Philippine Journal of Science 142 (1): 69-76, June 2013 ISSN 0031 - 7683 Date Received: ?? Feb 20??

Morphometric Analysis and DNA Barcoding of Bactroceraoccipitalis(Bezzi) and B. philippinensis Drew and Hancock (Diptera: ) from and del Norte

Michael Leonardo C. Delomen1,*, Merlyn S. Mendioro1 and Ma. Genaleen Q. Diaz1

1Institute of Biological Sciences, College of Arts and Sciences, University of the Los Banos, College, 4031, Philippines

Morphometric analysis and DNA barcoding using the 5’ region of the mitochondrial cytochrome c oxidase subunit 1 (mtCOI) gene were carried out to distinguish the fruit flies occipitalis (Bezzi) and B. philippinensis Drew and Hancock. Adult flies and larvae were collected from Cavite and Davao del Norte, Philippines. The larvae were reared to adulthood at the Ecology Laboratory, University of the Philippines Los Baños; adults were then identified along with field- collected adult specimens. Each specimen was assigned with a numerical rating from zero to six through morphological examination, using the diagnostic key originally described by White and Hancock in 1997. For morphometric analysis, ratings zero and six were used for B. occipitalis and B. philippinensis, respectively. The following structures were analyzed: lengths of abdominal tergites/ sternites, femora, tibiae, and metatarsals; as well as wing length and width. Morphometric results showed significant differences (via t-test at α = 0.05%) in tergite III, mid-femur, and the metatarsals of the 2nd and 3rd leg pairs. For DNA barcoding, genomic DNA was extracted from specimens assigned with every rating (0-2 = B. occipitalis, 3 = intermediate/hybrid, 4-6 = B. philippinensis). Using customized primers, mtCOI was amplified, sequenced, and analyzed. A phylogenetic tree was constructed using the Neighbor Joining method. mtCOI clustering did not support morphological ratings, with B. occipitalis, hybrid, and B. philippinensis samples grouped together. Low bootstrap values at certain branches suggested the lack of phylogenetic differentiation among morphological species delineations. Pairwise distances of consensus sequences ranged from zero to 0.033, which were lower than the standard threshold of 0.5% utilized for species delineation in fruit flies. Therefore DNA barcoding failed to delineate B. occipitalis and B. philippinensis.

Key Words: Dacinae, mitochondrial cytochrome c oxidase I gene, Neighbor Joining, Tephritidae

INTRODUCTION region (Drew 2004).Members of the Bactrocera dorsalis (Hendel) species complex, the larval stages of which are The dacine fruit flies (Diptera: Tephritidae: Dacinae) are a frugivorous, caused significant damages(in some cases group of economically important insect pests in Southeast complete loss) in fruit production in Asia (Drew and Asia and the Pacific (Clarke et al. 2005), with the genus Hancock 1994). The consequences of these losses include Bactrocera (629 species) distributed in India, Southeast reduction of plant-based nutrients for the local residents Asia (including the Philippines), Australia and the Pacific and profit loss for farmers and landowners. Export markets *Corresponding author: [email protected] are hugely affected due to quarantine restrictions imposed by destination countries (Drew and Romig 1996). Pest

69 Philippine Journal of Science Delomen et al.: Morphometric Analysis and DNA Barcoding of Fruit Flies Vol. 142 No. 1, June 2013 Bactrocera occipitalis (Bezzi) and B. philippinensis Drew and Hancock management programs such as fruit-bagging, trapping formed a monophyletic clade (Zhang et al. 2010). and are used to combat fruit infestations. These methods are labor-intensive and DNA barcoding was proposed by Hebert et al. (2003a) expensive. Enforcement of quarantine inspections is also as a means of using sequence similarities to identify expensive. The capacity for inducing economic damage species. DNA barcoding of involves the analysis enabled by its high fecundity, extensive dispersion of sequence diversity at the 5’ end of mtCOI. The variation capability, and polyphagous diet, makes Bactrocera in this sequence is expected to be low within a species species one of the most serious threats to horticultural and high between different species (Hebert et al. 2003b; production and trade (Allwood and Leblanc 1996). Hebert and Gregory 2005). It has been described as a step towards an “encyclopedia of life” (Savolainen et al. The B. dorsalis species complex contains 75 species, 2005), indicating its potential application in the quick including B.occipitalis (Bezzi) and B. philippinensis identification and taxonomical cataloging of species. (Drew and Hancock), two sympatric species infecting mainly guava and mango in the Philippines. Local pest Hebert et al. (2004) used DNA barcoding to show management typically uses the sterile insect technique and that the previously identified lepidopteran species chemical toxins for pest control, which requires correct Astraptesfulgerator was actually a complex of 10 mostly identification of the pest species. sympatric species occurring in northern Costa Rica. A similar study in the region used mtCOI sequences to Conventional identification methods use morphological correctly identify three families (Hesperiidae, Sphingidae, features, such as exoskeletal and wing markings, aculeus and Saturniidae) of tropical lepidopterans (Hajibabaei et al. (female) and aedeagus (male) lengths (White and 2006). Smith et al. (2006) used DNA barcoding to clearly Hancock 1997). Iwaizumi et al. (1997) compared the discriminate between 17 parasitoid fly species (Belvosia aculeus/aedeagus lengths in several Bactrocera species, spp.), an insect group that had been difficult to differentiate concluding that B. philippinensis had longer aedeagi than due to morphological similarities. Furthermore, the 17 B. occipitalis. Iwahashi (1999) used the same method Belvosia species count was increased to 32 as DNA to distinguish B. occipitalis and B. philippinensis found barcoding confirmed the presence of cryptic species. The in Island, Philippines. Iwaizumi (2004) used DNA barcoding of Madagascar ant populations (Smith et aculeus/aedeagus lengths to identify B. occipitalis and al. 2005) confirmed hyperdiversity in these organisms, B. philippinensis collected infruit samples (including providing critical taxonomic knowledge for conservation guava, mango, citrus , santol, papaya, sugar apple planning on the island. and velvet apple) exported from the Philippines that were intercepted by Japanese plant quarantine department. DNA barcoding holds potential in delineating closely Using morphological characters for identifying fruit fly related species. The high mutation rate of mitochondrial larvae, however, is time-consuming, because larval keys DNA leads to the accumulation of differences in related are not available and larvae have to be reared to adults. The species, aiding in their differentiation. This study aims to lack of capability in identifying fruit fly larvae presents a integrate mtCOI barcoding and morphometric analysis serious issue for custom pest inspection, which typically to delineatethe closely related B. occipitalis and B. requires more or less immediate conclusion. philippinensis. Due to limitations in morphology-based , biochemical/genetic techniques may provide more rapid and accurate alternatives (Haymer et al. 1994). Armstrong MATERIALS AND METHODS et al. (1997) proposed the use of restriction fragment length polymorphisms (RFLP) in PCR-amplified ribosomal DNA Sample Collection (rDNA) for quick identification. PCR-RFLP analysis on a Adult flies and larvae were collected from mango fruit 1.2 kb region of the mitochondrial DNA of B. occipitalis orchards in Cavite and Davao del Norte, Philippines. and B. philippinensis, as well as other members of the Female adult flies were trapped using yeast/malathione B. dorsalis complex, showed species-specific banding solution,while male flies were trapped using methyl patterns on agarose gels (Muraji and Nakahara 2002). eugenol/malathione solution. Collected individuals were The mitochondrial cytochrome c oxidase 1 gene (mtCOI) stored in 95% ethanol before identification and DNA of B. occipitalis and B. philippinensis has also been extraction. Infested fruit samples containing larvae were analyzed (Chen et al. 2005), showing a 96.8% interspecific obtained both from the tree and the ground (fruit drops). similarity in the gene sequence. Analysis of mtCOI and Samples were sent to the Insect Ecology Laboratory 16s rDNA established 7 species in the B. dorsalis species (IEL), Crop Protection Cluster, College of Agriculture, complex (B. dorsalis, B. carambolae, B. kandiensis, B. University of the Philippines Los Baños, where rearing musae, B. occipitalis, B. papayae, and B. philippinensis) and identification were conducted.Adult samples were

70 Philippine Journal of Science Delomen et al.: Morphometric Analysis and DNA Barcoding of Fruit Flies Vol. 142 No. 1, June 2013 Bactrocera occipitalis (Bezzi) and B. philippinensis Drew and Hancock then inspected using a dissecting microscope (Euromex Genomic DNA extraction and mtCOI amplification STO). Six morphological diagnostic characters (Table From both studied locations, three samples from each 1) were examined, with each character scored as either morphological rating category (B. occipitalis, hybrid, a 0 or a 1. The scores for each character are then and B. philippinensis) were chosen for barcoding summed; a total score of 0-2 identifies an adult as B. analysis. Genomic DNA was extracted using the occipitalis, 3 as intermediate (hybrid), and 4-6 as B. QiagenDNeasy® (Qiagen, Hilden, Germany) blood philippinensis. Females were differentiated from males and tissue kit.Extracted DNA was then subjected to via the presence of ovipositor. Specimens were subjected PCR using primers designed from mtCOI fragment to morphometric analysis and then stored in ethanol prior sequences derived by Chen et al. (2005) for B. occipitalis to DNA extraction. and B. philippinensis.The forward primer had the sequence 5’-GGAATAGACGTTGATACTCGTGC-3’, while the reverse primer had the sequence5’- Table 1. Modified morphology rating system from White and AACTGTGTTCAGCTGGAGG-3’ (base 8 – base 635 Hancock (1997) to distinguish Bactrocera occipitalis and B. philippinensis. of the 5’ region of mtCOI). The PCR profile consisted of pre-denaturation (94°C for 5 min) and 35 cycles at 94°C SCORE AND ITS CORRESPONDING for 1 min, 56°C for 1 min, 72°C for 2 min; followed by CHARACTER DESCRIPTION a final extension at 72°C for 7 min. The PCR mix (total 0 1 volume = 25 μL) consisted of 1x PCR buffer (2.5 μL), 0.8 mM of dNTP mix (2.5 μL), 0.5 μM of each primer (2.5 1 medial line on broad narrow abdominal tergite 4 μL for each primer), 0.5 U of TaqDNA polymerase (0.1 μL), 50-100 ng template DNA (2 μL), and sterile nanopure 2 transverse dark band very broad at most narrow on abdominal tergite 3 posteriously posteriously water (12.9 μL). PCR products were then visualized using electrophoresis on a 1% agarose gel. Positive 3 lateral marks on broad and absent or a abdominal tergite 4 rectangular narrow triangle amplification products were then purified using Qiaquick PCR Purification Kit. Purified products were then sent to 4 ground color around red black prescutellar setae Macrogen Inc., South Korea for sequencing on an Applied Biosystems 3730 XL DNA Analyzer (Applied Biosystems 5 ground color around yellow black frontal setae by Life Technologies, Carlsbad, California, Unites States). Sequences were analyzed by BLAST, showing homology 6 costal band on fore- deep and narrow and not with mtCOI sequences previously derived from members wings clearly below vein R2+3 extending of the Bactrocera dorsalis complex (B. dorsalis, B.

below R2+3 philippinensis, B. carambolae, and B. papayae).

Morphometric Analysis Sequence Analysis From both studied locations, thirty female and thirty male Sequences were aligned using ClustalW2 (Larkin et al. samples rated for 0 and 6 were obtained. Rating 0 was 2007) and submitted to GenBank (accession numbers chosen as it contained all six diagnostic traits defining B. KC787182 – KC787217).Sequences were used to occipitalis. Likewise rating 6 was chosen as it contained construct a Neighbor Joining (NJ) tree using the program all six diagnostic characters defining B. philippinensis. MEGA 5 (Tamura et al. 2011). GenBank sequences The lengths of six abdominal tergites and six abdominal for Bactrocera dorsalis (DQ822877) and B. papayae sternites were measured using a dissecting microscope, (GQ152152) were also included as outgroup. Bootstrap a ruler, a stage micrometer and ImageJ (available at analysis (500 replications) was conducted and bootstrap http://rsb.info.nih.gov/ij; developed by Wayne Rasband, values >50 were marked on the notes. Consensus sequences National Institutes of Health, Bethesda, MD). Wing for each morphological rating category were derived to length and width were measured. Femoral, tibial, and summarize sequence data. Pairwise genetic distances and metatarsal lengths were also obtained for all leg pairs; the overall mean distance between consensus sequences the average length of the two legs of a pair was obtained. were computed using MEGA 5. In a survey of mtCOI All measurements were in mm. Means were calculated sequences differences among members of Diptera, the and divided with the length of the radial-medial (r-m) average genetic difference is 0.9% within a species and cross vein of the wing yielding ratios. This is to cancel 2.1% between two species (Hebert 2003b). Therefore we out variations in body size among individual flies. Mean decided to use 0.5% sequence difference to determine ratios were then compared using t-test at α = 0.05%. species delineation between Bactrocera species. Morphometric data showing significant differences between the two species are shown on Tables 2 and 3.

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RESULTS AND DISCUSSION Other morphological structures, like antennae dimensions, may also be analyzed. Morphometric Analysis Most of the morphological structures measured for the mtCOI analysis samples show no significant differences between B. A 556 bp sequence (base 8 – base 564) in the 5’ region occipitalis and B. philippinensis. For female samples, of mtCOI was amplified and sequenced. BLAST analysis statistically significant differences (t-test at α = 0.05) were show that sequences derived from all samples showed high observed between the average lengths of tergite 3, the homology with mtCOI sequences from other members of femur of the 2ndleg pair, the metatarsus of the 2ndleg pair, the Bactrocera species complex. and the metatarsus of the 3rd leg pair. The measurements are shown in Table 2. A similar trend was seen in the male For all samples, high sequence similarity was observed samples (Table 3). between the three replicates for all taxonomical ratings, as well as derived consensus sequences. Intraspecific All male and female samples of B. occipitalis collected divergences ranged from 0-4.0%. from Cavite and Davao del Norte have larger tergite 3 to crossvein ratios than those in B. philippinensis;while The NJ tree (Figure 1) shows strong to moderate bootstrap for the femur/crossvein ratios of the 2nd leg pair, and the supports for three clusters; the internal branches, however, metatarsal/crossvein ratios of the 2nd and 3rd leg pairs, had low bootstrap support and were condensed. COI B. philippinensis have larger values than B. occipitalis clustering does not support morphological delineations (Tables 2 and 3). The measurements of these structures are between the two Bactrocera species, with haplotypes suggestive of a morphometric difference between the two representing both species and intermediate forms species. It is recommended to further increase the number intermingled within several weakly supported clusters. of samples to validate the current delineation. Sampling The pairwise comparisons of genetic distance were done from other infested sites in the Philippines as well as in to determine relationships between similar taxonomical other South East Asian countries may also prove helpful. ratings found in different provinces (Table 4). Genetic

Table 2. Mean ratios (mean measurement/mean radial-medial crossvein length) of counterpart structures of thirty female Bactrocera spp. samples from Cavite and Davao del Norte, Philippines. Morphological Structure Location/Rating tergite 3 femur (2nd leg pair) metatarsal (2nd leg pair) metatarsal (3rd leg pair) Cavite/0 (B.occipialis) 2.2500 ± 0.0606a 3.9167 ± 0.0648a 2.8214 ± 0.0254a 2.8374 ± 0.0305a Davao/0 (B. occipitalis) 2.29857± 0.0615a 3.9267 ± 0.0664a 2.8434 ± 0.0305a 2.8671 ± 0.0346a Cavite/6 (B. philippinensis) 2.0085 ± 0.0724b 4.1054 ± 0.0817b 3.1785 ± 0.0885b 3.1071 ± 0.0877b Davao/6 (B.philippinensis) 2.0105 ± 0.0718b 4.0194 ± 0.0874b 3.2143 ± 0.0890b 3.1423 ± 0.0925b

Values are expressed as mean ±SD.

Values within a column followed by a common letter are not significantly different (P>0.05) according to Welch's t-test.

Values within a column followed by different letters are significantly different (P<0.05) according to Welch's t-test.

Table 3. Mean ratios (mean measurement/mean radial-medial crossvein length) of counterpart structures of thirty male Bactrocera spp. samples from Cavite and Davao del Norte, Philippines. Morphological Structure Location/Rating tergite 3 femur (2nd leg pair) metatarsal (2nd leg pair) metatarsal (3rd leg pair) Cavite/0 (B.occipialis) 2.2507 ± 0.0606a 3.9375 ± 0.0691a 2.8216 ± 0.0254a 2.8314 ± 0.0254a Davao/0 (B. occipitalis) 2.2515 ± 0.0596a 3.9276 ± 0.0712a 2.8278 ± 0.0305a 2.8333 ± 0.0305a Cavite/6 (B. philippinensis) 1.9643 ± 0.0730b 4.1052 ± 0.0507b 3.1429 ± 0.0858b 3.0357 ± 0.0776b Davao/6 (B.philippinensis) 1.9286 ± 0.0728b 4.1167 ± 0.0504b 3.1387 ± 0.0847b 3.0714 ± 0.0817b

Values are expressed as mean ±SD.

Values within a column followed by a common letter are not significantly different (P>0.05) according to Welch's t-test.

Values within a column followed by different letters are significantly different (P<0.05) according to Welch's t-test.

72 Philippine Journal of Science Delomen et al.: Morphometric Analysis and DNA Barcoding of Fruit Flies Vol. 142 No. 1, June 2013 Bactrocera occipitalis (Bezzi) and B. philippinensis Drew and Hancock

Figure 1. Neighbor Joining (NJ) tree of divergence among Bactrocera specimens from Cavite and Davao del Norte, Philippines (location [replicate]_species_rating).

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Table 4. Pairwise distances between Bactrocera occipitalis (Bezzi)and Bactrocera philippinensis Drew and Hancock from Cavite and Davao del Norte, Philippines. C0 D0 C1 D1 C2 D2 C3 D3 C4 D4 C5 D5 C6 D6 C0 D0 0.0018 C1 0.0018 0.0036 D1 0.0018 0.0000 0.0036 C2 0.0018 0.0036 0.0000 0.0036 D2 0.0316 0.0296 0.0335 0.0296 0.0335 C3 0.0000 0.0018 0.0018 0.0018 0.0018 0.0316 D3 0.0316 0.0296 0.0335 0.0296 0.0335 0.0036 0.0316 C4 0.0000 0.0018 0.0018 0.0018 0.0018 0.0316 0.0000 0.0316 D4 0.0036 0.0018 0.0054 0.0018 0.0054 0.0316 0.0036 0.0316 0.0036 C5 0.0036 0.0018 0.0054 0.0018 0.0054 0.0316 0.0036 0.0316 0.0036 0.0000 D5 0.0000 0.0018 0.0018 0.0018 0.0018 0.0316 0.0000 0.0316 0.0000 0.0036 0.0036 C6 0.0054 0.0036 0.0072 0.0036 0.0072 0.0335 0.0054 0.0296 0.0054 0.0018 0.0018 0.0054 D6 0.0018 0.0000 0.0036 0.0000 0.0036 0.0296 0.0018 0.0335 0.0018 0.0018 0.0018 0.0018 0.0036 Mean 0.01+0.002 Distance C1 – Cavite rating 1 D1 – Davao rating 1 C2 – Cavite rating 2 D2 – Davao rating 2 intermediate C3 – Cavite rating 3 D3 – Davao rating 3 C4 – Cavite rating 4 D4 – Davao rating 4 Bactrocera philippinensis C5 – Cavite rating 5 D5 – Davao rating 5 C6 – Cavite rating 6 D6 – Davao rating 6

distances range from 0.002% to 0.032%. The overall mean Cognato (2006) stated that varying mutation rates in distance was is 0.01%. None of these values exceeded different species makes the use of standard percent DNA the divergence threshold of 0.5% for delineating between differences questionable in predicting species boundaries. species.Species delineation is also not supported even if a The validity of using mtCOI as a genetic barcode is still more stringent divergence standard of 0.1% is used.The a source of debate among taxonomists and geneticists. observed distances are very low, supporting the results of The use of barcoding to classify organisms into large taxa the NJ tree: that the mtCOI gene region analyzed fails to (such as classes and orders) is arguable at best. However, distinguish between B. occipitalis and B. philippinensis. the use of DNA barcoding for identification of samples in the field may still be very useful. If DNA can be extracted Our barcoding results raise the possibility that B. from fruit fly larvae from infested fruits, pest species can occipitalis and B. philippinensis are actually the same be identified accurately without the need to wait for adults species. The diversity of the morphological characters to be reared. Such a practice is ideal for identification in can be attributed to phenotypic plasticity. Variations connection with quarantine procedures, provided a clear of morphological traits in response to environmental genetic marker is developed. In light of this, the 5’ 556 conditions have been documented in other , e.g., base pair region of mtCOI fails to serve as a barcode the hymenopteran Pnigaliosoemius showed diverse larval to distinguish between identified B. occipitalis and B. appearances under varying rearing temperatures (Bernardo philippinensis specimens infesting mango orchards in et al. 2007). Predator-induced phenotypic plasticity has Cavite and Davao del Norte. However, it is possible that been documented in cotton aphids (Mondor et al. 2004), genetic variations may exist in regions further downstream while pigmentation plasticity in Drosophila has been well of the mtCOIgene. Analysis of other genes such as documented (Chipindale et al. 2002; Ayrinhac et al. 2004). mtCOII, 16S rDNA, and white eye to analyze Bactrocera It is possible that morphological differences between B. species may also be useful. These genes were used to occipitalis and B. philippinensis are due to phenotypic support the possibility of a new genus Zeogodacus, which plasticity, especially when considered with the occurrence is currently recognized as a subgenus under Bactrocera of hybridization and similarity in the mtCOI sequence. (Krosch et al. 2012). Molecular differences between

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