Morphometric Analysis and DNA Barcoding of Fruit Flies Bactrocera occipitalis (Bezzi) and B. philippinensis Drew and Hancock (Diptera: Tephritidae) from Cavite and Davao 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 Philippines Los Banos, College, Laguna 4031, Philippines
corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.
ABSTRACT
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 Bactrocera 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 Insect 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.
INTRODUCTION
The dacine fruit flies (Diptera: Tephritidae: Dacinae) are a group of economically important insect pests in Southeast Asia and the Pacific (Clarke et al. 2005), with the genus Bactrocera (629 species) distributed in India, Southeast Asia (including the Philippines), Australia and the Pacific region . . . . . . . . . . . .
REFERENCES
ALLWOOD LJ, LEBLANC L. 1996. Losses caused by fruit flies (Diptera: Tephritidae) in seven Pacific island countries. In: AJ Alwood and Drew RAI, eds. Management of Fruit Flies in the Pacific, ACIAR proceedings No. 76 Canberra: Australian Centre for International Agricultural Research. 267p.
ARMSTRONG KF, CAMERON CM, FRAMPTON ER. 1997. Fruit fly (Diptera: Tephritidae) species identification: a rapid molecular diagnostic technique for quarantine application. Bulletin of Entomological Research 87: 111-118.
AYRINHAC A, DEBAT V, GIBERT P, KISTER AG, LEGOUT H, MORETEAU B, VERGILINO R, DAVID JR. 2004. Cold adaptation in geographical populations of Drosophila melanogaster: phenotypic plasticity is more important than genetic variability. Functional Ecology 18(5): 700-706.
BERNARDO U, PEDATA PA, VIGGIANI P. 2007. Phenotypic plasticity of pigmentation and morphometric traits in Pnigalio soemius (Hymenoptera: Eulophidae). Bulletin of Entomological Research 97: 101-109.
CHEN R, YIN W, ZHANG R. 2005. Realtime qualitative PCR for the inspection and identification of Bactrocera philippinensis and Bactrocera occipitalis (Diptera: Tephritidae) using SYBR Green Assay: The Raffles Bulletin of Zoology 53(1): 73-78.
CHIPPINDALE AK, LEROI AM, KIM SB, ROSE MR. 2002. Phenotypic plasticity and selection in Drosophila life-history evolution. I. Nutrition and the cost of reproduction. Journal of Evolutionary Biology 6(2): 171-193.
CLARKE AR, ARMSTRONG KF, CARMICHAEL AE, MILNE JR, RODERICK GK, YEATES DK. 2005. Invasive phytophagous pests arising through a recent tropical evolutionary radiation: The Bactrocera dorsalis complex of fruit flies. Annual Review of Entomology 50: 293- 319.
COGNATO AI. 2006. Standard percent DNA sequence difference for insects does not predict species boundaries. Journal of Economic Entomology 99(4): 1037-45.
DREW RAI. 2004. Biogeography and Speciation in the Dacini (Diptera: Tephritidae: Dacinae). Bishop Museum Bulletin in Entomology 12: 165-178.
DREW RAI, ROMIG MC. 1996. Overview - Tephritidae in the Pacific and Southeast Asia. In: Management of Fruit flies in the Pacific, ACIAR Proceedings No. 76 Canberra: Australian Centre for International Agricultural Research. 267p.
DREW RAI, HANCOCK DL. 1994 . The Bactrocera dorsalis complex of fruit flies (Diptera: Tephritidae: Dacinae) in Asia. Bulletin of Entomological Research Supplement No 2: 1-68.
HAJIBABAEI M, JANZEN DH, BURNS JM, HALLWACHS W, HEBERT PDN. 2006. DNA barcodes distinguish species of tropical Lepidoptera. Proceedings of the NationalAcademy of Sciences 103: 968-971.
HAYMER DS, TANAKA T, TERAMAE C. 1994. DNA probes can be used to discriminate between tephritid species at all stages of the life cycle (Diptera: Tephritidae). Journal of Economic Entomology 87(3): 741-746.
HEBERT PDN, GREGORY TR. 2005.The promise of DNA barcoding for taxonomy. Systematic Biology 54: 852-859.
HEBERT PDN, CYWINSKA A, BALL SL, DEWAARD JR. 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences 270: 313-321.
HEBERT PDN, RATNASINGHAM S, DEWAARD JR. 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society B: Biological Sciences 270(Suppl.): 96-99.
HEBERT PDN, PENTON EH, BURNS JM, JANZEN DH, HALLWACHS W. 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the NationalAcademy of Sciences 101: 14812-17. http://rsb.info.nih.gov/ij
IWAHASHI O. 1999. Distinguishing between two sympatric species, Bactrocera occipitalis and B. philippinensis (Diptera: Tephritidae), based on the aedeagal length. Annals of the Entomological Society of America 92: 182-187.
IWAIZUMI R. 2004. Species and host record of the Bactrocera dorsalis complex (Diptera: Tephritidae) detected by the plant quarantine of Japan. Applied Entomology and Zoology 39(2): 327-333.
IWAIZUMI R, MASASHI K, IWAHASHI O. 1997. Correlation of length of terminalia of males and females among nine species of Bactrocera (Diptera, Tephritidae) and differences among sympatric species of B. dorsalis complex. Annals of the Entomological Society of America 90: 664-666.
KROSCH MN SCHUTZE MK, ARMSTRONG KF, GRAHAM GC, YEATES DK, CLARKE AR. 2012. A molecular phylogeny for the Tribe Dacini (Diptera: Tephritidae): Systematic and biogeographic implications. Molecular Phylogenetics and Evolution 64(3): 513-523.
LARKIN MA, BLACKSHIELDS G, BROWN NP, CHENNA R, McGETTIGAN PA, McWILLIAM H, VALENTIN F, WALLACE IM, WILM A, LOPEZ R, THOMPSON JD, GIBSON TJ, HIGGINS DG. ClustalW and ClustalX version 2. Bioinformatics 23(21): 2947-48.
MONDOR EB, ROSENHEIM JA, and ADDICOTT JF. 2004. Predator-induced transgenerational phenotypic plasticity in the cotton aphid. Oecologia 142(1): 104-108.
MURAJI M, NAKAHARA S. 2002.Discrimination among pest species of Bactrocera (Diptera: Tephritidae) based on PCR-RFLP of the mitochondrial DNA. Applied Entomology and Zoology 37(3): 437-446.
NAKAHARA S, MURAJI M. 2008. Phylogenetic analyses of Bactrocera fruit flies (Diptera: Tephritidae) based on nucleotide sequences of the mitochondrial COI and COII genes. Research Bulletin of Plant Protection Japan 44: 1-12.
SAVOLAINEN V, COWAN RS, VOGLER AP, RODERICK G, LANE R. 2005. Towards writing the encyclopaedia of life: an introduction to DNA barcoding. Philosophical Transaction of the Royal Society B: Biological Sciences 360: 1805-11.
SMITH MA, WOODLEY NE, JANZEN DH, HALLWACHS W, HEBERT PDN. 2006. DNA barcodes reveal cryptic host-specificity within the presumed polyphagous members of a genus of parasitoid flies (Diptera: Tachinidae). Proceedings of the National Academy of Sciences 103: 3657-62.
SMITH MA, FISHER BL, HEBERT PDN. 2005. DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar. Philosophical Transaction of the Royal Society B: Biological Sciences 360: 1825-34.
TAMURA K, PETERSON D, PETERSON N, STECHER G, NEI M, KUMAR S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28(10): 2731-39.
WHITE IM, HANCOCK DL. 1997. CABIKEY to the Indo-Australasian Dacini fruit flies. CAB International, Wellingford. CD-ROM.
ZHANG B, LUI YH, WU WX, WANG ZL. 2010. Molecular phylogeny of Bactrocera species (Diptera: Tephritidae: Dacini) inferred from mitochondrial sequences of 16S rDNA and COI seqeuences. Florida Entomologist 93(3): 369-377.
