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Phylogeny of Economically Important Insect Pests That Infesting Several Crops

Species in Malaysia

Conference Paper · September 2014

DOI: 10.1063/1.4895288

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Phylogeny of economically important insect pests that infesting several crops speciesin MalaysiaSiti Zafirah Ghazali, Badrul Munir Md. Zain, and Salmah Yaakop

Citation: AIP Conference Proceedings 1614, 707 (2014); doi: 10.1063/1.4895288 View online: http://dx.doi.org/10.1063/1.4895288 View Table of Contents: http://scitation.aip.org/content/aip/proceeding/aipcp/1614?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Dynamics relationship between stock prices and economic variables in Malaysia AIP Conf. Proc. 1605, 822 (2014); 10.1063/1.4887696 Determination of Opiinae parasitoids (Hymenoptera: Braconidae) associated with crop infesting Bactrocera spp.(Diptera: Tephritidae) using COI and Cyt b sequences AIP Conf. Proc. 1571, 331 (2013); 10.1063/1.4858678 Comparison of the financial performance of Islamic and conventional bank in Malaysia during and after economiccrisis AIP Conf. Proc. 1557, 247 (2013); 10.1063/1.4823913 Perception of auditory temporality in several species of birds J. Acoust. Soc. Am. 61, S62 (1977); 10.1121/1.2015816 Parasitic Infestations in Acoustical Materials J. Acoust. Soc. Am. 11, 375 (1940); 10.1121/1.1902151

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Phylogeny of Economically Important Insect Pests that Infesting Several Crops Species in Malaysia

Siti Zafirah Ghazali, Badrul Munir Md. Zain and Salmah Yaakop

School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia 43600 Bangi, Selangor, Malaysia.

Abstract. This paper reported molecular data on insect pests of commercial crops in Peninsular Malaysia. Fifteen insect pests (Metisa plana, Calliteara horsefeldii, Cotesia vestalis, Bactrocera papayae, Bactrocera carambolae, Bactrocera latifrons, Conopomorpha cramella, Sesamia inferens, Chilo polychrysa, Rhynchophorus vulneratus, and Rhynchophorus ferrugineus) of nine crops were sampled (oil palm, coconut, paddy, cocoa, starfruit, angled loofah, guava, chili and mustard) and also four species that belong to the fern’s pest (Herpetogramma platycapna) and storage and rice pests (Tribolium castaneum, Oryzaephilus surinamensis and Cadra cautella). The presented phylogeny summarized the initial phylogenetic hypothesis, which concerning by implementation of the economically important insect pests. In this paper, phylogenetic relationships among 39 individuals of 15 species that belonging to three orders under 12 genera were inferred from DNA sequences of mitochondrial marker, cytochrome oxidase subunit I (COI) and nuclear marker, ribosomal DNA 28S D2 region. The phylogenies resulted from the phylogenetic analyses of both genes are relatively similar, but differ in the sequence of evolution. Interestingly, this most recent molecular data of COI sequences data by using Bayesian Inference analysis resulted a more-resolved phylogeny that corroborated with traditional hypotheses of holometabolan relationships based on traditional hypotheses of holometabolan relationships and most of recently molecular study compared to 28S sequences. This finding provides the information on relationships of pests species, which infested several crops in Malaysia and also estimation on Holometabola's order relationships. The identification of the larval stages of insect pests could be done accurately, without waiting the emergence of adults and supported by the phylogenetic tree.

Keywords: Pest, identification, molecular phylogeny, cytochrome oxidase subunit I (COI) gene, 28S gene.

INTRODUCTION

Holometabola or Endopterygota are the most successful lineage of living organisms [1]. Holometabola comprise over 80% of insect species, more than 50% of animal lives and the most successful group of organisms [2]. Holometabola insects undergo a complete development consist of larvae, pupae and adult stages. The reason of this success has been proven due to the development of complete metamorphosis within their life cycles. Norris [3] revealed that metamorphosis allow the partitioning of the environmental resources in life history and roles such as feeding and dispersal between morphological specialized immature and adult stages. Holometabola includes insect pest of medical and economic importance e.g. biting flies bees, leaf beetles and weevils [4]. Among the many species of insects that on this earth, not more than 1% can be classified as a pest [5]. Nowadays, many types of economically important crops are threatened by serious pest problems worldwide including Malaysia. Invertebrate pests are the most ubiquitous, damaging and responsible for economic losses worldwide.

Previously, many studies concerning relationships within Holometabola insect group has been done based on morphological characteristics. Phylogenetic hypothesis by Hennig [6] was widely accepted and has been re-examined by Kristensen [1,7] using morphological data. Several molecular studies using different marker have been conducted [4, 8-12]. But, none of these studies use pest in their dataset. Kristensen [13] has divided Holometabola into three major clades: Neuropterida (= Hennig's Neuropteroidea) + Coleoptera, Hymenoptera + Mecopterida, and Mecopterida (= Hennig's Mecopteroidea) (FIGURE 1).

The 2014 UKM FST Postgraduate ColloquiumAIP Conf. Proc. 1614, 707-712 (2014); doi: 10.1063/1.4895288

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FIGURE 1. Kristensen's [14] phylogeny of insect orders based on morphology, including common supraordinal names. Dotted lines mark areas of questionable relationships.

In this study, we sequenced a 700 bp of cytochrome oxidase subunit I (COI) and 500 bp of 28S ribosomal DNA D2 region using 39 individuals of dominant insect pests of several commercial plants. Maximum parsimony (MP) and Bayesian Inference (BI) tree topology have been generated based on the genetic data and compared to the previous holometabola trees. The identity of species has been confirmed by referring to their clustering in the phylogeny [15]. The objective of this study to infer phylogenetic relationships among selected pest species using mitochondrial DNA sequences, COI and ribosomal RNA 28S D2 region sequences and discuss their relationship based on holometabolous insect orders.

MATERIALS AND METHODS

Thirty-nine individuals of pest species of 11 crops species viz. oil palm, coconut, paddy, cocoa, starfruit, angled loofah, guava, chili and mustard including pest of storage rice and fern, Angiopteris evecta (G. Forst.) Hoffm have been collected from various localities in Peninsular Malaysia. The pests (mostly larval stages) were brought to the laboratory for molecular work.

DNA was extracted from specimens using the commercial kit DNeasy Blood and Tissue kit (Qiagen, Valencia, California, U.S.A) according to the manufacturer instructions. Every insect pest species have been extracted from the larval stages and only a small amount of tissue from adult stages. Polymerase chain reaction (PCR) have been carried out in 50 μl volumes containing, 25 μL MyTaqTM Red Mix (Bioline, United Kingdom), 2.0 μL each of 10 pmol/μL primer and 6.0 μL of DNA samples (6ng/μL). Two pairs combination of primers, designed by Folmer [16] was used to amplify COI genes. Primers designed by Belshaw & Quicke [17] and Campbell et al. [18] were used to amplify 28S forward and 28S reverse sequences respectively. PCR has been carried out using “Eppendolf Mastersycler” (Eppendolf, Netherler Hinz GmBH, Hamburg) with the following cycles: 94 °C for 1 min as initial denaturation followed by 35 cycles of 94 °C 1 min, 45-50 °C for 30 s 72 °C for 1 min and 72 °C for 10 min as final extension. PCR products have been checked on electrophoresis using 1.5% agarose gel in 1X TAE buffer. The products have been purified with QIAquick PCR purification kits (Qiagen, Valencia, California, U.S.A). The purified samples have been sent for sequencing analysis to First Base Sdn. Bhd., Selangor, Malaysia.

Sequence of pest (ingroups) including outgroup sequence (Ixodes scapularis) have been edited using BioEdit 5.0.9 [19] and ClustalW [20]. The COI mtDNA sequences have been aligned using software MEGA6 [21] while 28S rRNA D2 region sequences have been aligned using MEGA6 and continued with manual alignment for secondary structure. The analysis of nucleotide composition, the overall transition and transversion ratio and pairwise genetic distances based on Kimura two-parameter [22] have been analysed using MEGA6 software. Barcode of Life Database (BOLD) and Basic Local Alignment Tool (BLAST) have been used to compare the homology of obtained species with the available sequences in the GenBank (NCBI). Maximum parsimony (MP) has been performed with


PAUP 4.0b 10 using the 1000 stepwise addition replicates in a heuristic search [23], with the tree bisection reconnection (TBR) branch swapping method for branch-swapping algorithm. Bayesian Inference was initially performed with PAUP 4.0b10 [23] to get the AIC (Akaike Information Criterion) and best model for further analysis with MrBayes 3.1.2 [24]. Confidence levels of the maximum-parsimony (MP) with bootstrap values of ≥ 70% were considered as sufficiently resolved topologies [25] and those of 50%-70% as tendencies. For the Bayesian Inference, posterior probabilities of ≥ 98% were considered as significant [26].

RESULTS

Molecular data successfully identified 15 species of economically important insect pests consists of three order, 12 genera, namely: Metisa plana, Cadra cautella, Calliteara horsfieldii, Bactrocera papayae, B. carambolae, and B. latifrons, Sesamia inferens, Chilo polychrysa, Herpetogramma platycapna, Plutella xylostella, Conopomorpha cramella, Tribolium castaneum, Oryzaephilus surinamensis, Rhynchophorus ferrugineus and R. vulneratus. Each pest clustered together in a same clade and supported by low bootstrap and posterior probability value (FIGUREs 2 (a), 2 (b), 3 (a), 3 (b)). The association of insect pests with the similar host plant of crop showed in the specific clade; oilpalm = M. plana and C. horsfieldii, coconut = R. ferrugineus and R. vulneratus, cocoa = C. cramella, rice = S. inferens and C. polychrysa, angled loofah and guava = B. papayae, starfruit = B. carambolae, chili = B. latifrons. Pest species of storage rice and fern also showed in the clade; storage rice = T. castaneum and O. surinamensis, fern (A. evecta) = H. platycapna.

A total of 700 bp and 500 bp fragments have been obtained from the multiple alignments of COI and 28S genes, respectively. Final length (maximum length) of 28S sequences after alignment with consideration to secondary structure of each species is 1465bp. Among the aligned COI sequence sites, 322 (46%) sites were variable and 304 (43%) sites were informative for parsimony analysis and for 28S sequence sites, 514 (35%) sites were variable and 371 (25%) sites were informative for parsimony analysis. Sequence analysis indicated that COI sequences showed more variable and parsimony informative compared to 28S sequences. Furthermore, the conserve sites of COI constituted of 378 (54%) compared to 28S of 765 (52%) conserve sites.

The MP tree for COI dataset has been constructed from the heuristic search, with TL (tree length) = 1015, CI (consistency index) = 0.4660, HI (homoplasy index) = 0.5340 and RI (retention index) = 0.3846. While the MP tree for 28S dataset generated, with TL (tree length) = 1013, CI (consistency index) = 0.7631, HI (homoplasy index) = 0.2369 and RI (retention index) = 0.8765 respectively. In the Bayesian Inference, the general time reversible model (Tavaré, 1986) with gamma distributed rates and invariant positions (GTR + I + G) for the COI partition, the general time reversible model with gamma distributed rates (GTR + G) for the 28S partition have been determined. The Bayesian Inference for both partitions was successfully divides the pest sequences into three highly supported monophyletic groups. (FIGURE 3 (a), 3 (b)).

The 50% majority rule consensus trees of COI gene of MP analysis indicated that each order: Coleoptera, Lepidoptera and Diptera formed a monophyletic (Fig. 2a) moderately supported with bootstrap value (59% - 100%). Trees of BI analysis also indicated that each order: Coleoptera, Lepidoptera and Diptera formed a monophyletic (FIGURE 3 (a)) supported with high posterior probability value (0.97 - 1.00). MP tree separated clade Lepidoptera with a Coleoptera and Diptera. In the analysis, Coleoptera and Diptera become a sister clade, but lowly supported with only 33% of bootstrap value. Meanwhile, BI analysis tree topology obtained from COI gene has been successfully separated clade Coleoptera with a Mecopterida (Diptera + Lepidoptera) supported with high (1.00) posterior probability. Lepidoptera and Diptera formed a sister clade and supported with low (0.60) with posterior probability in Bayesian inference tree. All the pest species have been successfully clustered together in MP and BI tree. Meanwhile, both trees of 28S gene were unable to cluster all the pest species together. MP and BI tree topology of 28S gene (FIGURE 2 (b), 3 (b)) separated clade Diptera with a (Coleoptera + Lepidoptera) clade with bootstrap support, 100% and posterior probability, 1.00. Order Coleoptera and Diptera formed sister order and supported with 100% bootstrap value and 0.93 posterior probability in both trees.


FIGURE 2. (a) Bootstrap 50% majority rule consensus tree obtained from maximum parsimony analysis of COI sequences (including one outgroup sequence). Numbers at nodes indicate posterior probabilities above 70% and different branch color

indicate different pest order. (b) Bootstrap 50% majority rule consensus tree obtained from maximum parsimony analysis of 28S sequences (including one outgroup sequence). Numbers at nodes indicate posterior probabilities above 70% and different branch

color indicate different pest order.

FIGURE 3. (a) Bayesian posterior probability tree of COI mtDNA gene sequences (including one outgroup sequence). Numbers

at nodes indicate posterior probabilities above 0.7 and different branch colors indicate different pest orders. (b) Bayesian posterior probability tree of 28S mtDNA gene sequences (including one outgroup sequence). Number at nodes indicate posterior

probabilities above 0.7 and different branch colors indicate different pest orders.


DISCUSSION

The dominant pest species in this study are from the order Coleptera, Lepidoptera and Diptera that infesting various economically important crops especially during their larval stages [27-29]. The larval stages are often challenging to identify at quarantine inspection sites. The confirmations of the pest species are very important as preliminary data and highly necessary in biological control programs. Therefore COI marker can be especially useful in identification of insects at immature stages that are not morphologically distinct, without the necessity for delay in waiting for emergence of the adult specimens [30]. In this paper the larvae specimen of the similar pest species has been successfully achieved by clustering the species in the same clade in the COI tree topologies (FIGUREs. 2 (a), 3 (a)). MP and BI trees of COI gene was successfully clustered each pest species into a sub-clade at the end of the node. This finding also supports the suitability of COI gene as the core of a global bioidentification system for animals. DNA barcoding using short, standardized gene regions as internal species tags designed to provide rapid, accurate, and automatable species identifications [31].

Bayesian tree of COI genes agreed well with the traditional hypotheses of holometabolan relationships previous molecular-based phylogenetic study [32]. Our data support a phylogenetic placement for order Coleoptera, Hymenoptera and Diptera that was first suggested in the early 1800s. Our data also supported with recently molecular study. Whiting [33] study phylogenetic relationships among the holometabolous insect orders inferred from 18S ribosomal DNA, 28S rDNA and morphological characters. In this study, Coleoptera certainly sit outside of Mecopterida (Trichoptera, Lepidoptera, Mecoptera, Siphonaptera, Strepsiptera and Diptera). Nuclear phylogenomic data sets [32] found that 4 of the 11 holometabolan orders included as (Hymenoptera + (Coleoptera + (Diptera +Lepidoptera))). This result support the formation of Lepidoptera and Diptera as sister group while Coleptera placed outside these clade. The differences between two analytical methods (MP and BI) are due to the high among site rate heterogeneity found in insect mitochondrial genomes which is properly modelled by Bayesian methods but results in artifactual relationships under parsimony [12]. In addition, Bayesian analyses converged on a single topology whereas parsimony analyses supported a range of relationships with poor nodal support. These results supported with Cameron [12] that accurate inference of inter-ordinal relationships using mitochondrial genome data will require sophisticated analytical methods. These results also suggested that COI marker as a potential candidate gene to study higher taxonomic level in the presence of sophisticated analytical method. Although there are a lot of study that proposed the usefulness of 28S in higher taxonomic study, but the tree generated in this study are contradict with the previous findings. Hovmoller et al. [34] study the Paleoptera problem at the basal Pterygote phylogeny inferred from 18S and 28S rDNA sequences and also encounter the similar problem while analyze 28S region. In their study, the phylogenetic tree was poorly resolved compared to other 18S region. As a conclusion, the phylogenetic tree well resolved using COI marker compared to 28S marker.

ACKNOWLEDGEMENTS

Special thanks to Mr. Mohd Syukri Ahmat, Mrs. Nursuhana Dahari and Mr. Khairul Fadzelin Zulkifli, officers of the Malaysian Palm Oil Board (MPOB), Mrs. Suhana Yusof officer of the Malaysian Agricultural Research and Development Institute (MARDI), Mrs. Azlina Zakaria officer of the Sime Darby Berhad, and Ms. Saripah Bakar from the Malaysian Cocoa Board (LKM) for their help in insects sampling. This research was fully supported by GGPM-2012-021.

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Phylogeny of economically important insect pests that ......Determination of Opiinae parasitoids...

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