Genetic delineation of sibling species of the pest fruit fly Bactocera (Diptera: Tephritidae) using microsatellites

Using a large set of microsatellites, the genetic relationships between three closely related Australian fruit fly species, Bactrocera tryoni (Froggatt), B. neohumeralis (Hardy) and B. aquilonis(May) were investigated. Bactrocera tryoni and B. neohumeralis are sympatric, while B. aquilonisis allopatric to both. The sympatric species, B. tryoni and B. neohumeralis, were found to be genetically distinct. It is likely that despite differences in mating time between these two species, some gene flow still occurs. In contrast, the sibling species B. tryoni and B. aquilonis were found to be closely related, despite allopatry. The level of genetic divergence was similar to that found within eastern Australian populations of B. tryoni. Consideration of all available genetic data suggests that this similarity is not due to recent (i.e. within the last 30 years) displacement of B. aquilonis by B. tryoni from the B. aquilonis region (north-western Australia). Instead the data suggests that, at least in the areas sampled, asymmetrical hybridization may have occurred over a longer timescale.     

The likely fate of hybrids of Bactrocera tryoni and Bactrocera neohumeralis

Bactrocera tryoni (Froggatt) and B. neohumeralis (Hardy) (Diptera: Tephritidae) are sympatric species which hybridise readily in the laboratory yet remain distinct in the field. B. tryoni mates only at dusk and B. neohumeralis mates only during the day, but hybrids can mate at both times. We investigated the inheritance of mating time in successively backcrossed hybrid stocks to establish whether mating with either species is more likely. The progeny of all backcrosses to B. tryoni mated only at dusk. The majority of the progeny of the first and a minority of the progeny of the second backcross to B. neohumeralis also mated at dusk, but the third successive B. neohumeralis backcross produced flies that mated only during the day. This trend towards dominance of the B. tryoni trait was also reflected in a diagnostic morphological character. We discuss the possible genetic background for these phenomena and propose that unidirectional gene flow might explain how the two species remain distinct in the face of natural hybridisation.     

Close genetic similarity between two sympatric species of tephritid fruit fly reproductively isolated by mating time

Abstract.— Two sibling species of tephritid fruit fly, Bactrocera tryoni and B. neohumeralis , occur sympatrically throughout the range of B. neohumeralis in Australia. Isolation between the two species appears to be maintained by a difference in mating time: B. tryoni mates at dusk, whereas B. neohumeralis mates during the middle of the day. A morphological difference in humeral callus color also distinguishes the two species. Despite clear phenotypic evidence that B. tryoni and B. neohumeralis are distinct species, genetic differentiation as measured by four markers–nuclear DNA sequences from the white gene and the ribosomal internal transcribed spacer (ITS2), and mitochondrial DNA sequences from the cytochrome b (cytb) and cytochrome oxidase subunit II (COII) genes–is very small. Minor fixed differences occur in the ITS2 sequence, however, in all other cases the two species exhibit a high level of shared polymorphic variation. The close genetic similarity suggests either that speciation has occurred very rapidly and recently in the absence of any mitochondrial DNA sorting or that the sharing of polymorphisms is due to hybridization or introgression. A third species within the tryoni complex, B. aquilonis , is geographically isolated. Bactrocera aquilonis is also genetically very similar, but in this case there is clear differentiation for the mitochondrial loci. The three species form a group of considerable interest for investigation of speciation mechanisms.     

Genetic manipulation of the circadian clock's timing of sexual behaviour in the Queensland fruit flies, Dacus tryoni and Dacus neohumeralis

ABSTRACT. Mating in Dacus tryoni is restricted to dusk, whereas that of a sibling species, Dacus neohumeralis , occurs in the middle of the day. The timing of sexual behaviour in both species is determined by an interaction between a circadian clock and light intensity. In D. tryoni peak mating responsiveness is at the time of dusk, and the optimal light intensity for mating is approximately 91x. In D.neohumeralis peak responsiveness is in the middle of the day, and the optimal light intensity for mating is greater than 10 000 lx. The two species were crossed and the time of mating and response to light intensity of F₁, F₂ and backcross progeny determined. The circadian clock set a mating phase ('gate') as narrow in F₁ flies as in their parents, suggesting the circadian timing mechanism to be common between the two species. The results indicate that the genetic mechanism controlling timing is independent of that controlling response to light intensity, and that both genetic mechanisms are complex.     

DNA microsatellite analysis of naturally occurring colour intermediates between Bactrocera tryoni (Froggatt) and Bactrocera neohumeralis (Hardy) (Diptera: Tephritidae)

Abstract  The sympatric tephritid fruit flies Bactrocera tryoni (Froggatt) (Queensland fruit fly) and B. neohumeralis (Hardy) differ in time of mating and for the colour of the humeral callus ('shoulder pad'), which is typically entirely yellow in B. tryoni and typically entirely brown in B. neohumeralis . Field collections in sympatric regions usually include at least 1% of individuals whose humeral calli show mixed patches of yellow and brown ('intermediates'). Over 40 years, a number of studies have debated the possibility that these intermediates are interspecific hybrids. In the present study, we have used microsatellites to show that few if any of these intermediates are hybrids. Instead, most variation humeral callus appears to be confined to one species, B. tryoni . We discuss these results in the context of geneflow between the two species and suggest directions for future research.     

The Effects of Selection for early (day) and late (dusk) Mating lines of Hybrids of Bactrocera tryoni and Bactrocera neohumeralis

Bactrocera neohumeralis and Bactrocera tryoni are closely related tephritid fruit fly species. B. neohumeralis mates throughout the day (in bright light) and B. tryoni mates at dusk. The two species can also be distinguished by the colour of their calli (prothoracic sclerites) which are brown and yellow, respectively. The F1 hybrids can mate both in bright light just before dusk and during dusk and have calli that are partly brown and partly yellow. The F2 hybrids have a wider range of callus patterns and mating occurs more widely in the day as well as at dusk. We directly selected hybrid stocks for mating time, creating 'early' (day-mating) and 'late' (dusk-mating) lines. As an apparently inadvertent consequence, the two types of line respectively had predominantly brown and predominantly yellow calli and thus came to closely resemble the original two species in both behaviour and appearance. Lines that were evenly selected (half for day and half for dusk) essentially retained the mating pattern of F2 hybrids. Selection for callus colour alone also affected the distribution of mating times in a predictable way. We propose a genetical model to account for the results and discuss them in the light of the apparent maintenance of species integrity in nature.     

Attributes pertinent to overwintering potential do not explain why Bactrocera neohumeralis (Hardy) (Diptera: Tephritidae) does not spread further south within the geographical range of B. tryoni (Froggatt)

Abstract  The geographical range of Bactrocera neohumeralis does not extend as far south as that of its sibling species, B. tryoni . However, there was no evidence of any difference between the two species in terms of physiological limitation to southerly spread when comparisons were made of low temperature torpor thresholds of adults, survival time of adults at −4°C and development rates of all stages in either warm or cool regimes. The survival schedule of the two species was similar in the laboratory and also in the moderately cold conditions experienced by caged cohorts that were exposed to winter field temperatures between late April and early November at Richmond, New South Wales (500 km south of the usual southerly limit of B. neohumeralis ). Overwintered cohorts of both species laid similar numbers of eggs in September in terms of eggs per emerged female (an indicator of the reproductive potential). However, because the proportion of B. tryoni surviving to the period of 1–15 September was less than half that for B. neohumeralis , the production per surviving female was more than double in B. tryoni . The possibility of the southerly spread of B. neohumeralis being limited by an Allee effect is discussed.     

Australian Distribution of 17 Species of Fruit Flies (Diptera: Tephritidae) Caught in Cue Lure Traps in February 1994

We report the first widespread survey of tephritid fruit flies attempted in a single time period. 1,471 cue lure traps caught 17 species, and extensions to previously recorded geographical ranges were detected for seven of them: Bactrocera tryoni, B. neohumeralis, B. frauenfeldi, B. aeroginosa, Dacus absonifascies, D. aequalis and D. newmani. the traps also unexpectedly caught several B. cacuminata and also both males and females of Dirioxa pornia and Ceratitis capitata. the geographical variation in the relative abundance of B. tryoni and B. neohumeralis in the region of their co-occurrence was in substantial agreement with earlier estimates. the regional variation in abundance of B. tryoni in the eastern states was in accordance with the predictions of a published bioclimatic model. Furthermore, the spread of this species (expected from the model) to several locations in the Northern Territory is recorded here for the first time.     

Comparison of two polymorphic sites in the clock gene cryptochrome in the Taiwan strain of the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae): a possible quick method to estimate the mating time of trapped invading flies

For successful sterile insect technique (SIT), synchronized copulation between invaded females and sterilized males is required. Understanding the mating time of the invaded strain is an aid in synchronizing and thus improving the effectiveness of SIT. We previously demonstrated a relationship between variation at two sites of a circadian clock gene cryptochrome (cry) (cry1212 and cry1865) and circadian behavior in the melon fly Bactrocera cucurbitae (Coquillett). Here we investigated the relationship in two other populations, Taiwan1 (T1) and Taiwan2 (T2), which may re-invade Okinawa. The results showed that T1 exhibited a lower frequency of the S-type allele, which was observed in early mating flies in the strains in Okinawa, than T2 at the site of cry1212. In addition, T1 showed a longer circadian period than T2. We also noted that the cry1212 site showed higher amino acid sequence conservation than cry1865 by comparing CRY1 among five insect species. These results suggest that genotyping of only the cry1212 site of trapped flies enables an immediate estimate of the mating time of the B. cucurbitae population from Taiwan and that cry1212 would be more likely to be involved in determining the mating time than cry1865.     

THESIS ABSTRACT

Geographical distribution, seasonal abundance and habitat preference of fruit fly species (Diptera: Tephritidae) in South-East Queensland, with special reference to Bactrocera tryoni (Froggatt) and Bactrocera neohumeralis (Hardy) . S. Raghu.     

Bactrocera tryoni and closely related pest tephritids--molecular analysis and prospects for transgenic control strategies

Bactrocera tryoni is a serious pest of horticulture in eastern Australia. Here we review molecular data relevant to pest status and development of a transformation system for this species. The development of transformation vectors for non-drosophilid insects has opened the door to the possibility of improving the sterile insect technique (SIT), by genetically engineering factory strains of pest insects to produce male-only broods. Transposition assays indicate that all five of the vectors currently used for transformation in non-drosophilid species have the potential to be useful as transformation vectors in B. tryoni. Evidence of cross mobilization of hobo by an endogenous Homer element emphasises the necessity to understand the endogenous transposons within 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 the potential of RNAi to modify the sex ratio of resultant broods. An understanding of how pest status is determined and maintained is 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.     

The period gene and allochronic reproductive isolation in Bactrocera cucurbitae

Clock genes that pleiotropically control circadian rhythm and the time of mating may cause allochronic reproductive isolation in the melon fly Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Flies with a shorter circadian period (ca. 22 h of locomotor activity rhythm) mated 5 h earlier in the day than those with a longer circadian period (ca. 30 h). Mate-choice tests demonstrated significant pre-mating isolation between populations with short and long circadian periods. Pre-mating isolation did not occur when the mating time was synchronized between the two populations by photoperiodic controls, indicating that reproductive isolation is due to variations in the time of mating and not any unidentified ethological difference between the two populations. We cloned the period (per) gene of B. cucurbitae that is homologous to the per gene in Drosophila. The relative level of per mRNA in the melon fly exhibited a robust daily fluctuation under light : dark conditions. The fluctuation of per expression under dark : dark conditions is closely correlated to the locomotor rhythm in B. cucurbitae. These results suggest that clock genes can cause reproductive isolation via the pleiotropic effect as a change of mating time.     

The effect of photoperiod on the circadian rhythm of mating responsiveness in the fruit fly, Dacus tryoni

ABSTRACT. The daily fluctuation in mating responsiveness of Dacus tryoni was recorded through a range of light cycles. Evidence was obtained of strict control of the fluctuation by a circadian clock. The phase setting of the fluctuation in the different light cycles was in conformity with Aschoff's (1965) generalizations about circadian rhythms, and had no relationship to overt sexual behaviour occurring during the cycles. The amplitude of the fluctuation in mating responsiveness varied between cycles, suggesting possible variation in the amplitude of the oscillation of the circadian clock.     

Spatial and circadian regulation of cry in Drosophila

In Drosophila, cryptochrome (cry) encodes a blue-light photoreceptor that mediates light input to circadian oscillators and sustains oscillator function in peripheral tissues. The levels of cry mRNA cycle with a peak at approximately ZT5, which is similar to the phase of Clock (Clk) mRNA cycling in Drosophila. To understand how cry spatial and circadian expression is regulated, a series of cry-Gal4 trans-genes containing different portions of cry upstream and intron 1 sequences were tested for spatial and circadian expression. In fly heads, cry upstream sequences drive constitutive expression in brain oscillator neurons, a novel group of nonoscillator cells in the optic lobe, and peripheral oscillator cells in eyes and antennae. In contrast, cry intron 1 drives rhythmic expression in eyes and antennae, but not brain oscillator neurons. These results demonstrate that intron 1 is sufficient for high-amplitude cry mRNA cycling, show that cry upstream sequences are sufficient for expression in brain oscillator neurons, and suggest that cry spatial and circadian expression are regulated by different elements.     

Host preference and host suitability in an egg-pupal fruit fly parasitoid, Fopius arisanus (Sonan) (Hym., Braconidae)

The relative oviposition rate of the parasitoid Fopius arisanus (Sonan) was investigated across three frugivorous tephritid species, Bactrocera tryoni Froggart, Bactrocera jarvisi (Tryon) and Bactrocera cucumis French. Choice and no-choice tests were both used. The suitability of these three species for sustaining larval development and survival to the adult stage was also assessed. Fopius arisanus parasitized all three tephritid species, regardless of the method of exposure, but showed stronger preference for B. tryoni and B. jarvisi over B. cucumis. Superparasitism was extremely rare. Successful development of F. arisanus varied across host species. Bactrocera tryoni yielded significantly more parasitoids than B. jarvisi, but no wasps emerged from B. cucumis puparia. Tests were set up in replicated trials, but results were not homogeneous across trials. We discuss the host relationships of F. arisanus with reference to this variation and in relation to host suitability for larval development.     

Pleiotropic effect, clock genes, and reproductive isolation

 The mechanism by which a clock gene pleiotropically controlling life history and behavioral traits causes reproductive isolation is explained using a model species, the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Melon flies mate once a day, at dusk. The population selected for life history traits exhibits correlated responses in the time of mating during the day. For example, the fly populations selected for faster (slower) development have an earlier (later) time of mating. A circadian rhythm controls the time of mating. The circadian periods in constant darkness were about 22 h in lines selected for a short developmental period and about 31 h in lines selected for a long developmental period. The data on crosses between the selected lines indicated that the developmental period is controlled by a polygene, whereas the circadian period may be controlled by a single clock gene. These results suggest a clock gene pleiotropically controls developmental and circadian periods in the melon fly. Reproductive isolation may often evolve as an indirect (pleiotropic) consequence of adaptation to different environments or habitats. For example, niches that are temporally or seasonally offset can select organisms with different developmental characteristics. These developmental differences can inadvertently cause reproductive isolation by a variety of means including shifts in mating activity patterns. The difference in time of mating between populations selected for developmental period translated into significant prezygotic isolation, as measured by mate choice tests. If the mating time between populations differed more than 1 h, the isolation index was significantly higher than zero. These findings indicate that premating isolation can be established by a pleiotropic effect of a clock gene. There are many examples in which the difference in timing of reproduction prevents gene flow between populations, such as the egg spawning time in marine organisms, the flowering time in angiosperms, and the time of mating in insects. In such organisms, if genetic correlations between circadian rhythm and reproductive traits exist, multifarious divergent selection for life history traits would often accelerate the evolution of reproductive isolation through clock genes. Natural populations may diverge in reproduction time through drift, direct natural selection for time of reproduction, or as a by-product effect of genetic correlations. In any case, clock genes are keys in reproductive isolation. Received: January 31, 2002 / Accepted: July 29, 2002 Acknowledgments I am grateful to Tetsuo Arai, Akira Matsumoto, Takashi Matsuyama, Toru Shimizu, Aya Takahashi, Teiichi Tanimura, Tetsuya Toyosato, and Yasuhiko Watari for useful discussion, and to the responsible editor and two anonymous reviewers for helpful suggestions. I also thank Yoshihiko Chiba, Norio Ishida, Emi Koyama, Kazuhiko Sakai, and Takaomi Sakai for useful information. My work on speciation has been supported by a Grant-in-Aid for Scientific Research (KAKENHI 14340244) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Drosophila CRY is a deep brain circadian photoreceptor

cry (cryptochrome) is an important clock gene, and recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cry(b), inhibits circadian photoresponses. Restricting CRY expression to specific fly tissues shows that CRY expression is needed in a cell-autonomous fashion for oscillators present in different locations. CRY overexpression in brain pacemaker cells increases behavioral photosensitivity, and this restricted CRY expression also rescues all circadian defects of cry(b) behavior. As wild-type pacemaker neurons express CRY, the results indicate that they make a striking contribution to all aspects of behavioral circadian rhythms and are directly light responsive. These brain neurons therefore contain an identified deep brain photoreceptor, as well as the other circadian elements: a central pace-maker and a behavioral output system.     

Faster (or slower) developers have a shorter (or longer) circadian period in Bactrocera cucurbitae

A clock controlling circadian rhythm may relate to another clock controlling development time. The relationship between the two clocks remains open to discussion. Genetic correlation between developmental and circadian periods is observed in two Dipteran species (Bactrocera cucurbitae and Drosophila melanogaster), whereas it is not observed in a Coleopteran species (Callosobruchus chinensis). No studies, however, are available that report on the phenotypic correlation between the two traits. In the present study, free‐running circadian periods are compared between individuals that develop fastest and slowest within a population of B. cucurbitae. The measurement of circadian periods is replicated using five populations of B. cucurbitae with different geographical and rearing histories. The results demonstrate that the flies developing more slowly have significantly longer circadian periods compared with the flies developing more quickly in two of five populations examined in B. cucurbitae, and thus the phenotypic correlation is dependent on population.