identification of africanized honeybees

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  • Journal of Chromatography A, 1096 (2005) 6975

    Identification of Africanizedul N.y, Stillwersity, P



    Gas chrom potentiAfricanized drocarexocrine gla am conretention tim ed to idof the genotype. The pattern recognition GA searched for features in the chromatograms that optimized the separation of the European andAfricanized honeybees in a plot of the two or three largest principal components of the data. Because the largest principal components capturethe bulk of the variance in the data, the peaks identified by the pattern recognition GA primarily contained information about differencesbetween gas chromatograms of European and Africanized honeybees. The principal component analysis routine embedded in the fitnessfunction of the pattern recognition GA acted as an information filter, significantly reducing the size of the search space since it restrictedthe search tofocused on tcorrectly aresimilar to asmart one 2005 Else

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    0021-9673/$doi:10.1016/jfeature sets whose principal component plots showed clustering on the basis of the bees genotype. In addition, the algorithmhose classes and/or samples that were difficult to classify as it trained using a form of boosting. Samples that consistently classifynot as heavily weighted as samples that are difficult to classify. Over time, the algorithm learns its optimal parameters in a manner

    neural network. The pattern recognition GA integrates aspects of artificial intelligence and evolutionary computations to yield a-pass procedure for feature selection and classification.vier B.V. All rights reserved.

    ttern recognition; Genetic algorithms; Classification; Chemical taxonomy; Feature selection


    ized honeybees are descendants of African beesto Brazil by scientists attempting to breed a hon-

    er adapted to the South American tropics. Theoneybee that resulted from the interbreeding of theEuropean bee with the newly imported African

    red to as the Africanized bee, has since dominateda of much of South and Central America. In 1990,ized honeybee appeared outside the small southof Hildago [1]. In the past 14 years, Africanized

    have spread to southern California, Arizona, Newvada, and Oklahoma. The success of Africanized

    ding author. Tel.: +1 405 744 5945; fax: +1 405 744 6007.dress: (B.K. Lavine).

    honeybees in supplanting the European honeybee populationhas been attributed to a variety of biological and behaviorfactors and is one of the most successful introgressions everdocumented.

    Africanized honeybees have received considerable atten-tion in the popular press. Many stories have stressed theaggressive behavior of this bee and the inherent danger thatAfricanized bees pose for both man and domestic animals.In addition, Africanized honeybees also have the potentialto alter agricultural practices and significantly increase thecost of bee-pollinated food products. Honeybees account for80% of all insect pollination activity in the United States.They pollinate more than 100 different agricultural productsincluding many fruits and vegetables, forage plants, whichare important in production of meat and dairy products, andoil seed crops. The United States Department of Agriculture

    see front matter 2005 Elsevier B.V. All rights reserved..chroma.2005.06.049Barry K. Lavine a,, Meha Department of Chemistry, Oklahoma State Universit

    b Mathematics & Computer Science, Clarkson Univ

    Available online 11 July

    atography and pattern recognition methods were used to develop ahoneybees. The test data consisted of 237 gas chromatograms of hynds of European and Africanized honeybees. Each gas chromatogre windows. A genetic algorithm (GA) for pattern recognition was ushoneybeesVora b

    ater, OK 74078-3071, USAotsdam, NY 13699, USA

    al method for differentiating European honeybees frombon extracts obtained from the wax glands, cuticle, andtained 65 peaks corresponding to a set of standardizedentify features in the gas chromatograms characteristic

  • 70 B.K. Lavine, M.N. Vora / J. Chromatogr. A 1096 (2005) 6975

    estimates that 20 billion dollars worth of agricultural prod-ucts are dependent on the European honeybee for pollination[2]. If Africanized honeybees appear in the United States inthe same fthey couldagricultura

    To contStates, it wtification. Tand easy tohoneybeesUnited Stahoneybee iprocedureapproximabee specimmorphomepopulationwhich is oofficials. Ahas recentare neededmethod.

    Our prematographcuticular hyare fully Ahoneybeeshave also sgenotype inthe linear lheavily Afand moderyet fully Acan be difdifferences

    In thefrom the wEuropeancapillary c(GA) for pfeatures inAfrican gefeatures inof the Eurotwo or threBecause thof the variarecognitiondifferencesThe princithe fitnessan informasearch spawhose prinbasis of g

    those classes and/or samples that were difficult to classify asit trained using a form of boosting. Samples that consistentlyclassify correctly are not as heavily weighted as samples that

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    MS exal gasan Afatogorm in which they dispersed throughout Brazil,have deleterious effects on all aspects of the USl economy influenced by bee pollination.rol the spread of Africanized bees in the Unitedill be necessary to develop a program of stock cer-his program can only be implemented if a reliableuse method for the identification of Africanized

    is developed. Currently, the method used by thetes Department of Agriculture for Africanizeddentification is morphometric analysis [3]. Thisemploys a linear discriminant developed fromtely 20 body measurements to identify individual

    ens as Africanized or European. However,tric analysis cannot determine if a given beeis in the initial stages of becoming Africanized,f great interest to Federal and State regulatorylthough a polymerase chain reaction based assayly been developed [4], more selective primers

    to ensure accurate genotyping when using this

    vious work using packed column gas chro-y and the linear learning machine [5] to analyzedrocarbons of insects has shown that bees whichfricanized can be differentiated from Europeanon the basis of their hydrocarbon profiles [6]. Wehown that it is possible to identify the AfricanF1 hybrids [7]. Using gas chromatography and

    earning machine, we have also demonstrated thatricanized (i.e., bees that are fully Africanized)ately Africanized honeybees (bees that are notfricanized but possess many of the African traits)ferentiated from European honeybees based onin their hydrocarbon profiles [8].present study, hydrocarbon extracts obtainedax glands, cuticle, and exocrine glands of 238

    and Africanized honeybees were analyzed byolumn gas chromatography. A genetic algorithmattern recognition [911] was used to identifythe gas chromatograms characteristic of the

    notype. The pattern recognition GA searched forthe chromatograms that optimized the separationpean and Africanized honeybees in a plot of thee largest principal components [12] of the data.e largest principal components capture the bulknce in the data, the peaks identified by the pattern

    GA primarily contained information aboutbetween European and Africanized honeybees.

    pal component analysis routine embedded infunction of the pattern recognition GA acted astion filter, significantly reducing the size of thece since it restricted the search to feature setscipal component plots showed clustering on theenotype. In addition, the algorithm focused on

    are doptim

    2. E





    Hand esoak72 h.of ausingfractstreapriorgas c


    Hbeesfusedi.d. =50 toThea HPdetecusingpresedieneto thcapilretenfromGCtypicfromchromt to classify. Over time, the algorithm learns itsrameters in a manner similar to a neural network.



    rbon extracts were obtained from 294 adults. Of the 294 foragers, 128 were Africanized hon-the other 166 were European. The Africanized

    were collected from colonies in Costa Rica, Peru,eru, Honduras, and Mexico. Many of the coloniesnated as moderately or heavily Africanized bythese sites based on a field test for colony defenseuropean honeybees were collected from managedaintained in the United States. They represented aommercially available US stocks.

    e preparation

    rbons were extracted from the wax gland, cuticle,e gland of individual whole bee specimens by firstividual specimens in pesticide grade hexane for

    ydrocarbons were isolated from the soak by meansgel syringe column (silica Sep-Pac, Millipore)

    cide grade hexane as the eluent. The hydrocarbons collected and concentrated to dryness under aitrogen. It was reconstituted with 50l of hexanelysis by capillary column gas chromatography ortographymass spectrometery (GCMS).

    hromatographic analysis

    rbon extracts obtained from individual wholeanalyzed on a 25-m 5% phenyl methyl siliconea capillary column (Hewlett-Packard Ultra 2,m), which was temperature programmed fromat 7/min and then from 200 to 250 C at 1/min.

    romatographic experiments were performed oninstrument equipped with a flame ionization

    CMS analysis was also performed in this studyinnigan OWA 1020 automated GCMS. Thenormal and branched chain alkanes, alkenes, andrevealed in the extract. GC peaks correspondingkanes were used as