Abstract
The CRB (coconut rhinoceros beetle) haplotype was classified into CRB-S and CRB-G, based on the presence of single nucleotide polymorphisms (SNPs) in the mitochondrial cox1 gene. Mitochondrial genomes (mitogenomes) are the most widely used genetic resources for molecular evolution, phylogenetics, and population genetics in relation to insects. This study presents the mitogenome CRB-G and CRB-S which were collected in Johor, Malaysia. The mitogenome of CRB-G collected from oil palm plantations in 2020 and 2021, and wild coconut palms in 2021 was 15,315 bp, 15,475 bp, and 17,275 bp, respectively. The CRB-S was discovered in coconut and oil palms in 2021, and its mitogenome was 15,484 bp and 17,142 bp, respectively. All the mitogenomes have 37 genes with more than 99% nucleotide sequence homology, except the CRB-G haplotype collected from oil palm in 2021 with 89.24% nucleotide sequence homology. The mitogenome of Johor CRBs was variable in the natural population due to its elevated mutation rate. Substitutions and indels in cox1, cox2, nad2 and atp6 genes were able to distinguish the Johor CRBs into two haplotypes. The mitogenome data generated in the present study may provide baseline information to study the infection and relationship between the two haplotypes of Johor CRB and OrNV in the field. This study is the first report on the mitogenomes of mixed haplotypes of CRB in the field.
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Introduction
Oryctes rhinoceros (L.) (Coleoptera: Scarabaeidae: Dynastinae), known as the coconut rhinoceros beetle (CRB), is a severe agricultural pest found in coconut and other palm trees throughout Asia and the South Pacific. CRB is an endemic insect pest of coconuts in Asia, ranging from West Pakistan to India, Ceylon, Burma, Hainan, Hong Kong, Formosa, Peninsular Malaysia, Indonesia, and the Philippines1, and the Pacific Islands2. Oryctes rhinoceros nudivirus (OrNV) is an endemic entomopathogenic virus affecting both the adults and immature stages of the CRB3. It was found in Malaysia in 1963 and introduced to the Pacific Islands to suppress the population of CRB2. However, OrNV has been reported failure to control the new invasive CRB in Guam (2007), Papua New Guinea (2009), Hawaii (2013), the Solomon Islands (2015), and more recently in New Caledonia and Vanuatu4. The intolerant of CRB to OrNV infection could be haplotype dependent5.
The CRB was classified into two haplotypes, CRB-S and CRB-G, based on the presence of single nucleotide polymorphisms (SNPs) in the mitochondrial cox1 gene in 20175. The haplotype CRB-S was susceptible to OrNV5, while the haplotype CRB-G was tolerant to OrNV. Later, CRB-PNG haplotype was identified in Fiji, Samoa, Papua New Guinea, Tonga, and the Solomon Islands in 20216. The susceptibility of CRB-PNG towards OrNV infection is not reported. Different haplotypes of CRB vary in their tolerance to OrNV infection. Mixture of CRB haplotypes in the field may affect the successful use of OrNV as a biological control measure in controlling the CRB in the field.
The cox1 gene and several other mitochondrial genes are routinely used as a universal barcoding region to identify CRB5,7. The presence of a single SNPs found in the partial cox1 gene amplicon has been used to determine the CRB-G haplotype in the Pacific Islands in the early 1900s5,6. However, such partial sequencing data may be challenging to distinguish the true mitochondrial lineages. The insect mitogenomes are widely used for investigating insect health, comparative and evolutionary genomics8,9,10, and molecular evolution studies due to the features of maternal inheritance7,11,12,13,14,15,16,17. The first mitogenome of CRB was reported in the Solomon Islands and has confirmed the CRB which was tolerant to OrNV infection was CRB-G haplotype5,7. To date, the mitogenome of CRB-S with susceptibility to OrNV infection and CRB-PNG with unclear pathogenicity has yet been reported.
CRB has long been reported to infest oil palm and coconut trees in Malaysia. Johor is the second largest oil palm planted area18 and the largest coconut planted area in Peninsular Malaysia19. Four types of OrNV have been detected in the local CRB20. The type A OrNV was detected in many places in Malaysia while the type B OrNV was detected in Selangor, Perak and Johor. Type C and D were localized OrNV in Sabah and Kelantan, respectively. Although a high incidence of CRB infestation was reported in Johor21, no research has been conducted to reveal the haplotype of CRB and their interaction with OrNV in the field. This paper reports the mitogenome of CRB haplotypes and their OrNV incidence in the oil palm and coconut palms in Johor, Malaysia. This comparative mitogenome study could aid in the biosecurity and control effort against this invasive pest in Malaysia.
Results
Mitochondrial genome assembly
The mitochondrial genomes of Johor CRB were successfully assembled. The Johor CRB from oil palm and coconut plantations contained different sizes of mitogenomes (Table 1). Two groups of mitogenomes were found in the oil palm and coconut CRBs. The first group consists of Johor CRB (oil palm Johor CRB 2020, oil palm Johor CRB 2021 and coconut Johor CRB 2021) with mitogenome size approximately 15,315 bp to 15,484 bp while the second group consists of Johor CRB (oil palm Johor CRB 2021 and coconut Johor CRB 2021) with mitogenome size around 17,200 bp. Among the Johor CRBs examined, the smallest mitogenome (15,315 bp) was recorded in the oil palm Johor CRB 2020 (ON764799) while the biggest mitogenome (17,275 bp) was found in the coconut Johor CRB 2021 (ON764801).
The mitogenome of Johor CRBs was high A + T bias. Among the Johor CRB with smaller mitogenomes, oil palm Johor CRB 2020 (ON764799) and coconut Johor CRB 2021 revealed similar range of A, C, G, and T content. Approximately 250 × coverage was recorded in the mitogenome of oil palm CRB 2020 (ON764799) containing 39.5% A, 18.8% C, 10.0% G, and 31.7% T. The mitogenome of coconut Johor CRB (OP694175) contained 39.2% A, 18.9% C, 10.1% G and 31.8% T with 840 × coverage. The mitogenome of the oil palm Johor CRB collected in 2021 (ON764800) was slightly bigger than those collected in 2020, which was 15,475 bp (approximately 265 × coverage) with 35.8% A, 21.7% C, 12.3% G, and 30.3% T. The second group of Johor CRB with bigger mitogenome contained similar A (39.3%), C (18.6%), G (9.9%) and T (32.2–32.3%) content.
Mitochondrial genome annotation
The mitogenome of Johor CRBs contained 13 protein-coding genes (PCGs), two ribosomal RNA genes, and 22 transfer RNA genes (Tables 2, 3, 4, 5 and 6). All PCGs started with a regular initiation codon (ATN). A total of 10 out of 13 PCGs had conventional stop codons (TAG or TAA) while three other genes, such as atp6, cox3, and nad5, had an incomplete stop codon (TAT). The annotation of all mitogenomes revealed 37 genes, with the trnI and trnQ genes rearranged in the following order: control region-trnQ-trnI-trnM-nad2 instead of control region-trnI-trnQ-trnM-nad2 in invertebrates.
The haplotypes of Johor CRB were confirmed as CRB-G and CRB-S by in silico digestion. The Johor CRB-G had generated three fragments (253 bp, 138 bp, and 92 bp) while the Johor CRB-S generated four fragments (181 bp, 138 bp, 92 bp, and 72 bp). The Johor CRB from oil palm and coconut plantations contained both haplotypes G and S.
The mitogenome of oil palm Johor CRB-G 2020 (GenBank accession number: ON764799) revealed one substitution in the nad1 gene while the mitogenome of oil palm Johor CRB-G 2021 (GenBank accession number: ON764800) contained many substitutions in nad5, nad4, nad4L, nad6, cob, and nad1 genes (Table 7). The mitogenome of coconut Johor CRB-G 2021 (GenBank accession number: ON764801) had no substitution nor indel. The coconut Johor CRB-S 2021 (GenBank accession number: OP694175) and oil palm Johor CRB-S 2021 (GenBank accession number: OP694176) had substitution and indels found in nad2, cox1, cox2, atp6, nad5, nad4, nad4L, nad6, cob and nad1 genes.
A total of 13 PCGs are presented in Table 8. SNPs were detected in cox1, cox2, atp6 and nad2 genes using muscle alignment plug in Geneious Prime version 2023.0 (Fig. 1). A total of 4 SNPs was detected in cox1 gene, 1 SNP in cox2 gene, 3 SNPs in atp6 gene, and 6 SNPs in nad2 gene. The SNPs presented in Fig. 1 could differentiate the CRB-G and CRB-S significantly.
SNP locations found in (A) cox1, (B) cox2, (C) nad2 and (D) atp6 genes of Johor CRB mitogenomes.
Mitochondrial genome visualization
The mitogenome of all Johor CRBs collected in the present study featured a gene-packed section and a control region, also known as the D-loop region which contained components required for transcription and replication. It contained 13 PCGs, two rRNA genes, and 22 tRNA genes. Among the 13 PCGs, 9 PCGs (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6, cob) were encoded in the majority strand (J strand) while 4 PCGs (nad5, nad4, nad4L, nad1) were encoded in the minority strand (N strand) (Fig. 2).
Circular map of the mitogenome of Johor CRB. The position and orientation of 13 PCG genes (green), 22 tRNA (pink), two rRNA genes (red), and the control region (black).
Phylogenetic analysis
The phylogenetic analysis presented the relationship between the mitogenome of Johor CRBs and other members of the subfamily Dynastinae. The 23 datasets of mitogenomes of scarab beetles with Trogidae and Geotrupidae as the outgroup were aligned without removing redundant sequences or trimming end gaps from the alignment. The yielded alignment of the aligned mitogenomes sequence was 26,220 bp. Tree construction was inferred from Bayesian phylogenetic analysis using HKY85 model with an equal rate variation setting carried out in Geneious version 2023.0.2. Posterior probabilities were calculated over 2.0 × 106 generations. The Bayesian tree showed the more robust phylogeny tree of scarab beetles which has successfully separated Family Scarabaeidae as one clade per subfamily with a posterior probability of 100% (Fig. 3).
Bayesian inference of phylogenetic tree for Scarabaeidae, with the outgroups Family Trogidae and Geotrupidae, constructed using MrBayes plugin Genious prime version 2023.0.222.
The mitogenome of Johor CRBs was compared to the complete mitogenome of CRB-G from the Solomon Islands (GenBank accession number: MT457815). The percent of sequence identity of the mitogenomes of Johor CRBs (GenBank accession number: ON764799, ON764801, OP694175, and OP694176) was around 99% except the Johor CRB-G collected from the oil palm in 2021 (GenBank accession number: ON764800) was 89.24% (Table 8).
OrNV confirmation and symptoms
The gDNA of Johor CRB samples (n = 30) confirmed the presence of OrNV by PCR amplification (Fig. 4). The presence of OrNV in the Johor CRB-G samples collected from oil palm (n = 5) and coconut (n = 3) plantations was detected with a target band of 945 bp. However, the OrNV was not detected in the Johor CRB-S haplotype samples (n = 22).
Uncropped and unadjusted images of gel. Images of agarose gel of gel electrophoresis of the DNA amplification product to detect OrNV in Johor CRB samples. M: CSL-MDNA 1 kb ladder, (1) oil palm Johor CRB-G 2020, (2) oil palm Johor CRB-G 2021, (3) coconut Johor CRB-G 2021, (4) coconut Johor CRB-S 2021, (5) oil palm Johor CRB-S 2021. The PCR product was run on 1% agarose gel in 1 × TAE buffer (w/v) added with DNA stain (Canvax, Brightmax) at 65 V for 40 min.
The Johor CRB-G with positive OrNV detection (Fig. 5C) had a milky white body with bigger translucent abdomen than those Johor CRB-S with negative OrNV detection which had beige abdomen (Fig. 5D,E). The diseased oil palm Johor CRB-G 2020 exhibited prolapsed rectum in general (Fig. 5A) while those diseased oil palm Johor CRB-G collected in 2021 displayed a swollen abdomen without prolapsed rectum (Fig. 5B). The diseased coconut Johor CRB-G also exhibited similar symptoms to those of oil palm Johor CRB-G 2021 except the translucent abdomen was much smaller in size (Fig. 5C).
Symptoms of Johor CRB with OrNV infection. (A) Diseased oil palm Johor CRB-G 2020 with milky white abdomen and prolapsed rectum, (B) diseased oil palm Johor CRB-G 2021 with swollen milky white abdomen, (C) diseased coconut Johor CRB-G 2021 with swollen milky white abdomen but smaller in size, (D) coconut Johor CRB-S 2021 with negative OrNV infection, and (E) oil palm Johor CRB-S 2021 with negative OrNV infection.
Discussions
This study reported the mitochondrial genome of CRBs collected in oil palm and coconut plantations in Johor, Malaysia. Two different haplotypes, namely CRB-G and CRB-S, were discovered in similar breeding sites. It indicates an overlapping population of different haplotypes in one breeding site. These haplotypes have different length of mitogenome either within or between haplotypes. The mitogenome size of oil palm Johor CRB-G 2020 and 2021, coconut Johor CRB-G 2021, coconut Johor CRB-S 2021, oil palm Johor CRB-S 2021 was 15,315, 15,475, 17,275, 15,484 and 17,142 bp, respectively. The Johor CRB-G and CRB-S contained similar mitogenome size compared to CRB (unknown haplotype) from Taiwan (15,339 bp)16 but smaller than those CRB-G from Solomon Island (20,898 bp)7 and other Coleoptera species, Protaetia brevitarsis (20,319 bp)23, and O. nasicornis (20,396 bp)24. The difference in the mitogenome size are primarily due to the size variation of the non-coding region25. In general, the mitogenome has a non-coding region (NR) with AT-rich hairpin structures, tandem repetitions, and unusual patterns26,27,28. The largest NR of O. rhinoceros was identified as a putative control region (CR)7. Previous studies reported that the mitochondrial genome could be highly polymorphic even across individuals of the same species29.
The control region (CR) of Johor CRB-G and CRB-S contained extraordinarily high A + T composition which is often referred as “A + T-rich area” in insects30. This non-coding region involved in the initiation of mtDNA transcription and replication31,32,33. It demonstrates a high rate of nucleotide change, divergence of primary nucleotide sequences, and diverse fragment length between species and individuals34.
To date, the mitogenome of Johor CRB-S presented in this study is the first report of CRB haplotype S in the world. Both mitogenomes of the Johor CRB-G and CRB-S have a full feature of 37 genes: ATPase subunits 6 and 8 (atp6 and atp8), cytochrome oxidase subunits 1 to 3 (cox1-cox3), cytochrome b (cob), NADH dehydrogenase subunits 1–6 and 4L (nad1-6 and nad4L); small and large subunit rRNAs (rrnL and rrnS); and 22 transfer RNA (tRNA), which are the characteristics of metazoan mitogenomes35,36. Metazoan mitogenomes show diversity in several aspects, including length, tRNA secondary structure, gene order, the number and internal structure of regulatory areas, and sequence variation35,37,38. These characteristics can reveal the evolutionary links between species at high and low taxonomic levels8.
The mitogenomes of Johor CRBs contained standard gene order of insects, except for three tRNAs presenting the "tQ-tI-tM" order instead of the "tI-tQ-tM" order8. The trnQ gene precedes the trnI gene in the mitogenomes of Johor CRB collected from oil palm and coconut (Fig. 1). It is similar to the complete mitogenome of CRB from the Solomon Islands7 and Taiwan16. The trnI and trnQ genes were also found rearranged in the mitogenomes of all Hymenoptera species39 and were reported in flat bugs (Hemiptera, Aradidae)40. tRNA gene rearrangement had been observed in Lepidoptera and Neuropteran14,41. The tRNA rearrangement between the CR and cox1 happened in Johor CRBs, and it has been proposed that this region may act as a "hotspot" for tRNA rearrangement39.
The mitogenomes of Johor CRB-G and CRB-S contained 13 PCGs with a regular initiation codon (ATN). A total of 10 PCGs ended with common stop codons (TAG or TAA) while three other genes, such as atp6, cox3, and nad5 had an incomplete stop codon T, which is similar to the mitogenome of CRB-G from Solomon Islands7. Other lepidopteran mitogenomes featured incomplete stop codons, which are prevalent among their mitogenomes42.
Substitutions and indels in the PCGs indicate mutation in the mitogenomes. Based on the mauve alignment, substitutions and indels present in the mitogenome of Johor CRB-G and Johor CRB-S haplotypes, except the coconut Johor CRB-G. The coconut Johor CRB-S 2021 (OP694175) and oil palm Johor CRB-S 2021 (OP694176) have substitutions and indels found in 10 genes: nad2, cox1, cox2, atp6, nad5, nad4, nad4L, nad6, cob, and nad1 genes when compared to all Johor CRB-G in this study and Solomon Islands. The Johor CRB-G contained substitutions and indels only in 6 genes: nad5, nad4, nad4L, nad6, cob, and nad1. Among the Johor CRB-G, the oil palm Johor CRB-G 2021 (GenBank accession number: ON764800) contained many substitutions in nad5, nad4, nad4L, nad6, cob, and nad1 genes.
Mitochondrial DNA (mtDNA) genes such as cox1 and cox2 had been used in designing universal primers for DNA barcoding of invertebrates43. The presence of SNPs in cox1 gene has been used to categorize the haplotype of CRB from Guam, Solomon Islands5. In Orthoptera, cox2 gene was used to identify the orthopteroid insects44. In the present study, SNPs were detected in both the cox1 and cox2 genes of Johor CRB-G and Johor CRB-S. Four fixed base change was found in cox1 gene, and one fixed base change was found in cox2 gene that could possibly distinguish the Johor CRB-S group from the Johor CRB-G. For example, in cox1 gene, the substitutions were located at nucleotide position 318 (G > A), 723 (T > C), 906 (C > T) and 1,080 (T > C) within the sequence fragments examined. The Johor CRB-G has more SNPs in cox1 gene as compared to the partial cox1 gene of CRB-G from Solomon Islands. An A > G transition at nucleotide position 426 was detected in the cox2 gene of Johor CRB-G. In addition, the nad2 and atp6 genes showed 6 and 3 nucleotide substitutions in the Johor CRB-S group, respectively. In nad2 gene, the substituitions were located at nucleotide position 333 (T > C), 591 (T > C), 642 (A > G) within the sequence fragments examined while in atp6 gene, the substituitions were located at nucleotide position 64 (C > T), 207 (C > T), 542 (C > T) within the sequence fragments examined. The cox1, cox2, nad2 and atp6 genes were able to distinguish the Johor CRB-S from Johor CRB-G as well as the CRB-G from Solomon Islands.
The control region (CR) of Johor CRB-G and CRB-S contained extraordinarily high A + T composition which is often referred as "A + T-rich area" in insects30. This non-coding region involved in the initiation of mtDNA transcription and replication31,32,33. It demonstrates a high rate of nucleotide change, divergence of primary nucleotide sequences, and diverse fragment length between species and individuals34. The Johor CRB collected from the stump of coconut had a clear white body colour whereas the Johor CRB collected from decayed oil palm was white with a hint of light brown colour. Differences in the environment and food nutrition may influence a phenotypic change45. In general, the Johor CRB-G and Johor CRB-S were phenotypically similar. However, different haplotypes of Johor CRBs collected from the same sampling sites had exhibited different susceptibility towards OrNV infection. The CRB-G haplotypes collected from oil palm and coconut were confirmed positive to OrNV detection and infection. However, the CRB-S haplotype collected both from oil palm and coconut were confirmed negative to OrNV detection and infection. Even though Johor CRB-G and Johor CRB-S were found in the same sampling sites, only Johor CRB-G were susceptible to OrNV infection. The CRB-G and CRB-S from Johor Malaysia had exhibited different response to OrNV infection compared to those CRB-G and CRB-S reported in Pacific Islands5. This could be due to variation in the virulence of OrNV isolate from different geographical regions46. There were two OrNV strains, OrNV Kluang and OrNV Batu Pahat, were detected in Johor CRB-G47. The OrNV isolates found in Johor, Malaysia may have different virulence than those OrNV Solomon Islands isolate towards CRB-G.
Johor CRB-G exhibited different symptoms of OrNV infection. The oil palm CRB-G 2020 showed chronic lethal OrNV infection with swollen midgut and prolapsed rectum as reported in OrNV-infected CRBs2,48,49. In contrast, the oil palm CRB-G 2021 and coconut CRB-G 2021 did not have prolapsed rectum. Symptomatic infections were shown by the clinical signs and high level of viral particle production, to which the insect succumbs or survives depending on the state of its immune system50. OrNV-infected CRBs will exhibit a prolapsed rectum when they are severely infected51.
Melolonthinae, Cetoniinae, Dynastinae and Rutelinae were used in the phylogenetic analysis of scarabaeidae species. Previous study reported that the subfamily of Melolonthinae was paraphyletic while Cetoniinae was more closely linked to Dynastinae and Rutelinae52,53,54. The present study showed that the Dynastinae formed a monophyletic group as a clade while the Cetoniinae and Rutelinae formed sister clades that established a basal split with Melolonthinae. This result was similar to another previous study of two mitogenomes of scarab beetles16. However, our finding provides more robust support for branch nodes in which almost all branch nodes are equal to one. The Dynastinae, Cetoniinae, Rutelinae, and Melolonthinae are phytophagous group while the Scarabaeinae are coprophagous group52,53. The present Bayesian tree has successfully confirmed the correlation of the subfamily to their feeding habits.
The phylogenetic analysis has confirmed the oil palm Johor CRB-G 2020 (ON764799), the coconut Johor CRB-G 2021 (ON764801) and the CRB-G from Solomon Islands (MT457815) were monophyletic. On the other hand, the oil palm Johor CRB-S (OP694175), coconut Johor CRB-S (OP694176) and CRB from Taiwan (unconfirmed haplotype: NC059756) had a common ancestor. The oil palm Johor CRB-G 2021 (ON764800) revealed a separate ancestor from other Johor CRB-Gs. Although the BLAST result of the oil palm Johor CRB-G 2021 revealed a low (89.24%) sequence homology, it was grouped with other Johor CRB-Gs by in silico digestion. This indicates the oil palm Johor CRB-G 2021 has a unique mitogenome of CRB-G and is considered as an unrecognized haplotype of CRB-G.
Conclusions
Two haplotypes of CRB were discovered in the oil palms and wild coconut in Johor, Malaysia. Both haplotypes can be found in the same sampling sites in the field. The Johor CRB-G samples were prone to OrNV infection while the Johor CRB-S were resistant to OrNV infection. The mitogenome of Johor CRBs was variable in the natural population due to its elevated mutation rate. Substitutions and indels in cox1, cox2, nad2 and atp6 genes were able to distinguish the Johor CRBs into two haplotypes. Further investigation is needed to study the relationship between the two haplotypes of Johor CRB and OrNV infections in the field.
Materials and methods
Ethics statement
No specific permits were required for the insect specimen collection in this study. All experiments were performed in accordance with relevant named guidelines and regulations. All sequenced insects are common species in Malaysia and are not included in the “Red List of Mammals for Peninsular, Malaysia version 2.0.
Sample collection
Oil palm CRB-G 2020 (GPS coordinate: 2.0248117446899414, 103.25872039794922) and oil palm CRB-G and CRB-S 2021 (GPS Coordinate 2,0,310,530, 103,2,703,850) were collected from decayed palms in a private oil palm plantation in Kluang, Johor. Coconut CRB -G and CRB-S 2021 were collected from wild coconut trees in Batu Pahat, Johor (GPS Coordinate: 1.720853, 103.053085). The distance between the oil palm and the coconut sampling location was more than 50 km. The field studies did not involve endangered or protected species. 3rd instar larvae were extracted at Laboratory of Insect Pathology, Department of Plant Protection, Universiti Putra Malaysia, Serdang, Selangor.
DNA extraction of CRB
Insect gut tissue was cut and washed with two times diluted 1 × PBS. The gut tissue was subjected to DNA extraction using a modified protocol of NucleoBond® RNA Soil (MachereyNagel GmbH & Co., Germany). Briefly, approximately 1–1.5 g sample was suspended in 3.2 ml Lysis Buffer E1 and divided into four portions. Each portion (~ 800 µl) was transferred into a 2 ml NucleoSpin® Bead Tubes Type A. 100 µl of buffer OPT was added to the mixture, followed by 100 µl of phenol: chloroform: isoamyl alcohol (25:24:1 v/v). The sample was lysed by bead beating for 5 min at 2280 rpm on a mechanical cell disruptor. The sample tubes were then centrifuged for 2 min at 14,800 rpm. The supernatant of different tubes was pooled into a 15 ml centrifuge tube to a final volume of 2.5 ml. An aliquot of 313 µl of binding Buffer E2 was added, and the tube was inverted five times, then incubated for 2 min at room temperature. The tube was then centrifuged for 2 min at 6000 rpm. The supernatant was transferred into a NucleoBond® RNA Column (including a filter) pre-equilibrated with 12 ml of equilibration Buffer EQU. The supernatant was loaded into the centre of the filter. The filter was washed with 6 ml of Buffer E3; the flow through and filter were then discarded. The NucleoBond® RNA Column without a filter was washed with 8 ml of Buffer E4. The column was transferred to a fresh 50 ml tube, and the DNA was eluted with 5 ml of elution buffer EDNA. The first eluted DNA was mixed with 3.5 ml of isopropanol. The mixture was then loaded into a NucleoSpin® Finisher Column and centrifuged for 2 min at 6000 rpm. The column was washed with 1 ml Buffer E5, followed by drying using centrifugation at 6000 rpm for 2 min. Finally, the DNA was eluted with 100 µl of RNase-free H2O. DNA was subjected to RNase treatment at 37 °C for 30 min and then precipitated with phenol: chloroform: isoamyl alcohol extraction, followed by ethanol precipitation. Lastly, the DNA pellet was dissolved in 50 µl of RNase-free H2O.
DNA quality check
The quality of the DNA samples was confirmed prior to Next-generation sequencing (Supplementary Table 1). Two methods in quality control of DNA samples were used. Method 1: DNA degradation and potential contamination was assessed on 1% agarose gel. Method 2: the DNA concentration was determined using a Qubit® 2.0 Fluorometer and the Qubit® dsDNA Assay Kit (Life Technologies, CA, USA). The sample with OD values between 1.8 and 2.0, and DNA concentration greater than one µg was used to construct a library. The samples were sent for Next-generation sequencing using the Illumina platform at Novogene Co., Ltd. Singapore.
Library construction
A total of 1 µg of DNA sample was used as input material for library preparation. Libraries were generated using the NEBNext® Ultra™ D.N.A. Library Prep Kit (NEB, USA). The index codes were added to attribute sequences to each sample. The DNA sample was fragmented by sonication to a size of 350 bp. Then, the DNA fragments were end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification. Finally, the PCR products were purified (AMPure XP system), and libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer and quantified using real-time PCR.
Illumina sequencing
The clustering of the index-coded samples was performed using cBot Cluster Generation System. After cluster generation, the library preparation was sequenced on an Illumina NovaSeq6000 platform, and paired-end reads were generated.
Mitogenome assembly, annotation, and analysis
The quality of raw reads was inspected with FastQC v.0.11.955. Low-quality reads (Q ≤ 28) were removed with fastp v.0.20.156. The mitogenome was assembled with MitoZ v2.4-alpha57. Mitogenome annotation was performed with the Mitos2 web server (http://mitos2.bioinf.uni-leipzig.de/index.py) with parameters as follows: Reference: "RefSeq 89 Metazoa" and genetic code: "5 Invertebrate". The contig was imported to Geneious prime version 2023.0.2, and the mitogenomes were finally visualized with the Geneious prime version 2023.0.2.
In silico digestion
The assembled cox1 gene sequences were aligned with the CRB cox1 gene (526 bp) obtained from the GenBank using the MAFFT alignment with the default setting parameters in Geneious Prime version 2023.0.2. The alignment was further trimmed to reduce gaps, yielding a 526-bp sequencing fragment. The trimmed sequence was cut with MseI restriction enzyme and RFLP pattern was analysed for confirmation of CRB haplotypes.
Phylogenetic analysis
The phylogenetic tree was constructed with additional taxa (complete or partially complete mitogenome data) available at the NCBI (Table 9). Sixteen species from five Scarabaeidae subfamilies (Dynastinae, Rutelinae, Cetoniine, Melolonthinae, and Scarabaeinae) and outgroups from the superfamily of Scarabaeoidea (Family Trogidae and Geotrupidae) were compared. Each mitochondrial genome was aligned using MAFFT58,59 with default parameter settings in Geneious Prime version 2023.0.2. (https://www.geneious.com). The phylogenetic tree construction was inferred from Bayesian phylogenetic analysis using the HKY85 substitution model with an equal variation setting carried out in Geneious Prime version 2023.0.2 (https://www.geneious.com). The posterior probability was calculated with a 1,000,000-chain length and burn-in length of 100,000 using molecular clock computation with uniform branch length gamma 1 to 1,000,000.
Confirmation of OrNV infection
Briefly, a total 25 µl PCR reaction mixture was prepared by mixing 12.5 µl of PCR GoTaq® Green Master Mix, 2.5 µl of the forward and reverse primer, 2.5 µl of DNA template, and 5 µl autoclaved distilled water. The primers of OrV1564 was used for the OrNV confirmation. The PCR diagnosis was carried out under the following conditions: an initial denaturation of 95 °C for 2 min, and 35 cycles of denaturation at 95 °C for 30 s, annealing 50 °C for 45 s, and extension 72 °C for 1 min with a final extension at 72 °C for 5 min. Amplified DNA samples were run on 1% agarose gel prepared in 1 × TAE buffer at 68 V for 40 min.
Data availability
The assembled data are available on the website of NCBI with accession numbers: ON764799, ON764800, ON764801, OP694175, and OP 694176.
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Acknowledgements
The authors thank the Southeast Asian Ministers of Education Organization (SEAMEO)-Southeast Asian Regional Centre for Graduate Study and Research in Agriculture (SEARCA) for supporting Madam Erise Anggraini in pursuing her Ph.D. at Universiti Putra Malaysia under the SEARCA Scholarship. Also, the authors thank the field assistants of the Malaysian Agricultural Research and Development Institute (MARDI) for assisting in the collection of insect samples.
Funding
This research received no external funding. This research was a part of a SEARCA research grant for a scholar. The reference of the scholarship award letter was Ref. No. GBG19-1456.
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Conceptualization, E.A. and W.H.L.; Specimen collection and identification, E.A., and M.M.; Methodology and Experiments, E.A. and W.H.L.; Data analysis, E.A. and W.H.L.; writing, review, and editing, E.A., W.H.L., G.V., L.L.K., M.M.; funding acquisition, E.A., and W.H.L. All authors agreed to the published version of the manuscript.
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Anggraini, E., Vadamalai, G., Kong, L.L. et al. Variants in the mitochondrial genome sequence of Oryctes rhinoceros (Coleoptera: Scarabaeidae) infected with Oryctes rhinoceros nudivirus in oil palm and coconut plantations. Sci Rep 13, 16850 (2023). https://doi.org/10.1038/s41598-023-43691-w
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DOI: https://doi.org/10.1038/s41598-023-43691-w
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