which remained liquid after being set in the refrigerator for ± 30 minutes. The test pointed out that isolates could hydrolyze gelatin or proteins which
were large molecules into polypeptides and then became amino acids using extracellular proteolytic enzymes in the form of gelatinase and proteases.
The bacteria are able to grow using citrate as the only source of carbon, there will be a change in the color of the bacteria growing media on the surface so that it will turn blue due to the pH increase in the media.
The TSIA test, characterized by a yellow top and a yellow underside, demonstrated that lactose and sucrose could be fermented. If the upper part is red and the bottom is yellow, only glucose can be fermented by bacteria.
The test result based on the six isolates of bacteria produced the enzyme catalase. With respect to Lay’s suggestion, the catalase test was carried out to determine the presence of the catalase enzyme in bacterial isolates. Catalase is an enzyme that can catalyze the decomposition of hydrogen peroxide (H2O2) into H2O and O2. This test is very critical to determine the ability of bacteria to produce O2 by deciding hydrogen peroxide (H2O2) to defend them selves.
Table 1. Gram staining and biochemical tests ISOLATES
CODE
GRAM SA GELATIN SCA TSIA CATALASE
B1 (+) (-) (+) (-) (-) (+)
B2 (+) (-) (+) (+) (-) (+)
B3 (+) (+) (+) (-) (-) (+)
B4 (-) (-) (-) (+) (-) (+)
B5 (-) (-) (+) (+) (-) (+)
B6 (-) (-) (-) (+) (+) (+)
(B1) (B2)
(B3) (B4)
(B5) (B6) Figure 1: Gram staining bacterial from hindgut larvae
Identification of Bacterial Species Based on 16S rRNA
The PCR results were visualized on 1% agarose gel and stained with ethidium bromide. DNA amplification was subsequently purified and commercially sequenced to find out the bases of DNA sequences. Afterward, the sequence data was compared with GenBank data from The National Center database for Biotechnology Information (NCBI ) (http://www.ncbi.nlm.nih.gov), using the Basic Local Alignment Search Tool (BLAST) program.
The phylogenetic tree was designed by assigning a comparison of 16S rRNA sequences from other bacteria in the BLAST database tracking program, visualized by the ClustalW program. The
formation of phylogenetic trees wasconducted using the MEGA06 program. Phylogenic trees can be seen in the following figure.
Bacillus stratophericus
Figure 2. Phylogenic tree of the larval symbiont gene with CLUSTAL W and 1000 replications and GenBank.
Bacillus siamensis
Figure 3. Phylogenic tree of the larval symbiont gene with CLUSTAL W and 1000 replications and GenBank.
Bacillus cereus
Figure 4. Phylogenic tree of the larval symbiotic genes with CLUSTAL W and 1000 replications and GenBank
.
Haemophilus parainfluenzae
Figure 5. Phylogeny tree from larval symbiotic genes with CLUSTAL W and 1000 replication and GenBank.
Achromobacter xylosoxidans
Figure 6. Phylogeny trees from larval symbiotic genes with CLUSTAL W and 1000 replications and GenBank.
Alcaligenes faecalis
Figure 7. Phylogeny tree of the larval symbiotic genes with CLUSTAL W and 1000 replications and GenBank.
Identification of B6 isolates had a compatibility of up to 98% against Alcaligenes faecalis.This bacterium is aerobic, gram-negative and pigmented rod-shape. The characteristics distinguishes it from pseudomonas. This species is motile with one or more flagella. The bacteria from the Achromobacter family do not encapsulate and form spores, but grow slowly(Kavuncuoglu et al.,2010).
Identification of bacteria employedthe 16S rRNA to determine the composition of nucleotides because all species had different arrangements or sequences from each nucleotide chain (Thimin, Guanine, Adenine, Cytosin) so that the nucleotide arrangement could be seen or identified species or name of an organism. The results demonstrated that there were 6 bacterial isolates and identified molecularly, for species analysis using sequencing of 16S rDNA bacterial species, namely (B1) Bacillus stratosphericus, (B2) Bacillus siamensis, (B3) Bacillus cereus, (B4) Haemophilus parainfluenzae, (B5) Achromobacter xylosoxidans, and (B6) Alcaligenes faecalis.
B. stratospheric can be found at altitude in the stratosphere and is able to survive in unfavorable environmental conditions and nutrients. Hosseini et al (2014) Said that A strain of B. stratosphericus has been originally isolated from cryogenic tubes of atmospheric air sample. However, atmospheric cycling processes can establish its presence on earth. The vegetative cells of this bacterium have been found to have severalspecific properties such as the ability of catalase production, resistance to UV radiation, and potential to produce electricity. The properties of the B. stratosphericus spores are yet to be unknown.
Herein, some properties of the spores were figured out, and they were introduced as an efficient tool for silver nanoparticle synthesis. Our results are revealed that are vegetative cells of this strain were not able to synthesize silver nanoparticles; however, the spores are represented this ability during a short time.
B. stratospheric resistance in adverse conditions incorporates resistance to UV radiation and tolerance to halo with up to 17.5% NaCl and will not be damaged in the cell. These bacteria can grow at temperatures between 8°C and 37°C, and at pH 6-10. B. stratospheric is highly tolerant of Fe, Co, Ni, and Cu ions, and is quite tolerant of Zn(Raga et al., 2013) and Dunlap (2015) B. stratosphericus can produce biodetergent compatible lipase enzymes using coconut pulp to be used as a substrate that supplies carbon sources and triglycerides. Likewise, marheni's research (2019) states that B.
stratospheric can be used to increase the composting of oil palm empty bunches plus other bioactivators.
B. siamensis is a gram-positive, facultative anaerobic, rod-shaped and movable bacterium. Its colony on media is creamy whiteand grows at 37oC with 6-7 pH (Chen et al., 2016). B. siamensisis a bio- control bacterium producinglipopeptideswhich can potentially reduce the use of pesticides in agricultural areas and inhibit the development of Fusarium oxysporum fungi.
Bacilus cereus is a gram-positive rod shaped. This bacterium is a mesophilicorganism which can develop at temperatures of 30-350C and produce amylase enzymes and proteases, which can break down proteins and clot milk. In stressful situations such as the bad environment and the unsupported food availability, the bacterium will experience sporulation. The produced spores will be able to change back into vegetative cells, influenced by temperature, oxygen, nitrogen. Bacillus spp. memiliki kemampuan antagonis berupa antibiosis terhadap jamur (Abidin et al.,2015) and setiawan et al (2017) B. cereus bacteria is one of the agents pathogens that have great potential for used as a biological controller.
Bacteria has a specific host, no harmful to natural enemies of pests and other non-target objects, easybiodegradable by the Environment and can increased its pathogenicity by technique genetical
manipulation. Some pathogenic B. cereus strain has been developed to control disease pest prohibits strain Pr 1017 isolated from the sick Pristiphora erichsonii larvae.
Morphology of Haemophilus sp. is pleomorphic and sometimes looks like a small stem, a coccobacilli or a short filament. This bacterium is gram-negative and motile.Haemophilus spin habitsin facultative anaerobic atmosphere. H . influenzae is difficult grown and is a bacterium fastidious, which is bacteria growth requires nutrition and special environment. H. influenza grows at a temperature of 35- 37 C with CO2 concentration of 5% -10% (Sulistyaningsih et al., 2018)
The result of identification of the bacteria from the 5th isolate was Achromobacter xylosoxidans.
Achromobacter is an aerobic, a moving, non-fermenting, and gram-negative bacillus. Some researchers said that this bacterium causedinfebacteriaemia, pneumonia, bile duct infections, urinary tract infections, wound infections and peritonitis in human, but not in plants (Park et al., 2012)
Alcaligenes faecalis can be commonly found either in the land and water or in the digestive tract.
In addition, this bacterium can be detected in samples of human blood, urine, and feces (Latt et al., 2013) In biogeochemical cycles in waters, heterotrophic bacterium has a role as decomposers and is able to remineralize organic materials into simple inorganic components returned to the soil and atmosphere as nutrients. The study conducted by Grbavcic et al. (2011), showed that the results of the relative activity of lipase enzymes of more than 70% which were able to survive on 1% detergent exposure could be used as candidates to be formulated into biodiesel detergents.
CONCLUSION
In this study, isolation and identification of symbiotic bacteria from O. rhinoceros larvae in the OPEFB produced 6 species, namely Bacillus stratosphericus, Bacillus siamensis, Bacillus cereus, Haemophilus parainfluenzae, Achromobacter xylosoxidans and Alcaligenes faecalis. Biochemical testing of catalase pointed out that all the symbionic bacteria brought forth the catalase enzyme, extracellular proteolytic enzymes in the form of gelatinase and proteaase produced by B. stratosphericus, B.
siamensis, B. cereus and A. faecalis through biochemical gelatin testing. Lactose and glucose fragmentation through biochemical testing of TSIA was found in A. faecalis, and only B. cereus possessed the ability to hydrolyze starch through SA biochemical tests.
ACKNOWLEDGEMENT
The authors gratitude convey to the Directorate General of Higher Education, Ministry of Research, Technology, Higher Education of Indonesia. Through the University of Sumatera Utara’s Research DRPM. The authors also convey their gratitude the Faculty of Agriculture, Univeristy of Sumatera Utara for giving permission and facilities to conduct research.
Conflict of interest: The authors have declared that the results of this study original and there is no conflict with other parties
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