Characterization of Three Species of Thrips on Weeping Fig , Nutmeg , and Marine Seruni Plants Based on Mtcoi DNA Sequences

Thrips are widely reported as pests in vegetable crops. However, the existence of Phlaeothripidae members has a less concern in Indonesia. Phlaeothripidae is the only family of Tubulifera Suborder and some reports suggested that they had potential to be pests in several crops due to their ability to roll up and to make galls on leaves. The first step in pest management attempt is to identify the pest accurately and quickly, so the pest management can be on target and more efficient. One of the identification methods is the molecular identification using DNA barcoding techniques. This study aimed to characterize and to compare species thrips in banyan, nutmeg, and marine seruni based on their molecular characteristics. This research was conducted in Bogor and Kuningan. The process of molecular characterization consisteds of four steps DNA total extraction, amplification by using PCR, COI gene sequence, and data analysis. PCR programme was succesfully to amplified mtCOI gene fragment at 710 bp. The length of mtCOI gene of Gynaikothrips uzeli, Haplothrips ganglbaueri, and Pseudophilothrips ichini were 704, 686, and 702 bp dominated by A and T bases with nucleotide variation value of 27.8%. This results confirmed that molecular characterization using mtCOI gene mitochondrial had successfully supported the morphological data.

in species, such as in colour and size (Mehle & Trdan, 2012).When identifying adult thrips species using traditional, printed dichotomous keys, a series of questions must be answered in a fixed order, and this sequential approach can limit identification if an early character is not identifiable (Mehle & Trdan, 2012).Some identification keys have limited morphological characters and number of pictures (Brunner et al., 2002;Mound & Morris, 2007).In most cases, identification keys needed additional characteristics that can be used to identify the thrips more rapidly and correctly.
Thrips identification of the members of Phlaeothripidae family based on the molecular characteristics had never been conducted in Indonesia since the members were considered nonpests.Molecular assays can be applied to all life stages including the immature stages for which morphological identification to species is not possible (Ubaidillah & Sutrisno, 2009).This research aimed to identify and to compare molecular characteristics of three thrips species found in banyan, nutmeg, and marine seruni plants through nucleotides sequences of COI gene.The objectives were in the form of data of nucleotides sequences of COI gene which can be used as the additional characteristics to complete the morphological data in the process of thrips identification.

METHODS
This research was conducted from September 2014 to August 2015 in Bogor and Kuningan.Molecular identification was conducted in Plant Virology laboratory, Plant Protection Department, Faculty of Agriculture, Bogor Agricultural University Indonesia.

Sample Collection
The sample collection was carried directly by picking up the thrips on the flowers and plants of banyan, nutmeg, and marine seruni plants that had symptoms caused by the thrips attack.Then, the samples were sorted based on its morphological characteristics and was inserted to the eppendorf tube containing 96% alcohol.The samples were then inserted to the clips plastic with description.The location of sampling was marked by using Global Positioning System (GPS) to get the coordinate.
Slide preparation which has been modified based on Mound & Kibby (1998) method.Imago thrips was removed from the collecting fluid into clean 96% alcohol and attempt to open the wings and straighten the antennae using micro-

INTRODUCTION
Thrips typically feed on and damaged young, growing leaves and immature fruits, colour distortion and stunted growth.Their minute size often 0,5 to 5,0 mm.Among the 6680 thrips species described worldwide only 1% are known as pest species.They are divided into two suborders, i.e.Terebrantia with a blunt or angled body end, and Tubulifera in which the abdomen forms a tube at the end (Boror et al., 1996).There are around 3950 species including Tubulifera Suborder (Mound & Morris, 2007).
The identification to the thrips in Indonesia has been conducted by Sartiami & Mound (2013), as well as Subagyo ( 2014), but information about the existence of the Phlaeothripidaefamily in Indonesia is very limited.Khalsoven (1981) reported that there was one genus found in association with Graminae (genus of Haplothrips).In some countries, the report regarding the members of the Phlaeothripidae family has ever been reported to be predators dan galls former of plants, e.g., Gynripsikothrips flaviantennus Multon, Schedothrips orientalis Ananthakrishnan, Crotonothrips dantahasta (Ramakrishna), Thilakothrips babuli Ramakrishna dan Androthrips flavipes Schmutz.The members of Tubulifera Suborder that have been reported are Gynaikothrips uzeli that attacks banyan trees, G. ficorum that attacks bayleaf trees, and Haplothrips ganglbaueri that attacks weeds of Echinochloa crusgalli (Varadarasan & Ananthakrishnan, 1982).Whereas Pseudophilothrips ichini attacks papper plants (Held et al., 2005;Mound et al., 2010).
Additionally, their minute size and cryptic behaviour make them difficult to detect either in the field on in fresh vegetation trasported throught international trade.Consequently, many species have now spread from the original natural habitats an host to favourable new environments, including valuables crops (Brunner et al., 2002).In the identification of thrips, it is also important to understand their biology and to empower integrated pest management strategies.The identification of thrips has been based on external morphology (appearance) (Mehle & Trdan, 2012).Illustrated keys have a major advantage in that they graphically display the characters of interest.
However, there are many obstacles in the use of morphological identification key.Identification on thrips species by morphological examination is restricted to adult specimens, because there are no adequate key for identification for eegs, larvae or pupae (ISPM, 2010).Morphological appearance can vary in many ways with-pins.Place a drop of hoyers solution on the object glass with a diameter of 13 mm and place a thrips into this drop, ventral side uppermost, and gently lower a slide onto the drop.Invert the slide as soon as the solution has spread sufficiently.Place immediately into an oven at 35-40°C.Then leave for a few hours before attempting to study.When the mountant was dried, ring with nail varnish and labelled appropriately.Identification of trips conducted directly under a stereo microscope OLYMPUS CX21FSI with Dino-eye camera AM4232.Identification used key identification of Mound and Kibby (1998), Sartiami & Mound (2013) & Subagyo (2014).

Molecular Characterization DNA Extraction
Total genomic DNA was extracted from single thrips using the slightly modified protocol by Goodwin et al. (1994).Briefly, individual thrips imago were placed in eppendorf tube 1.5 mL, then it was added the CTAB bufer extraction 100 μL (CTAB 2%, NaCl 1.4 M, Tris-HCI 100 mM, EDTA 20 mM, and PVP 1% [-40ºC]), 1 μL of protainase K. Plastic grinders were used to crush the insect, an the tube were incubated at 65 o C for 3 minutes.The tube was added CI 100 μL with 24:1 ratio.It were vortexed for 3 minutes, and were centrifuged at 10 000 rpm for 15 minutes.The supernatant that had been formed was taken of 60 μL, and moved into new tube.Then, added sodium acetate 3 M (pH 5.2) 6 μL and 44 μL isopropyl.The homogenate was store -20 o C for 24 hours.After the preparation, the tube were centrifuged at 10.000 rpm for 10 minutes and the supernatant were disposed.The pellets was washed by using 100 μL of 80% ethanol and was centrifuged at 8000 rpm for 5 minutes.Final step, the supernatant was disposed and the pellets was dried for less than 1 hour and then resuspent using TE buffer 20 μL for subsequent PCR amplification.

Data Analysis
Direct sequenced at Genetica Science.Sequence were trimmed to aligned manually in BioEdit ver.7.0.9. then analyzed by using BLAST programme.Nucleotide matrix formation was conducted with Clustal x (1.83) software, while the phylogenetic tree from morfological data constructed by NTSys21, and sequence data constructed by Molecular Evolutionary Genetic Analisis (MEGA 6) software with UPGMA methode and bootstrap for 1000 replicates

RESULT AND DISCUSSION
Based on the results of this research, the thrips found in banyan, nutmeg, and marine seruni plants were the members of the Phlaeothripidae family.The morphological identification showed that the three samples belong to different species.They were Gynaikothrips uzeli (Zimmerman), Haplothrips ganglbaueri (Schmutz), and Pseudophilothrips ichini (Table 1).
G. uzeli sample had morphological characteristics such as: an antenna consisting of 8 segments, shape of the end of its stylet is curve and narrow, cheeks without setae, the pronotum usually with less than 5 paors of well-developed setae, forewings without venation with paralel sides, tergites with only 2 pairs of wing-retaining

Amplification and Alignment COI gene
The diagnostic PCR succesfull distinguished the three COI of thrips.Specimens with COI gene produced a single PCR product of ± 710 bp (Figure 1).The second band from G. uzeli (banyan tree), third band from H. ganglbaueri (marine seruni/Widelia biflora) and fourth band from P. ichini (nutmeg tree).This study succeed to identified thrips species by using COI gene.Based on the results there were three thrips sequences in which each sample had different lenght in nucleotide array.It was happened bacause the three samples from different taxonomy level in genus.As described Karimi et al. (2010) based on COI gene sequences, also successfully distinguished some of thrips species of four different genus.Further-more COI gene sequence possible identify unknown specimens by comparing their COI sequence and has been used for identification purposes in projects known as species barcoding.Basically, biodiversity and polymorphism can be seen from DNA sequences of certain fragments of an organism genome (Suryanto, 2001).
Sequence analysis is a technique considered as the best technique to see biodiversity in a group of organism.Basically, biodiversity and polymorphism can be seen from DNA sequences of certain fragments of an organism genome (Suryanto, 2001).The characteristics of DNA nucleotide of COI gene G. uzeli was 704 pb, but with the content of the lowest A base (Adenine) and T base (Thymine) of 70,60% followed by P. ichini 702 pb sequences with AT 73.03% composition.The height of AT composition was a special characteristic of nucleotide array of mtCOI gene on insects.A and T base have weak hydrogen bonds so that easy to changed (Liu & Beckenbench, 1992).Base on the results of nucelotide array of BLAST of G. uzeli in Indonesia has similarity value of similarity of 95% with the same species from China, H. ganglbaueri found has 92% with H.  ganglbaueri from Australia, P. ichini has 84% with P. ichini from Canada.It is so since the three samples took place in different taxonomy level (Table 2).Karimi et al. (2010) suggest that based on COI gene sequences, it had successfully distinguished some of thrips species of four different genus.
Base on the results of nucelotide array of BLAST of G. uzeli from Indonesia, it had the similarity percentage of 93% with the same species from China.H. ganglbaueri found in marine seruni (Widelia biflora) showed the similarity percentage of 92% with H. ganglbaueri species from Australia.Then, samples of P. ichini nutmeg tree indicated the similarity percentage of 84% with P. ichini from Canada (Table 2).
The results of DNA sequences alignment of COI gene of the three samples (Table 3) showed that the three species were taxonomically separated.It can be seen from the nucleotide array of mtCOI gene that had been successfully aligned in which there were differences in some points of nucleotide base sequences of each genus.The number of converse nucleotide of the three species was 622 pb and the nucleotide variation was 27.8% or 173 pb.That was the variation of the array of each sample.When there was variation of array occured in a nucleotide alignment with the old ones, it would show the mutation, including insertion, deletion, or rearrangement of genetic materials (Dharmayanti, 2011;Hoy, 2003).

Genetic Distance and Phylogenetic Tree Based on Gene Sequence
Based on the mtCOI gene sequences of three species, it can be analyzed genetic distance and phylogenetic construction.High value of genetic distance indicates that they are closely related.G. uzeli has genetic disctance value 6.2% with the same species from China.H. ganglbaueri 8.9% with the same species from Australia, while P. ichini 15.8% with P. ichini species from Canada (Table 4).
G. uzeli and H. ganglbaueri species showed hight similarity levels with haplotype in GenBank as showed low genetic distance value.It was quite different from P. ichini in which the similarity levels with haplotype in GenBank was low.However, according to Li et al. (2009), genetic distance within insect species was recieved with genetic distance value 0.05-0.23 or homology value 73-95%.It can be seen from Caligula japonica (Lepidoptera: Saturniidae) (Li et al., 2009), Diadegma (Hymenoptera: Ichneumonidae) (Wagener et al, 2006), and mosquitos (Cywinska et al., 2006).Ubaidillah & Sutrisno (2009), the presence of the differences of nucleotide sequence array occur due to the mutation.It can cause the change of nucleotide base which then change encoded amino acid (Hawkes et al., 2004).Our analyses clearly indicate that genetic differentiation happen because geographic isolation and genetic drift.According to Templeton (1998) genetic variation is distributed spatially within a species geographical range.Resricted gene flow and historical events can be interwined, and cladistical analyses can constructed their temporal juxta position, thereby yielding great insight into both the evolutionary history an population structure of the species.
Phylogenetics tree based on COI gene (Figure 3) showed that there were two main groups of thrips, P. ichini and G. uzeli.Claodgram construction was based on the DNA sequences of mtCOI gene demonstrating the separated classification with bootsrap value between in-group sequences (G.uzeli, H. ganglbaueri, P. ichini) and out-group sequence (Ceratothripoides brunneus).The first group was P. ichini with H. ganglbaueri as sister-grup with the bootstrap value of 84%.The second group was G. uzeli, from Indonesia classifying with G. uzeli from China with the bootsrap value of 100%.
This study investigated the utility of COI for identifying thrips species.As demonstrated in this work, there is a relationship between phylogeny and origin evolution of thrips species.We interpret our analyses of congruence phylogenetics tree as suggesting that the different data sets provide complementary information that is not contradictory.Crespi et al. (1998) result showed adult morphology data was not significant from sequences of COI gene so that can support the results of morphological identification of thrips species classification from Tubulifera (Phlaeothripidae).Karimi et al. (2010) also used COI gene to identified some of thrips species, the result showed there was closely relations of evolu-  Karimi et al. (2010) that used DNA of mtCOI gene to identify some of thrips species in which there was familiarity relations of evolution of each thrips species.The cladogram construction with out-group showed that there was a clear separation among three species.Molecular identification by using COI gene had successfully identified the three thrips species because the gene were conserve (Herbert et al., 2003).Karimi et al. (2010) added that DNA of COI gene is considered as a very great genetic marker in encoding DNA.The data of DNA sequence array of COI gene is also the completing data of biological data and morphological characteristics on insects.Beside the DNA nucleotide array of mtCOI gene, the morphological characteristics can also be used in supporting the results of identification using nucleotide characteristics of COI gene.Crespi et al. (1998) stated that the data from imago morphological characteristics was not different from the identification using DNA sequences of COI gene that can support the results of morphological identification of thrips species classification from Phlaeothripidae.

Figure 3 .
Figure 3. Thrips cladogram based on DNA sequnce nuclotide fragment of COI gene by using UPGMA method . Thermocycling was 5 minutes at 94 o C, an then with 35 cycles of 1 minute at 94 o C, 35 seconds at 52 o C, 1 minute 30 seconds 72 o C and 7 minutes at 72 o C. Visualization of DNA fragment as the result of electrophoresis amplification used agarose gel 1% in Tris-borate (TBE) buffer 0.5X with voltage of 50 volt for 50 minutes.PCR product were electrophoresis in 2% agarose gels.Gels were stained ethidium bromide for 15 minutes, visualized and photographed under UV light.

Table 1 .
Host plants and thrips species of the Phlaeothripidae found in research location.

Table 2 .
DNA BLAST of COI gene on three thrips species by using BLAST-N programme

Table 4 .
Genetic distance of DNA sequences of COI gene on three thrips species