Identification of Africanized Honey Bees Through Wing Morphometrics
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Apidologie 39 (2008) 488–494 Available online at: c INRA/DIB-AGIB/ EDP Sciences, 2008 www.apidologie.org DOI: 10.1051/apido:2008028 Original article Identification of Africanized honey bees through wing morphometrics: two fast and efficient procedures* Tiago Mauricio Francoy1, Dieter Wittmann2,MartinDrauschke3,Stefan Muller¨ 3,VolkerSteinhage3, Marcela A. F. Bezerra-Laure1,DavidDe Jong1, Lionel Segui Goncalves¸ 4 1 Depto. de Genética, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil 2 Institut für Nutzpflanzenwissenschaften und Ressourcenschutz, Universität Bonn, Bonn, Germany 3 Institut für Informatik III, Universität Bonn, Bonn, Germany 4 Depto. de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil Received 28 December 2007 – Revised 10 March 2008 – Accepted 19 March 2008 Abstract – Currently available morphometric and genetic techniques that can accurately identify African- ized honey bees are both costly and time consuming. We tested two new morphometric techniques (ABIS – Automatic Bee Identification System and geometric morphometrics analysis) on samples consisting of digital images of five worker forewings per colony. These were collected from 394 colonies of Africanized bees from all over Brazil and from colonies of African bees, Apis mellifera scutellata (n = 14), and Euro- pean bees, A. m. ligustica (n = 10), A. m. mellifera (n = 15), and A. m. carnica (n=15) from the Ruttner collection in Oberursel, Germany (preserved specimens). Both methods required less than five minutes per sample, giving more than 99% correct identifications. There was just one misidentification (based on ge- ometric morphometrics analysis) of Africanized bees compared with European subspecies, which would be the principal concern in newly-colonized areas, such as the southern USA. These new techniques are inexpensive, fast and precise. Africanized honey bee / morphometrics / geometric morphometrics analysis / ABIS / Apis mellifera / automatic identification 1. INTRODUCTION mellifera races, they are quite similar in ap- pearance; often beekeepers and the public only After the introduced African honey bee, became aware of their presence after stinging Apis mellifera scutellata Lepeletier, escaped incidents. Consequently, there has been con- from a university apiary in Brazil in 1957, siderable interest in methods for identifying it crossed with the previously-established Eu- Africanized honey bees so that proper man- ropean bee races (Kerr, 1967); the resul- agement decisions can be made. tant polyhybrid, named the ‘Africanized bee’ (Gonçalves, 1974), spread throughout most of Various methods have been developed to South and Central America by 1987. Subse- identify Africanized bees, including analyses quently, it invaded Mexico and reached the of isozymes (Contel et al., 1977; Del Lama USA in 1991 (Rinderer et al., 1993). Though et al., 1988), mitochondrial DNA polymor- the behavior of Africanized bees is consider- phisms (Hall and Muralidharan, 1989; Smith ably different from that of other European Apis et al., 1989; Sheppard et al., 1991a, b; Segura, 2000), cuticular hydrocarbons (Francis et al., Corresponding author: T.M. Francoy, 1985), and nuclear DNA (Hall, 1988; Clarke [email protected] et al., 2002; Whitfield et al., 2006). However, * Manuscript editor: Stefan Fuchs these biochemical and molecular methods Africanized bee morphometric identifications 489 require expensive reagents and laboratory tle user input and has greater precision than equipment. the original version (Steinhage et al., 2001). Morphometrics has been and continue to ABIS is a supervised classification method; be the most widely-used official methodol- i.e. the program must be trained with at least ogy for identifying Africanized honey bees, 20 specimens of each class (such as species, because of high practicability and low costs. race, or population). Currently, ABIS is able The first effective procedure, developed in to automatically identify and mark the land- the 1970s, consisted of manually measuring marks in the digital images of the wings. An- 25 characters of the wings, sternites and legs other improvement is the use of non-linear with an ocular micrometer; these measures discriminant analysis based on kernel func- were then analyzed with multivariate statis- tions (Roth and Steinhage, 1999), instead of tics to make the identifications (Daly and linear discriminant analysis, for the differ- Baling, 1978). This identification method was entiation of the groups. ABIS was able to later improved by incorporation of computer- discriminate European members of the gen- assisted measurements (Daly et al., 1982). A era Colletes, Andrena and Bombus (Steinhage simplification of these procedures resulted in et al., 2001) to the species level with a preci- the “Fast Africanized Bee Identification Sys- sion of 99.8%, and it correctly identified 94% tem” (FABIS) for preliminary identification of the bees in a comparison of honey bee sub- in the field (Rinderer et al., 1986). When- species (Drauschke et al., 2007; Francoy et al., ever colonies are suspected to be African- in press). ized based on FABIS, official identification in Another modern morphometric method that the USA is currently made with the USDA- is also very promising for shape studies is ID (United States Department of Agriculture geometrics morphometrics based on the de- identification) method (Rinderer et al., 1993). scription of shape in Cartesian coordinates This method uses 25 morphometric measure- (Bookstein, 1991). Though they are very use- ments, manually entered into a computer pro- ful for reconstructing shapes, since the relative gram, of five balsam-mounted parts of each position of landmarks is retained during analy- bee (fore- and hindwing, femur-tibia, basitar- sis (Rohlf and Marcus, 1993), the use of these sus of a hind leg and a sternite); though it techniques in honey bees has till now been re- has good precision, it requires skilled person- stricted to analysis of fluctuating asymmetry nel and several hours of preparation and anal- in studies of hybridization (Smith et al., 1997; ysis per 10-bee sample. These limitations and Schneider et al., 2003). We have found that this continuing reductions in the costs of molecular technique can be used to identify honey bee biology technology have turned DNA recog- subspecies; in a preliminary study, we were nition systems into an attractive alternative to able to identify 85% of the individuals in a morphometrics. comparison of European and Africanized bees However, recent advances in statistical (Francoy et al., in press). analysis and image recognition software have Based on these initial studies, we investi- made morphometric analysis more precise gated the utility of these two morphometric and practical for identifying species. Schröder techniques (ABIS and geometric morphomet- et al. (1995) developed a semi-automatic sys- rics analysis) for distinguishing Africanized tem for bee species recognition, based on honey bees collected from all regions of Brazil characteristics extracted from the forewings; from the African and European subspecies that this system was named ABIS (Automatic Bee gave origin to this polyhybrid. Identification System). The landmarks of this system were manually-plotted wing-vein junc- tions; after training the system with at least 2. MATERIAL AND METHODS 30 individuals per group, the identification of individuals was quite fast. Several improve- Worker bees from 394 colonies of Africanized ments were subsequently made to the origi- honey bees were collected from 24 different local- nal ABIS system; the new version requires lit- ities from 16 of the 24 states of Brazil (Tab. I). 490 T.M. Francoy et al. Table I. Localities (city and state) and number of network is used by this software as fingerprints for Africanized honey bee colonies sampled in Brazil bee species (Steinhage et al., 2001). The measures (in 1997). N = number of colonies sampled. made by ABIS are described in Drauschke et al. (2007). In a second step, a statistical classification Locality Latitude Longitude N is made using nonlinear kernel discriminant analy- Belém, PA 1˚ 26’S 48˚ 29’W 20 sis (Roth and Steinhage, 1999). São Luis, MA 2˚ 31’S 44˚ 18’W 20 For the geometric morphometrics analysis, 19 Fortaleza, CE 3˚ 46’S 38˚ 35’W 40 homologous landmarks were manually plotted at Baraúna, RN 5˚ 05’S 37˚ 36’W 6 the wing vein intersections (Fig. 1) using the soft- Serra do Mel, RN 5˚ 10’S 37˚ 02’W 8 ware tpsDig, version 2.04 (Rohlf, 2005); these Mossoró, RN 5˚ 11’S 37˚ 20’W 5 images were then Procrustes aligned (Bookstein, Severiano Melo, RN 5˚ 46’S 37˚ 57’W 5 1991). After alignment of the Cartesian coordinates Catolé do Rocha, PB 6˚ 20’S 37˚ 45’W 3 of the wings, the mean configuration of the bees Picos, PI 7˚ 05’S 41˚ 46’W 25 from a colony was used as a comparative parame- Crato, CE 7˚ 14’S 39˚ 25’W 22 ter and the identifications were made at the colony Araripina, PE 7˚ 33’S 40˚ 34’W 20 level. A forward stepwise analysis (tolerance 0.01; Aracaju, SE 10˚ 54’S 37˚ 03’W 20 F to enter 1.00) was carried out to determine classi- Tucano, BA 10˚ 58’S 38˚47’W 20 fication functions, followed by a canonical analysis Salvador, BA 12˚ 58’S 38˚ 30’W 13 and then a cross validation test to check the accu- Aquidauana, MS 20˚ 28’S 55˚ 47’W 19 racy of the equations in identifying the colonies. Ribeirão Preto, SP 21˚ 10’S 47˚ 51’W 18 São João da Boa Vista, SP 21˚ 59’S 46˚ 47’W 12 Rio de Janeiro, RJ 22˚ 54’S 43˚ 12’W 20 3. RESULTS Maringá, PR 23˚ 24’S 51˚ 55’W 10 Curitiba, PR 25˚ 25’S 49˚ 17’W 20 3.1. ABIS Florianópolis, SC 27˚ 35’S 48˚ 32’W 19 Santa Maria, RS 29˚ 41’S 53˚ 49’W 20 Alegrete, RS 29˚ 47’S 55˚ 47’W 14 We ran a cross validation test 30 times for Porto Alegre, RS 30˚ 02’S 51˚ 13’W 15 all the groups; each time 10% of the indi- viduals (randomly selected) were used as un- The Ribeirão Preto sample was collected in 2002.