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Insect Biochemistry and Molecular Biology 34 (2004) 167–176
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Bactrocera tryoni and closely related pest tephritids—molecularanalysis and prospects for transgenic control strategies

Kathryn A. Raphael a,�, Steven Whyard b, Deborah Shearman a, Xin An a,Marianne Frommer a

a Fruit Fly Research Centre, School of Biological Sciences, University of Sydney, New South Wales 2006, Australiab CSIRO Entomology, GPO Box 1700, Canberra, ACT 2615, Australia

Received 2 December 2002; received in revised form 1 April 2003; accepted 20 June 2003

Abstract

Bactrocera tryoni is a serious pest of horticulture in eastern Australia. Here we review molecular data relevant to pest statusand development of a transformation system for this species. The development of transformation vectors for non-drosophilidinsects has opened the door to the possibility of improving the sterile insect technique (SIT), by genetically engineering factorystrains of pest insects to produce male-only broods. Transposition assays indicate that all five of the vectors currently used fortransformation in non-drosophilid species have the potential to be useful as transformation vectors in B. tryoni. Evidence of crossmobilization of hobo by an endogenous Homer element emphasises the necessity to understand the endogenous transposonswithin a species. The sex-specific doublesex and yolk protein genes have been characterized with a view to engineering a female-specific lethal gene or modifying gene expression through RNA interference (RNAi). Data are presented which indicate thepotential of RNAi to modify the sex ratio of resultant broods. An understanding of how pest status is determined and maintainedis being addressed through the characterization of genes of the circadian clock that enable the fly to adapt to environmental cues.Such an understanding will be useful in the future to the effective delivery of sophisticated pest control measures.# 2003 Elsevier Ltd. All rights reserved.

Keywords: Bactrocera tryoni; Tephritid; Transformation vectors; Genetic sexing; SIT; Pest status

1. Pest fruit flies in Australia

Australia is an island continent with many different

habitats and climates. Close to one hundred species of

tephritid fruit flies are endemic to Australia (Drew,

1989), most occurring in the rainforests to the north of

the continent. With the introduction of cultivated fruit,

a few species have become horticultural pests, most

notably the Queensland fruit fly or Q-fly, Bactrocera

tryoni, which is the major pest in horticultural regions

of eastern Australia. This species has a very wide host-

fruit range, infesting almost every cultivated fruit

(Drew, 1989), as well as a wide bioclimatic potential

(Meats, 1981). Thus, with increasing movement of

people and produce, it has the potential to invade

other continents and islands.B. tryoni is part of a small species complex, the

tryoni complex of three named species, all endemic to

Australia. The sibling species Bactrocera neohumeralis

is also a serious pest, with the same or an extremely

similar host range but a narrower geographic range,

being confined to north-eastern coastal areas, entirely

within the range of B. tryoni Osborne et al., 1997;

Drew, 1989) (Fig. 1). B. neohumeralis has not been

observed to spread south into temperate fruit pro-

duction areas. B. tryoni and B. neohumeralis display an

apparently robust mating isolation mechanism, based

on time of mating, but hybrids resulting from forced

mating between the two species are viable and fertile

(Smith, 1979; Pike and Meats, 2002). The third named

species of the tryoni complex,Bactrocera aquilonis, occurs

in north-western Australia, geographically isolated

168 K.A. Raphael et al. / Insect Biochemistry and Molecular Biology 34 (2004) 167–176

from B. tryoni and B. neohumeralis (Fig. 1), and is not

considered a pest. B. aquilonis, like B. tryoni, mates at

dusk, and inter-specific matings result in viable and fer-

tile hybrids (Drew and Lambert, 1986).Outbreaks of B. tryoni, which occur in southern

fruit-growing areas (the Fruit Fly Exclusion Zone), are

eradicated by spraying and by conventional SIT with

mixed-sex releases. However, small populations appear

to have become established in some towns of south-

eastern New South Wales, outside the fly-free zone.

The species has become established in central Aus-

tralia, in and around Alice Springs, and pest fruit flies

are also found in Darwin, in northern Australia. It is

not known whether these Darwin pest infestations are

B. tryoni, hybrids between B. tryoni and B. aquilonis, or

a ‘‘noxious’’ strain of B. aquilonis. The spread of B.

tryoni outside its historical distribution has raised ques-

tions about the feasibility of area wide eradication of

pest fruit flies in central, northern and south-eastern

Australia, given the widespread and apparently con-

tinuous distribution of B. tryoni along the east coast

and its very high population numbers in Queensland.We have addressed these questions by population

analysis, initially using a set of six microsatellites (Kin-

near et al., 1998). The results show that the pest flies in

Alice Springs and Darwin are isolated from those of

the east coast. In particular, the flies from Alice

Springs show a low number of microsatellite alleles and

a lack of rare alleles, consistent with a single introduc-

tion, with no further exchange of genetic material from

other populations (Yu et al., 2001). Further, and sur-

prisingly, there is stable population subdivision (over

many years) within the east coast distribution of B.

tryoni, despite the high potential mobility of the species

(Yu et al., 2001). We propose that the genetic differen-

tiation between the Queensland and the more southern

populations is indicative of adaptation of southern flies

to the southern environmental conditions. A further 24

microsatellites have been isolated and mapped (Zhao

et al., 2003b; Wang et al., 2003), and are being used to

analyse isolated outbreaks in the Fruit Fly Exclusion

Fig. 1. Distribution of B. tryoni and B. neohumeralis in Australia. The distribution of B. neohumeralis is entirely within the distribution of B.

tryoni. The area circled indicates the approximate distribution of B. aquilonus, boundaries cannot be drawn for this species with precision on the

basis of current knowledge.

K.A. Raphael et al. / Insect Biochemistry and Molecular Biology 34 (2004) 167–176 169

Zone. Such outbreaks appear to arise most often fromsmall populations in surrounding towns, rather thanfrom the importation of infested fruit from the morenorthern distribution where population numbers arehuge (Sved et al., 2003; S. Gilchrist, unpublished).

2. Eradication protocols

The data showing stable population subdivision ofB. tryoni suggests that area wide eradication of outly-ing populations and outbreaks will be feasible, with alow frequency of recolonisation. However, efficient andenvironmentally-benign eradication protocols will berequired. Bait sprays have proved useful, but are moreeffective for suppression than eradication (Roessler,1989; Bateman, 1982). Cover sprays are becomingincreasingly unacceptable in Australia, particularly inurban areas. Therefore, SIT is the method of choice.For Medfly, Ceratitis capitata, SIT is considerablymore effective when only sterile males are released, andarea wide eradication has been implemented most suc-cessfully by male-only release using temperature-sensi-tive genetic sexing strains (Hendrichs et al., 1995;Robinson et al., 1999; Fisher, 2000). These strains weremany years in development and required the fortuitousidentification of a temperature-sensitive lethalmutation, and isolation of Y-autosome translocationswhich allowed the wild-type selectable marker to betranslocated to the Y chromosome (Franz et al., 1994).The Medfly genetic sexing systems have been developedfor extreme stability but nonetheless can break downdue to very low frequencies of male recombination,which become significant under mass-rearing con-ditions (Fisher and Caceres, 2000). For B. tryoni, thechromosomes have been described, and related to thoseof Medfly (Zhao et al., 1998). A visible marker (bentwings), located on chromosome 2 (Zhao et al., 2003b),has been found to be temperature sensitive at the eggand puparial stages (Meats et al., 2002). The bent wingsmutation could be used to create a genetic sexing sys-tem, by crossing bent wings females to males in whichthe wild-type gene has been translocated to the Y chro-mosome. When subjected to elevated temperature dur-ing development, most female progeny from thetranslocation strain would fail to emerge, and any sur-viving females would show an extreme bent-wingsphenotype and be unable to fly. However, to date,irradiation experiments have yielded no suitable trans-locations between the Y chromosome and chromosome2, stable enough for factory conditions (Meats et al.,2002).

During the years when genetic sexing strains werebeing developed and tested in Medfly, great advanceswere being made in the development of vectors andprotocols for transformation of insects other than Dro-

sophila. Transformation technology provides the possi-bility in the future of engineering factory strains thatcould be used to rear male-only broods for release inmany insect pest species. In the future, such strains,although modified by DNA transformation rather thanby chromosome manipulation, would not necessarilycontain integrated foreign DNA, as methods for tar-geted mutagenesis by homologous recombinationbecome more widely applicable (Rong, 2002). Thesemethods of targeted mutagenesis would involve recom-bination between an endogenous gene (for example, asex-determination gene) and an introduced DNA frag-ment from the same species modified to generate therequired changes in gene expression.

3. Transformation vectors

To date, five transposons have been used as trans-formation vectors in non-drosophilid insects (Handler,2001). The most widely used of these vectors are piggy-Bac, Hermes and Minos (Handler, 2001; Atkinson,2002). The piggyBac vector has proven the most versa-tile, having transformed more than a dozen species ofinsects spanning three orders (Handler, 2002), includ-ing three tephritid species: C. capitata (Handler et al.,1998), Bactrocera dorsalis (Handler and McCombs,2000), and Anastrepha suspensa (Handler and Harrell,2001). The Medfly, C. capitata, has also been trans-formed using Minos (Loukeris et al., 1995) and Hermes(Michel et al., 2001). To achieve stable transformationin other tephritids, such as B. tryoni or any of itsrelated species, it will be important to determine which,if any, of the currently available transposons would bemost effective. Interplasmid mobility assays have beeninstrumental in identifying transposons that have thepotential to serve as transformation vectors in speciesnot yet transformed (O’Brochta et al., 1994; Atkinsonet al., 2001). Such assays have shown that, in additionto piggyBac, Hermes and Minos, mariner and hobo arealso capable of accurate transposition in B. tryoni(Table 1). Hermes and piggyBac have equally hightransposition rates in Drosophila and B. tryoni, andeach transposon has been used to transform a range ofinsect species, including other tephritids. Therefore,both Hermes and piggyBac are excellent candidates toserve as transformation vectors in B. tryoni and relatedspecies. While the mobility frequencies of Minos, mari-ner, and hobo are low in Drosophila and B. tryoni, allthree transposons have been used successfully to trans-form Drosophila, albeit at lower transformation fre-quencies than those seen for piggyBac, Hermes, or thedrosophilid-specific transposon, P (Atkinson et al.,2001). Hence, any one of the five currently used trans-posons has some potential as a transformation vectorin the tryoni species complex.


An early attempt to transform B. tryoni with hobo,using the bacterial neomycin phosphotransferase II(NPT) gene as a selectable marker, was successful inproducing putative transgenics at a frequency of 9%, arate comparable to that of P elements in Drosophila(Atkinson et al., 1996). Molecular analyses indicated,however, that the integrations were not typical hobo-mediated integrations. In all five transgenic lineages,inverse PCR revealed that, while one end of the inte-grated DNA was delineated by a hobo inverted ter-minal repeat, the other junction point had plasmidDNA, indicating imperfect transposition (S. Whyard,unpublished). Similarly aberrant integrations of theHermes element have previously been observed in themosquito Aedes aegypti, where donor plasmid coin-tegrated with the transposon (Jasinskiene et al., 2000).The authors speculated that these imprecise Hermesintegrations may have resulted from interactions of theintroduced transposon with endogenous Hermes-likeelements in the mosquito genome. The mechanism forthe curious hobo integrations in B. tryoni is not fullyunderstood, although analyses of at least two of thefive transformed lines revealed that a native hobo-likeelement, Homer (Pinkerton et al., 1999), was within 1kb of the site of integration. As hobo and Homer share11/12 nucleotides of their inverted terminal repeatsequences and their transposase amino acid sequencesare ~70% identical, it is suspected that the two relatedelements may have interacted during integration. Ofgreater concern is the observation that the integrationswere not stable, with parts or all of the hobo elementdisappearing over subsequent generations. It is specu-lated that the integrated sequences were lost through across-mobilization of the hobo element by the puta-tively active Homer transposase. Cross-mobilizationhas previously been observed between hobo and the

related element Hermes in Drosophila (Sundararajanet al., 1999), where hobo transposase was observed tomobilize hobo and Hermes elements equally well,although Hermes rarely mobilized hobo. Other tephritidspecies harbour hobo-like transposons (Handler andGomez, 1996, 1997) and, in five tephritid species exam-ined, hobo was excised both in the presence andabsence of an exogenous source of hobo transposase(Handler and Gomez, 1996), suggesting that manytephritids possess potential hobo cross-mobilizing sys-tems. Interestingly, despite the cross-mobility observedbetween hobo and Hermes in Drosophila, no transposi-tions of Hermes have ever been detected in B. tryoniusing interplasmid mobility assays (Sarkar et al., 1997),which suggests that Hermes may still be safely used asa transformation vector for this species.In addition to a modest number (~20) of hobo-like

elements in its genome (Pinkerton et al., 1999), B.tryoni also has many (>1000) mariner-like elements,from a number of mariner family subgroups (Greenand Frommer, 2001). The mobility of the Mos1 (mari-ner) element in B. tryoni is considerably lower thanthat in Drosophila (Coates et al., 1995, 1997), possiblyindicating the presence of an endogenous repressionsystem, such as titration of the mariner transposase bynumerous defective elements or transposase subunitpoisoning (Lohe and Hartl, 1996; Hartl et al., 1997).Despite a similarly low transposition frequency in theyellow fever mosquito, Aedes aegypti, mariner was,nevertheless, used successfully to transform this speciesat a modest frequency of 4%, although the apparentabsence of mariner in this insect’s genome may havecontributed to its success as a transformation vector(Coates et al., 1998). However, an abundance of mari-ner elements does not necessarily hinder transformationof a species, as the silkmoth, Bombyx mori, and thehousefly, Musca domestica, contain numerous marinerelements, and both species have been transformedusing the Mos1 mariner element (Wang et al., 2000;Yoshiyama et al., 2000). It is therefore possible thatMos1 or a related mariner element could be used totransform B. tryoni, despite the abundance of such ele-ments present in its genome.Despite piggyBac’s success as a transformation vec-

tor for a range of insects, it too may face problems ofinstability or possible autoregulatory silencing fromendogenous piggyBac-like elements in some tephritids.Multiple piggyBac elements (with 95% DNA sequenceidentity to the original piggyBac derived from Tricho-plusia ni) have been observed in the Oriental fruit fly,B. dorsalis (Handler and McCombs, 2000), and relatedsequences have since been detected, although not fullycharacterized, in other related tephritids (Handler,2002). It has yet to be determined whether the piggy-Bac transgene integrations within B. dorsalis will besubject to future instability through cross-mobilisations

Table 1

Comparison of transposition rates of five transposons in Bactrocera

tryoni and Drosophila melanogaster

Transposon T
ransposition frequency (%) Reference
D
. melanogaster B. tryoni
hobo 0
.0019 0.00018 O ’Brochta et al.
(1994)

Hermes 0
.106 0.102 S arkar et al.
(1997)

mariner 0
.012 0.0009 C oates et al.
(1997)

Minos 0
.0016 0.00051 S . Whyard
(unpublished)a

piggyBac 0
.19 0.072 S . Whyard
(unpublished)b

a Transposition assays were performed as described in Klinakis

et al. (2000).b Transposition assays were performed as described in Lobo et al.

(1999).


using an endogenous piggyBac transposase. Thus, anytransposon to be considered as a transformation vectorin B. tryoni or related species must be fully assessed forits potential to be affected by endogenous elements.

4. Useful genes and markers

Eye-colour genes have provided useful transform-ation markers in Drosophila and Medfly (Rubin andSpradling, 1982; Loukeris et al., 1995). The white genehas been isolated from B. tryoni (Bennett and From-mer, 1998), but no white mutant has been isolated todate. An eye-colour mutant, lemon-eyes, with extremelypale eye colour on emergence, is located within onemap unit of the scarlet eye-colour gene, based on zerorecombination in a back-cross pedigree. A full lengthscarlet transcript is produced in the lemon-eyes mutantbut at a much reduced level compared to wild-type. Itseems likely that the lemon-eyes phenotype results froma mutation in a regulatory region of the scarlet gene,but the mutation has not yet been identified. However,scarlet may be a useful visible marker for transform-ation and, to that end, the gene has been characterised(Zhao et al., 2003a).

The genetic engineering of SIT release strains to pro-duce male-only broods may be possible by the manipu-lation of one or more sex-specifically expressed genes.Candidate genes that show sex-specific expressioninclude homologues of genes such as the yolk protein(yp) and chorion genes of D. melanogaster. The ypgenes in those dipteran insects studied to date havebeen shown to be expressed in a sex- and tissue-specificmanner. In D. melanogaster, the yp genes are the directtarget of the product of the sex-determination genedoublesex (dsx) (Burtis et al., 1991; Coschigano andWensink, 1993). Some of the regulatory elements whichdirect the female-specific expression of the yp1 and yp2genes in D. melanogaster have been identified withinthe intergenic region of these genes (Abel et al., 1992;Falb and Maniatis, 1992; An and Wensink, 1995;Lossky and Wensink, 1995; Erdman et al., 1996).Therefore the yp genes of B. tryoni were characterisedand compared with those of D. melanogaster and C.capitata, to identify female-specific regulatory cassettes(D. Shearman, unpublished). We conclude, fromsequence data and Southern blotting, that B. tryonicontains two pairs of genes homologous to the yp1 andyp2 genes of Drosophila. The arrangement of the yp1and yp2 gene pair is the same as that of D. melanoga-ster (Hovemann et al., 1981) and C. capitata (Rina andSavakis, 1991) and some putative regulatory elementshave been identified within the intergenic region of theB. tryoni genes.

The genes of the sex-determination pathway are ofparticular interest, as the genes uppermost in the path-

way are directly responsible for determination of maleand female somatic sex. Although not all the genes ofthe sex-determination pathway in B. tryoni have beenidentified, a number of genes have either been isolatedor their presence inferred. Male somatic sex in B. tryonihas been shown to be determined by the presence of adominant male determiner, which is carried on the Ychromosome (Meats et al., 2002). This mechanism ofsex determination has also been shown to operate inother members of the Bactrocera genus, namely Bac-trocera cucurbitae (McCombs et al., 1993), and Bac-trocera dorsalis (McCombs and Saul, 1995), as well asanother tephritid species, C. capitata (Zapater andRobinson, 1986). The dominant male determiner(designated M) holds the place of primary signal in thesex-determination pathways in all of the above species.This is in contrast to the mechanism of sex determi-nation which operates in D. melanogaster, where theprimary signal is the X:A ratio. While these mechan-isms of sex determination appear to be quite different,it has been proposed that they all fit a single principleand each may be described in terms of minor varia-tions on this principle (Nothiger and Steinmann-Zwicky, 1985; Dubendorfer et al., 1992; Marin andBaker, 1998; Shearman, 2002). The model of the primi-tive state of sex determination may be described interms of four main elements: the primary signal,maternal products, the key gene and the genetic doubleswitch. In D. melanogaster, the pathway consists of aregulatory cascade of genes, Sxl (the key gene), trans-former (tra) and transformer-2 (tra-2), and doublesex(dsx) (the genetic double switch), where the sex-specificexpression of Sxl is dictated by the X:A ratio (the pri-mary signal) (for reviews see MacDougall et al., 1995;Parkhurst and Meneely, 1994; Nothiger and Stein-mann-Zwicky, 1985; Schutt and Nothiger, 2000).Homologues of Sxl have been isolated from C. capitata(Saccone et al., 1998) and B. tryoni (S. Goodall and S.Whyard, pers. comm.) however in these species Sxl dis-plays different expression patterns to those found in D.melanogaster and the Sxl homologues do not appear tofunction in sex determination. This has led to thesuggestion that the next sex-specifically expressed genein the D. melanogaster hierarchy, tra, may fill the roleof the key gene in dipteran insects other than Droso-phila. The last gene in the Drosophila somatic sexdetermination pathway, the dsx gene, differs from theother genes in the pathway in that it shows active male-and female-specific transcripts. It has been suggestedthat the genetic double switch is the most ancient mem-ber of the sex-determination pathway and as such maybe a constant component of even the most primitiveinsect sex-determination pathways (Nothiger and Stein-mann-Zwicky, 1985; Dubendorfer et al., 1992; Marinand Baker, 1998; Shearman, 2002). Therefore, we choseto isolate the dsx gene from B. tryoni and have shown


that it too codes for male and female mRNAs, pro-duced by sex-specific splicing. Sequences with identityor high similarity to all regulatory elements which con-trol sex-specific splicing in D. melanogaster were alsoidentified in B. tryoni dsx, including a region in thefemale-specific fourth exon with high similarity to theD. melanogaster dsxRE (regulatory element) whichcontains the TRA/TRA-2 binding sites (Shearman andFrommer, 1998). This finding not only suggests thepresence of tra/tra-2 homologues in B. tryoni butwould further support the notion that tra is likely toact as the key gene in insects where Sxl does notappear to play a direct role in sex determination.

5. Modification of sex ratio

Two research groups have proposed the use of arepressible female-specific lethal genetic system to elim-inate females within either an SIT program, or as amethod to release transgenic fertile males that carry afemale-lethal construct (Heinrich and Scott, 2000; Tho-mas et al., 2000). Using a female-specific promoter(such as a yolk protein promoter) to regulate a tetra-cycline-repressible transcriptional activator (tTA), bothgroups demonstrated the sex-limited expression of alethality gene. A similar approach could be achievablein any insect, including tephritids, provided that spe-cies-specific regulatory elements and lethality genes orgenetic constructs are isolated. The yolk protein regu-latory elements, which have been isolated from B.tryoni, may form the basis for female-specificexpression of a transgene in this species. Alternately,the regulatory elements which control dsx sex-specificsplicing may be manipulated such that the effects of alethal gene are only observed in females of a line whichcarries an engineered dsx construct.

Recent developments in the field of RNA inter-ference (RNAi) technologies also hold considerablepromise for their application in pest insect control.RNAi is a phenomenon that occurs in a wide range oforganisms, by which exogenous double-stranded RNA(dsRNA) induces the degradation of mRNA sharingsequence homology with the dsRNA. RNAi could bedirected against sex-determination genes as a mean ofproducing only males in an improved genetic sex-sort-ing system. It is likely that closely-related dipteran spe-cies will not exhibit major changes in many of thegenes of the sex-determination pathway. This makesdsx, tra and M all suitable candidate genes for themodification of the sex ratio in dipteran species. Whiledsx appears to be most highly conserved across theorder Diptera, the modification of the expression ofthis gene alone would not likely alter sex ratios, as it isexpressed in both sexes. Preliminary efforts to useRNAi in B. tryoni to alter the expression of dsx in the

two sexes separately has however, provided interestingresults. We prepared dsRNA to the male- and female-specific exons of the B. tryoni dsx gene. Delivery of thedsRNA to developing B. tryoni embryos resulted in sig-nificantly altered sex ratios in those individuals thatsurvived to adulthood. Of the embryos injected withthe male-specific dsx dsRNA, few (15%) developed asphenotypic males, and over half of these were sterile.In contrast, most (78%) of the surviving embryos injec-ted with female-specific dsx dsRNA developed asphenotypic males, and most of the surviving femaleswere sterile (S. Whyard, unpublished). The increasedsterility may be the result of abnormal gonadal devel-opment in the affected individuals, which has beenobserved in houseflies treated with dsRNA specific tothe dsx gene (M. Hediger, pers. comm.).It is anticipated that complete elimination of either

sex could be achieved if dsRNA targeted against a sex-determination gene were expressed in transgenicinsects, using hairpin RNAi constructs (Kennerdell andCarthew, 2000; Tavernarakis et al., 2000). As both traand M are expressed earlier in the sex-determinationpathway than dsx, and their expression is sex-limited,they would be ideal targets for RNAi. M is a genewhose expression, when switched on, should directsomatic sexual development in chromosomal femalesdown the male pathway. Although the location of M isknown, the isolation of this gene has proven difficult,and to date M has not been isolated in any insect spe-cies. Ultimately, almost any gene critical for earlydevelopment could be targeted by RNAi and, if placedunder the control of a sex-limited gene promoter, thesex ratio of a population could be altered. Such geneticconstructs could revolutionize the production of gen-etic sexing strains of insects in SIT or other appliedinsect control methods.

6. Speciation and pest status

Superficially, the three species of the tryoni complex,B. tryoni, B. neohumeralis and B. aquilonis present afairly standard model for pest status—sibling speciesthat show different host-fruit preferences, and differentgeographic ranges and potential for invasion. But,genetically, the three species are extremely closelyrelated, more so than even the host races of Rhagoletis(Feder et al., 1988). B. tryoni and B. neohumeralis aredifferentiated by mating time (B. tryoni mates at dusk,whereas B. neohumeralis mates in bright light in themiddle of the day) and by a colour difference of thehumeral calli (yellow in B. tryoni and brown in B. neo-humeralis). In terms of DNA sequences tested, B.tryoni and B. neohumeralis are only differentiated bythree single-site nucleotide changes in the ribosomalITS 2 sequence (Morrow et al., 2000) and by differ-


ences in frequency of some polymorphic microsatellitealleles (Wang et al., 2003). We have found no fixedamino acid difference in any gene tested to date andsharing of polymorphisms in coding and non-codingregions is common (Morrow et al., 2000; An et al.,2002). We propose that the substantial shared poly-morphism is indicative of continuing genetic exchangebetween the two species.

The genetic basis for the difference in mating timebetween B. tryoni and B. neohumeralis has beendemonstrated by a study of the segregation of beha-vioural differences in hybrids (Smith, 1979). Further-more, the daily rhythm of mating activity in the flies iscontrolled by the endogenous circadian clock (Tychsenand Fletcher, 1971). Consequently, the genes involvedin the circadian clock are possible candidates for spe-cies differentiation. In Drosophila, period (per) was thefirst circadian gene to be identified and later cloned(Konopka and Benzer, 1971; Bargiello and Young,1984). The DNA sequence of per is highly variablebetween Drosophila species (Colot et al., 1988), and perplays a major role in setting up the behaviouralrhythms of locomotor activity and circadian eclosion(Yu et al., 1987; Konopka et al., 1989). Four genescomprise the core components of the central pace-maker: per, tim (timeless), clk (clock) and cyc (cycle)(reviewed in Young and Kay, 2001). The cryptochrome(cry) gene, a blue-light photoreceptor, mediates photo-entrainment of the endogenous clock to environmentallight–dark cycles (Emery et al., 1998). From studies inDrosophila and other organisms, the response to blue-light is strongly dependent on the concentration ofCRY protein (Emery et al., 2000; Lin et al., 1998).Since B. tryoni mates at dusk, when blue-light pre-dominates, we hypothesised that cry may have a role inthe mechanism mediating the effect of light intensity onmating behaviour.

The per and cry homologues have been isolated fromB. tryoni and B. neohumeralis (An et al., 2002; K.Raphael and X. An, unpublished). Sequence compar-isons revealed no differences in putative amino acidsequences for either per or cry. However, as for othergenomic regions, polymorphisms in coding and non-coding regions are shared between the two species.These data add to the picture of very close geneticsimilarity between these two pest species.

The mRNA expression profiles of per in the twoBactrocera species are identical (An et al., 2002). Bac-trocera per mRNA levels oscillate in abundance indiurnal light/dark cycles in both male and femaleheads and the male abdomen. Similar diurnal cyclingin per mRNA abundance is observed in D. melanoga-ster, L. cuprina and A. pernyi (Hardin et al., 1990;Warman et al., 2000; Sauman and Reppert, 1996).

In Drosophila splicing of an intron in the 30 UTR ofper is regulated by temperature and is believed to be a

mechanism by which flies adapt to seasonal changes in

temperature and day length (Majercak et al., 1999).

The potential role of temperature as a selection agent

in allowing the wider range of B. tryoni to colder

southerly latitudes led us to compare the effect of tem-

perature on per mRNA splicing in B. tryoni and B.

neohumeralis. No difference in the pattern of splicing in

response to temperature was observed between the two

species (An et al., 2002). However the response to tem-

perature differed to that of D. melanogaster. In both

Bactrocera species, abundance of the spliced transcript

was reduced compared to that of the unspliced tran-

script at low temperature, whereas low temperature

enhances splicing of the per 30 UTR in Drosophila

(Majercak et al., 1999).The very low level of genetic differentiation suggests

that the sibling Bactrocera species are defined, not by

widespread small differences throughout the genome,

but by differences in the genes that determine mating

isolation, speciation and climatic range, and thus pest

status. B. tryoni and B.neohumeralis are particularly

useful as a model system for identifying these genes

because hybrid flies can be readily obtained and scored

(Gibbs, 1968; Smith, 1979) and selected for species-spe-

cific behaviours (Pike and Meats, 2002). Gene trans-

formation will be particularly useful for testing the

functional significance of any differences identified.

Currently, it is not clear how such information will be

useful for pest insect control, but it is certain that an

increase in knowledge of how pest status is determined

and maintained will be useful in the future, as we

develop more sophisticated, environmentally-sound

protocols for suppression or eradication.

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