Takashi Matsuyama

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 clock gene cryptochrome of Bactrocera cucurbitae (Diptera: Tephritidae) in strains with different mating times

Differences in mating time between populations can give rise to premating reproductive isolation. Tephritid fruit flies exhibit large variation in mating time among intra- or inter-specific populations. We previously cloned the clock gene period from two strains of melon fly, Bactrocera cucurbitae; in one the individuals mate early during the day, whereas in the other the individuals mate later. These strains were originally established by divergent artificial selection for developmental time, ‘short’ and ‘long’, with early and late mating times, respectively. The deduced amino acid sequences of PERIOD proteins for these two strains were reported to be identical. Here we cloned another clock gene cryptochrome (cry) from the two strains, and found two stable amino acid substitutions in the strains. In addition, the allele frequency at the two polymorphic sites of cry gene correlated with the circadian locomotor period (τ) across strains, whereas the expression pattern of cry mRNA in the heads of flies taken from the short strain significantly differed from that from the long strain. These findings suggest that variation in the cry gene is related to differences in the circadian behaviour in the two strains, thus implying that the cry gene may have an important role in reproductive isolation.

Period Gene of Bactrocera cucurbitae (Diptera: Tephritidae) Among Strains with Different Mating Times and Sterile Insect Technique

Abstract The efficacy of sterile insect technique (SIT) depends on successful mating of released males with wild females. If the time of mating in a day of mass-reared and released males differs from those of wild females, the efficiency of SIT decreases. Therefore, understanding the molecular mechanisms controlling mating time of the target pests is particularly important for SIT. The period (per) gene, which has been considered as a key clock gene controlling the mating time of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae), was cloned from two strains having different times of mating during the day. DNA sites varied in the 5′ and 3′ untranslated regions and at synonymous sites, although protein sequences were identical. We also provide phylogenetic relationships among PER protein sequences of dipteran species including several tephritid pest species. The functional domains of PER in the melon fly are very similar to those in other tephritid species. A luciferase reporter assay showed that the melon fly PER can functionally complement that of Drosophila melanogaster (Meigen). The results implicate that the major genetic cause of the difference in circadian periods, and thus in reproductive isolation, is probably one or more other clock gene(s). Thus, the series of studies may provide a novel factor concerning genetic quality control of mass-reared insect pests for SIT, which depends on successful mating of released males and wild females.

Ultraviolet light-emitting diode (UV LED) trap the West Indian sweet potato weevil, Euscepes postfasciatus (Coleoptera: Curculionidae)

The West Indian sweet potato weevil Euscepes postfasciatus (Fairmaire) is a troublesome pest insect of sweet potato that originally came from the Caribbean, but is now expanding its distribution into the Pacific Islands. Although sterile insect techniques have been used against this pest in a demonstration experiment on Kume Island [Ohno et al. (2006) Kontyu to Shizen 41:25–30], effective methods of monitoring E. postfasciatus are scarce. It is necessary to detect the weevils at an early stage of invasion in uninvaded areas, and an attractant trap can be used to achieve this. Thus, we developed an ultraviolet (UV) light-emitting diode trap, invented a method for diffusing the light to attract more insects, and investigated the attractiveness of the light trap to E. postfasciatus under laboratory conditions. Our results indicate that diffused UV light has a higher potential to attract E. postfasciatus than direct UV light. Furthermore, sweet potato is an effective bait to use to capture the weevils attracted by UV light. Thus, E. postfasciatus can be trapped using diffused UV light and sweet potato bait.

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.

Host plants influence female oviposition and larval performance in West Indian sweet potato weevils Euscepes postfasciatus (Coleoptera: Curculionidae)

Euscepes postfasciatus (Fairmaire) is an invasive pest of the sweet potato (Ipomoea batatas) and is also parasitic to other wild host plants of the Ipomoea genus. The population density of E. postfasciatus is sometimes greater in Ipomoea pes-caprae L. than in Ipomoea indica (Burm. f.). We investigated the desirability of I. pes-caprae as a host plant for E. postfasciatus in terms of reproductive and developmental potential. Females laid fewer eggs on I. pes-caprae, and the eclosion of their larvae was delayed compared with on I. indica. Furthermore, the larval growth rate was slower on I. pes-caprae than on I. indica. These results suggest that I. pes-caprae is not always the preferred host for egg laying and growth rate in the early developmental stages. However, the larval survival rate after the initial period of development was markedly better on I. pes-caprae than on I. indica. The present simulation study demonstrated that the population density of E. postfasciatus on I. pes-caprae overwhelmed that on I. indica over generations. Comparing the two wild host plant species, I. pes-caprae outweighs I. indica with respect to total population growth, but reproduction on I. indica may be advantageous for the colonization of the new habitat.

Female preference did not evolve under laboratory conditions in the solanaceous fruit fly Bactrocera latifrons

Sterile insect technique (SIT) is widely used to eradicate target pest species. Its effectiveness depends on the ability of released sterile males to mate with wild females. In pest species with a female choice mating system, there is potential for female preference to evolve in the wild population in view of the resulting zero-fitness in females that mate with sterile males. The evolution of female preference against sterile males can, therefore, reduce the efficiency of the SIT as the likelihood decreases that sterile males will mate with wild females. We examined this experimentally by using the solanaceous fruit fly Bactrocera latifrons, a serious pest of solanaceous crops, by allowing female preference in mass-reared fruit flies to select between fertile and sterile males over 12 generations. However, this did not generate a rapid evolutionary response. Also the remating rate of females did not increase, even when the first mating partner was a sterile male. We discuss the implication of the results for the eradication project by the SIT program.