Scientific Notes 167

MOLECULAR IDENTIFICATION OF THE ECONOMICALLY IMPORTANT INVASIVE CITRUS ROOT WEEVIL DIAPREPES ABBREVIATUS (COLEOPTERA: )

MARINA S. ASCUNCE1, HERBERT N. NIGG2 AND ANNMARIE CLARK3 1USDA-ARS, Center for Medical, Agricultural and Veterinary Entomology, 1600 SW 23rd Drive, Gainesville, FL 32608

2University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850

3University of Florida, ICBR Genetic Analysis Laboratory, 1376 Mowry Road, Gainesville, FL 32610

Diaprepes abbreviatus L. is a polyphagous upon emergence. Thirty-two live larvae were col- weevil affecting more than 270 species of plants. lected by digging the roots of affected trees by T. The larvae feed on roots of the trees causing dam- Ellis and D. Kellum (San Diego County, CA) and age that can kill the plant. Originally from the M.S.A. Live, quiescent, and dead adult weevils Caribbean, this weevil was first found in the were included in the study. Larval and adult sam- United States in Apopka, Florida in 1964 (Woo- ples were preserved in 95% ethanol. druff 1964) and it currently infests 23 counties in Seventy-three DNA extractions were per- Florida. In 2000, D. abbreviatus was discovered in formed with a DNeasy Tissue Kit (Qiagen, Inc.) the Rio Grande Valley, Texas (Skaria & French following the manufacturer’s protocol for 2001) and in 2005 in southern California (Califor- tissue (Table 1). All sets of extractions were per- nia Department of Food and Agriculture, CDFA, formed in parallel with 1 blank extraction (extrac- 2007). tion negative-control: no sample). Two microliters Different control measurements, which are (µL) of the DNA extraction were used to conduct specific to a particular life stage of the weevil, are PCR-amplifications of a portion of the COI gene currently in use (McCoy et al. 2007). However, employing the primers s1541 (5’-TGAKCYG- when egg masses or larvae are found, the diagno- GAATASTAGGAICATC-3’; B. Crespi, Simon sis of infestation is often delayed due to lack of re- Fraser University) and a2411 (5’-GCTAAT- liable methods that allow one to identify non- CATCTAAAAACTTTAATTCCWGTWG-3’; Nor- adult stages. The objective of this study was to de- mark et al. 1999). All sets of PCR amplifications velop a method for species identification of imma- were performed in parallel with 1 blank reaction ture stages of D. abbreviatus based on DNA bar- (PCR negative-control: no DNA). Detailed PCR- coding technique. DNA barcoding consists of se- protocols can be found in Ascunce et al. (2008). quencing of a DNA segment from a specified re- Sixty-eight DNA samples were amplified success- gion of the genome, and the “barcode” sequences fully, and both extraction and PCR negative con- are compared to those available in a reference da- trols yielded no amplification products (no con- tabase to determine the species represented by tamination). Five failures included DNA from the sample. The mitochondrial gene cytochrome adult weevils that were dead, quiescent, placed in oxidase I (COI) is extensively used for barcoding 95% ethanol 2 h after collection or shipped in pro- of metazoans (Hebert et al. 2003). Moreover, COI pylene-glycol. These failures were likely due to sequences are the most commonly used for quar- degraded DNA or the presence of PCR-inhibitors. antine and forensic applications involving , Despite the wide range of DNA concentration val- Tetranychus mites (Lee & Lee 1997), Liriomyza ues obtained among the different samples, all pos- leafminers (Scheffer et al. 2001) and Calliphora itive amplifications were similar in intensity as- blowflies (Ames et al. 2006). In this study, a reli- sessed by visual inspection and provided similar able method based on PCR and sequencing of the quality sequences with the single exception of COI gene was developed to identify immature DP945. specimens of D. abbreviatus and to differentiate PCR-amplifications were purified by the them from another common root weevil, Panto- QIAquick PCR Purification Kit (Quiagen, Inc.) morus cervinus (Boheman) Kuschel. and sent to the ICBR DNA Sequencing Core at Eight egg-masses of D. abbreviatus were pro- the University of Florida for sequencing. Se- vided by S. Frazer at the Division of Plant Indus- quences of approximately 600 base pairs (bp) try (DPI) (Florida Department of Agriculture and were edited with Sequencher™ 3.1 (Gene Codes Consumer Services) rearing facility and kept at - Corporation, Inc.) and aligned with ClustalX (Th- 20°C. Another clutch of eggs collected at Fair- ompson et al. 1997). For the analysis of the COI banks Ranch (California) by D. Arena (CDFA) was sequence data, we used published D. abbreviatus reared by J. Bethke (University of California) and sequences (Genbank Acc. Numbers: EF042129- neonates were stored in 95% ethanol immediately EF042140) as reference sequences including the 3

168 Florida Entomologist 92(1) March 2009 5 5 e and when Result after 5 DNA concentration 3 COI-Haplotype . 2008). AL

ET

4 SCUNCE COI PCR (A DATABASE

N/S: not sequenced. [ng/µL] 3 Amount of buffer added for elution DNA. 2 REFERENCE

The amount of tissue used varied among specimens. Egg masses were 1 TO

Orange County. b COMPARISONS

(µL) DNA Concentration 2 AFTER

FOUND

Elution Buffer 1 The column “COI-PCR” indicates the presence (+) and absence (-) of PCR-amplification product. 4 Los Angeles County; and a HAPLOTYPES

mm larvamm larvamm larvamm larvamm larva 150mm larva 150mm larva 150 150 150 150 150 3.05 246.04 26.15 737.74 221.2 226.81 249.39 + + + + COI-2 Fuller rose COI + + Fuller rose beetle COI + COI-3 COI-3 COI-3 COI-3 AND

5 5 5 5 5 5 5 YIELDS

Life Stage Tissue Used and were rinsed with PBS 1X buffer before extraction. One single egg or entire egg-masses used. Larvae also varied in siz

2 EXTRACTION to 1 cm

2 , DNA & SAMPLES

OF

ESCRIPTION 1. D All collection sites in CA were San Diego County except as indicated & ABLE Sample ID Collection Site measured with a NanoDropTM 1000 spectrophotometer (Thermo Scientific). BLAST search: 95% similar to Fuller rose beetle COI sequence from GenBank (Accesion number: AY790876; Scataglini et al. 2005). DP965DP970 FLDP963 DPI, FLDP964 DPI, DP966 FL DPI, DP967 FL DPI, DP968 FL DPI, DP969 FL DPI, DP971 Egg FL DPI, DP972 Egg FL DPI, FLDP950 DPI, Egg FLDP953 DPI, EggDP954 Egg one egg CA Ranch, Fairbanks DP955 Egg one egg CA Ranch, Fairbanks DP956 neonate Egg CA Ranch, Fairbanks DP957 neonate Egg egg mass CA Ranch, Fairbanks DP958 neonate Egg egg mass CA Ranch, Fairbanks DP959 neonate Egg egg mass CA Ranch, Fairbanks 3 neonates neonate egg mass CA Ranch, DP947 Fairbanks 75 neonate neonate egg mass CA Ranch, Fairbanks 75 neonate neonate egg mass 150 neonate neonate egg mass 150 CA Oceanside, neonate egg mass 150 neonate 75 150 neonate 150 neonate 75 150 larva 75 150 75 1.51 150 75 1.01 15.59 75 107.95 75 45.96 60.91 75 0.21 28.44 21.51 19.53 1.27 + 27.31 9.08 + + 7.00 2.79 + 0.76 N/S + + 4.12 N/S COI-1 + 8.38 N/S + + + N/S COI-2 + + + N/S COI-1 + COI-2 + N/S + COI-2 COI-2 + N/S + N/S COI-2 COI-2 COI-2 COI-2 DP948DP949DP897 CA Oceanside, DP898 CA Oceanside, DP899 CA Encinitas, DP900 CA Encinitas, DP901 CA Encinitas, larva DP902 CA Encinitas, larva DP903 CA Encinitas, DP904 larva CA Encinitas, larva CA Encinitas, larva CA Encinitas, larva larva larva larva larva 5 mm larva 5 mm larva 5 mm larva 5 mm larva 150 150 150 150 405.61 851.10 111.01 184.53 + + + + COI-3 COI-3 COI-3 COI-3 possible a 5-8-mm segment was cut from the midsection; for small larvae anterior portion or whole specimen used. irregular in shape ranging from 6 mm T

Scientific Notes 169 5 . 2008). e and when Result after 5 AL

ET

DNA concentration 3 COI-Haplotype SCUNCE (A 4 DATABASE

COI PCR REFERENCE

TO N/S: not sequenced.

[ng/µL] 3 Amount of buffer added for elution DNA. 2 The amount of tissue used varied among specimens. Egg masses were 1 COMPARISONS

AFTER

Orange County. b FOUND (µL) DNA Concentration

2 HAPLOTYPES

Elution Buffer AND

1 The column “COI-PCR” indicates the presence (+) and absence (-) of PCR-amplification product. 4 Los Angeles County; and a YIELDS

EXTRACTION , DNA Life Stage Tissue Used and were rinsed with PBS 1X buffer before extraction. One single egg or entire egg-masses used. Larvae also varied in siz

2 SAMPLES

OF to 1 cm

2 & ESCRIPTION ) D ONTINUED 1. (C All collection sites in CA were San Diego County except as indicated & ABLE Sample ID Collection Site measured with a NanoDropTM 1000 spectrophotometer (Thermo Scientific). BLAST search: 95% similar to Fuller rose beetle COI sequence from GenBank (Accesion number: AY790876; Scataglini et al. 2005). DP905DP906DP907 CA Encinitas, DP922 CA Encinitas, DP923 CA Encinitas, DP924 CA Santa Fe, Rancho DP925 CA Santa Fe, Rancho DP926 larva larva CA Santa Fe, Rancho DP927 larva larva CA Santa Fe, Rancho DP928 larva larva CA Santa Fe, Rancho DP929 larva CA Santa Fe, Rancho DP930 larva CA 5 mm larva Santa Fe, Rancho DP931 5 mm larva larva CA 5 mm larva Santa Fe, Rancho DP932 5 mm larva larva CA 5 mm larva Santa Fe, Rancho DP933 5 mm larva larva CA Santa Fe, Rancho DP934 5 mm larva larva CA Santa Fe, Rancho DP935 5 mm larva 150 150 larva CA Santa Fe, Rancho DP936 5 mm larva 150 150 larva CA Santa Fe, Rancho DP937 5 mm larva 150 150 larva CA Santa Fe, Rancho DP938 5 mm larva 150 larva CA Santa Fe, Rancho DP939 5 mm larva 150 larva CA Santa Fe, Rancho 5 mm larva 150 larva CADP09 Santa Fe, Rancho 5 mm larva 150 larva CADP15 Santa Fe, Rancho 5 mm larva 150 larva 206.19 131.92DP18 5 mm larva 150 larva 241.61 184.10DP22 5 mm larva FL DPI, 150 208.70 349.05DP24 5 mm larva FL DPI, 150 364.34DP859 5 mm larva FL DPI, 150 102.74DP860 5 mm larva FL DPI, 150 124.62DP861 5 mm larva FL DPI, 150 CA San Diego, 264.35DP862 150 CA San Diego, + 169.15DP863 + 150 Adult CA San Diego, + 403.42 + 150 Adult CA San Diego, + + 99.03 150 Adult CA San Diego, 238.27 Adult + COI-3 COI-2 Adult 318.99 Adult + COI-3 COI-2 Adult 175.25 Adult + COI-3 one leg COI-2 235.16 Adult + one leg COI-2 Adult + one leg 424.7 Fuller rose beetle COI one leg 354.23 + one leg COI-2 one leg 263.73 one leg COI-3 one leg + + 328.4 COI-3 one leg + COI-2 one leg 75 + 75 COI-2 + 100 COI-3 75 100 COI-3 + 75 + 100 COI-2 75 + 100 COI-2 100 + COI-2 COI-2 COI-3 55.35 COI-2 134 10.25 95.89 150.92 5.43 161.62 25.13 2.51 23.44 + + + + + + COI-1 + + COI-2 COI-3 + COI-1 + COI-1 COI-3 COI-1 COI-3 COI-3 COI-3 possible a 5-8-mm segment was cut from the midsection; for small larvae anterior portion or whole specimen used. irregular in shape ranging from 6 mm T

170 Florida Entomologist 92(1) March 2009 . 2008). e and when Result after 5 AL

ET

DNA concentration 3 COI-Haplotype SCUNCE (A 4 DATABASE

COI PCR REFERENCE

TO N/S: not sequenced.

[ng/µL] 3 Amount of buffer added for elution DNA. 2 The amount of tissue used varied among specimens. Egg masses were 1 COMPARISONS

AFTER

Orange County. b FOUND (µL) DNA Concentration

2 HAPLOTYPES

Elution Buffer AND

1 The column “COI-PCR” indicates the presence (+) and absence (-) of PCR-amplification product. 4 Los Angeles County; and a YIELDS

EXTRACTION , DNA Life Stage Tissue Used and were rinsed with PBS 1X buffer before extraction. One single egg or entire egg-masses used. Larvae also varied in siz

2 Adult one leg 100 70.03 — N/S Adult one leg 100 38.65 + COI-2 SAMPLES

CA OF to 1 cm

, 2 a & CA , b ESCRIPTION ) D ONTINUED 1. (C All collection sites in CA were San Diego County except as indicated & ABLE Sample ID Collection Site DP943DP944DP945 CA Carlsbad, DP946 CA Carlsbad, DP961 CA Carlsbad, CA Carlsbad, CA Oceanside, Adult-deadmeasured with a NanoDropTM 1000 spectrophotometer (Thermo Scientific). Adult-dead Adult-dead head & thorax Adult-dead head & thorax Adult-dead head & thorax two legs 100 one leg 100 100 100 100 4.98 9.85 220.55 7.26 21.89 — — + N/S N/S failed to sequence + + COI-3 COI-2 BLAST search: 95% similar to Fuller rose beetle COI sequence from GenBank (Accesion number: AY790876; Scataglini et al. 2005). DP962 Dana Point DP864DP865DP866 CA San Diego, DP940 CA San Diego, DP941 CA San Diego, DP942 CA Encinitas, DP960 CA Encinitas, Adult CA Encinitas, Adult Huntington Beach Adult Adult Adult one leg Adult-quiescent one leg one leg one leg one leg one leg 100 100 100 100 100 100 23.35 16.75 1.58 18.67 12.09 4.60 + + — + — COI-3 COI-3 + N/S COI-3 N/S COI-3 possible a 5-8-mm segment was cut from the midsection; for small larvae anterior portion or whole specimen used. irregular in shape ranging from 6 mm T Scientific Notes 171 mitochondrial haplotypes described for the spe- We thank S. Frazer (DPI), and personnel from cies in the United States (Ascunce et al. 2008). All San Diego County and the CDFA for providing samples (eggs and adults) from the DPI colony samples. We are grateful to D. Shoemaker had haplotype COI-1, with the exception of DP15, (USDA-ARS-CMAVE), K. Hoffman (CDFA) and which had haplotype COI-2 (Table 1). Of the 52 M. Heaney (University of Florida) for comments sequences obtained from California samples, 23 that improved this manuscript. This research was sequences were identical to haplotype COI-2 and supported by a contract from the CDFA to M.S.A., 26 to haplotype COI-3 (Table 1). The 3 remaining the Florida Agricultural Experiment Station, and sequences obtained from larvae were different a T-STAR grant from the USDA to H.N.N. (17%) from D. abbreviatus sequences, but identi- cal to each other. A BLAST search of the unknown SUMMARY sequence (866 bp) was performed to get an idea of what these samples may be. The highest percent We developed a sensitive barcoding technique similarity (95%) was obtained from the COI se- based on the PCR-amplification and sequencing quence for the Fuller rose beetle, Pantomorus of the mitochondrial COI gene to use in identifica- cervinus (Accession number: AY790876). This re- tion of eggs, larvae, and adults of the citrus root sult is not surprising since these 2 root weevils weevil, D. abbreviatus. This molecular tool pro- share hosts (Woodruff & Bullock 1979; McCoy et vides accurate species identification for manage- al. 2007). ment and quarantine decisions of this pest. Morphological description of larvae was used to differentiate 4 species of citrus weevils in- cluding D. abbreviatus, P. cervinus, Pachnaeus REFERENCE CITED opalus Oliver, and Germar AMES, C., TURNER, B., AND DANIEL, B. 2006. The use of (Beavers & Woodruff 1971). Accurate taxonomic mitochondrial cytochrome oxidase I gene (COI) to identification requires highly trained personnel differentiate two UK blowfly species-Calliphora vic- and it is time consuming, and morphological ina and Calliphora vomitoria. Forensic Sci. Int. 164: characters cannot identify cryptic or unknown 179-182. species. Inter-specific genetic tests were de- ASCUNCE, M. S., ERNST, J., CLARK, A., AND NIGG, H. N. scribed to differentiate D. abbreviatus and 2008. Mitochondrial nucleotide variability in inva- Pachnaeus litus egg-masses based on enzyme sive populations of the root weevil, Diaprepes abbre- electrophoresis (Jones et al. 1984) and PCR of viatus (L.) (Coleoptera) of Florida and preliminary assessment of Diaprepes from Dominica. J. Econ. the nuclear locus coding for the 18S ribosomal Entomol. 101: 1443-1454. RNA gene (18S rRNA) combined with restric- BEAVERS, J. B., AND WOODRUFF, R. E. 1971. A field key tion fragment length polymorphisms (PCR- for separating larvae of four species of citrus weevils RFLPs) (Weathersbee et al. 2003). A shortcom- in Florida (Coleoptera: Curculionidae). Florida Div. ing of enzyme electrophoretic techniques is that Plant Ind. Entomol. Circ. No. 112: 1-2; 4 fig. they are time consuming and the preservation HEBERT, P. D. N., CYWINSKA, A., BALL, S. L., AND DEW- of the sample is critical since biochemical mark- AARD, J. R. 2003. Biological identifications through ers are sensitive to degradation. One drawback DNA barcodes. Philos. Trans. R. Soc. London B. Biol. of both enzyme electrophoresis and PCR-RFLP Sci. 270: 313-321. JONES, L. F., BEAVERS, J. B., AND SCHROEDER, W. J. methods for the identification of unknown spec- 1984. Identification of weevil egg masses (Co- imens is uncertainty of the negative results leoptera: Curculionidae) by enzyme electrophoresis. (i.e., generation of a different electrophoresis or Florida Entomol. 67: 573-575. RFLPs pattern than expected). In such cases, LEE, M. L., AND LEE, M. H. 1997. Amplified mitochon- one can only conclude that the observable pat- drial DNA identify four species of Tetranychus mites tern is not representative of the genetic varia- (Acarina: Tetranychidae) in Korea. Korean J. Appl. tion that the assay was designed to recognize. Entomol. 36: 30-36. Another limitation of these methods is that MCCOY, C. W., ROGERS, M. E., FUTCH, S. H., GRAHAM, J. they cannot provide data regarding the geo- H., DUNCAN, L. W., AND NIGG, H. N. 2007. 2007 Flor- ida Citrus Pest Management Guide: Citrus Root graphic origin of the weevils. Barcoding over- weevils. Series of the University of Florida, Entomol- comes these limitations by providing sequence ogy and Nematology Department ENY-611. data that can be compared to a previously gen- NORMARK, B. B., JORDAL, B. H., AND FARREL, B. D. erated COI-sequence reference database for D. 1999. Origin of haplodiploid beetle lineage. Philos. abbreviatus (Ascunce et al. 2008). The COI-hap- Trans. R. Soc. London B. Biol. Sci. 266: 2253-2259. lotype composition of new infestations of imma- SCATAGLINI, M. A., LANTERI, A. A., AND CONFALONIERI, ture and adult stages of D. abbreviatus can be V. A. 2005. Phylogeny of the Pantomorus-Naupactus monitored with this procedure. While sequenc- complex based on morphological and molecular data ing could be expensive ($15/reaction) currently (Coleoptera: Curculionidae). Cladistics 21: 131-142. SCHEFFER, S. J., WIJESEKARA, A., VISSER, D., AND HAL- availability of high-through technology allows LET, R. H. 2001. Polymerase Chain Reaction-Restric- the process of hundreds of samples per day and tion Fragment-Length polymorphism method to dis- lowers the costs to $2.5/sample. tinguish Liriomyza huidobrensis from L. langei 172 Florida Entomologist 92(1) March 2009

(Diptera: Agromyzidae) applied to three recent leaf- aprepes abbreviatus and Pachnaeus litus (Co- miners invasions. J. Econ. Entomol. 94: 1177-1182. leoptera: Curculionidae) egg masses: PCR-Restric- SKARIA, M., AND FRENCH, J. V. 2001. Phytophthora dis- tion-Fragment-Length Polymorphism and Species- ease of citrus associated with root weevils in Texas. Specific PCR amplification of 18S rDNA products. Phytopathol. 91, S203. Ann. Entomol. Soc. Am. 96: 637-642. THOMPSON, J. D., GIBSON, T. J., PLEWNIAK, F., JEAN- WOODRUFF, R. E. 1964. A Puerto Rican weevil new to MOUGIN, F., AND HIGGINS, D. G. 1997. The ClustalX the United States (Coleoptera: Curculionidae). Flor- Windows Interface: flexible strategies for multiple ida Div. Plant Ind. Entomol. Circ. No. 30: 1-2; 1 fig. sequence alignment aided by quality analysis tools. WOODRUFF, R. E., AND BULLOCK, R. C. 1979. Fuller’s Nucleic Acids Res. 24: 4876-4882. rose weevil Pantomorus cervinus (Boheman) in Flor- WEATHERSBEE III, A. A., BULLOCK, R. C., PANCHAL, T. ida (Coleoptera: Curculionidae). Florida Div. Plant D., AND DANG, P. M. 2003. Differentiation of Di- Ind. Entomol. Circ. No. 207: 1-4; 3 fig.