Masayoshi Watada

Prevalence of a Non-Male-Killing Spiroplasma in Natural Populations of Drosophila hydei

ABSTRACT Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.

Host range of Asobara japonica (Hymenoptera: Braconidae), a larval parasitoid of drosophilid flies

We studied the host range of Asobara japonica, a larval‐pupal parasitoid of drosophilid flies. Habitat selection was found to be an important determinant of host range in this parasitoid; it attacked drosophilid larvae breeding on banana and mushrooms, but seldom attacked those breeding on decayed leaves. This parasitoid was able to use diverse drosophilid taxa as hosts. Attack by A. japonica sometimes killed hosts at the larval stage, and therefore parasitoid larvae also died. Drosophila elegans and D. busckii suffered particularly high larval mortality due to the attack by A. japonica (in the latter species only when young larvae were attacked). Many individuals of D. subpulchrella also died at the pupal stage without producing parasitoids when they were parasitized at the late larval stage. In contrast, D. bipectinata, D. ficusphila, D. immigrans, D. formosana and D. albomicans were resistant to attack: large proportions of the larvae of these drosophilid species grew to adulthood, even in the presence of parasitoids. On the basis of phylogenetic information, we concluded that phylogenetic position has only limited importance as a factor determining whether a species is suitable as a host for A. japonica, at least within the genus Drosophila.

Ecological, morphological and molecular studies on Ganaspis individuals (Hymenoptera: Figitidae) attacking Drosophila suzukii (Diptera: Drosophilidae).

Ganaspis individuals parasitizing Drosophila suzukii (Matsumura), a pest of fruit crops, were examined for host use and molecular and morphological differences from those attacking D. lutescens Okada and some other Drosophila species that breed on fermenting fruits. Wild cherry fruits were collected in the suburbs of Tokyo, and drosophilid pupae obtained from these fruits were examined for parasitism. Drosophila suzukii was the only drosophilid species infesting fresh wild cherry fruits, and Ganaspis individuals were the major parasitoids attacking D. suzukii in wild cherry fruits. In parasitism experiments, these Ganaspis individuals parasitized D. suzukii larvae in fresh cherry fruits, but did not parasitize those in Drosophila medium. In addition, they did not parasitize larvae of some other fruit-feeding Drosophila species even when these occurred in fresh cherry fruit. These Ganaspis individuals parasitizing D. suzukii were different from those parasitizing D. lutescens and some other drosophilids in nucleotide sequences of the COI gene, as well as in ITS1 and ITS2. They were also different in forewing and antenna morphology, although they showed some overlap in morphological traits. They are tentatively assigned as the suzukii- and lutescens-associated types of G. xanthopoda Ashmead. In the present field survey, Leptopilina japonica Novković & Kimura and some Asobara species were also observed to attack D. suzukii larvae in wild cherry fruit.

P elements and P-M characteristics in natural populations of Drosophila melanogaster in the southernmost islands of Japan and in Taiwan

In order to study P element dynamics in natural populations of Drosophila melanogaster, 126 isofemale lines were examined from seven of the southernmost islands of Japan (the Sakishima Islands) and from Taiwan. Gonadal dysgenesis (GD) tests showed large divergences in the P-M phenotypes (P inducing and P repressing abilities) between the island populations. The P-M characteristics of each population, however, had not greatly changed in the past 15 years. Their genomic P element profiles are highly similar, consisting mostly of full-size P and of KP elements. We found no clear relationship between phenotype and genomic P element composition.

Negative Effects of Low Temperatures on the Vertical Transmission and Infection Density of a Spiroplasma Endosymbiont in Drosophila hydei

Maternally transmitted endosymbionts of the genus Spiroplasma infecting several species of Drosophila are known to cause selective death of male offspring (male killing). The male-killing trait is considered to be advantageous for maternally transmitted endosymbionts. However, a non-male-killing spiroplasma is present in Japanese populations of Drosophila hydei at high frequencies (23–66%). This spiroplasma is phylogenetically closely related to the male-killing spiroplasma infecting other Drosophila species. It is unknown why this spiroplasma is maintained in its host populations despite its inability to cause male killing. We examined the susceptibilities of the spiroplasma in D. hydei to four different temperatures (28, 25, 18, and 15°C). Diagnostic PCR revealed that vertical transmission of the spiroplasma was nearly perfect at 28 and 25°C, partially suppressed at 18°C, and completely blocked at 15°C. Furthermore, quantitative PCR demonstrated that offspring treated at 18°C exhibited dramatically lower densities of spiroplasma (i.e., approximately one-tenth) compared to offspring treated at 28 and 25°C. Considering the low temperatures during winter in Japan, some unknown advantageous effects of the spiroplasma that compensate for the failure of vertical transmission are suggested to act in natural populations of D. hydei.

Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species.

The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting “enhancer” elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony’s transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer’s activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits.

Multiple origins of Hawaiian drosophilids: Phylogeography of Scaptomyza Hardy (Diptera: Drosophilidae)

Scaptomyza is a highly diversified genus in the family Drosophilidae, having undergone an explosive radiation, along with the Hawaiian‐endemic genus Idiomyia in the Hawaiian Islands: about 60% of 269 Scaptomyza species so far described are endemic to the Hawaiian Islands. Two hypotheses have been proposed for the origin and diversification of Hawaiian drosophilids. One is the “single Hawaiian origin” hypothesis: Scaptomyza and Idiomyia diverged from a single common ancestor that had once colonized the Hawaiian Islands, and then non‐Hawaiian Scaptomyza migrated back to continents. The other is the “multiple origins” hypothesis: Hawaiian Scaptomyza and Idiomyia derived from different ancestors that independently colonized the Hawaiian Islands. A key issue for testing these two hypotheses is to clarify the phylogenetic relationships between Hawaiian and non‐Hawaiian species in Scaptomyza. Toward this goal, we sampled additional non‐Hawaiian Scaptomyza species, particularly in the Old World, and determined the nucleotide sequences of four mitochondrial and seven nuclear genes for these species. Combining these sequence data with published data for 79 species, we reconstructed the phylogeny and estimated ancestral distributions and divergence times. In the resulting phylogenetic trees, non‐Hawaiian Scaptomyza species were interspersed in two Hawaiian clades. From a reconstruction of ancestral biogeography, we inferred that Idiomyia and Scaptomyza diverged outside the Hawaiian Islands and then independently colonized the Hawaiian Islands, twice in Scaptomyza, thus supporting the “multiple origins” hypothesis.

Population dynamics of a maternally-transmitted Spiroplasma infection in Drosophila hydei

A maternally-inherited spiroplasma endosymbiont of Drosophila hydei does not exert apparent phenotypes on both sexes of its host and is prevalent in natural populations of D. hydei. Our previous experiments using a laboratory stock of D. hydei revealed that low temperatures (such as 15°C and 18°C) dramatically lower the vertical transmission rates of this spiroplasma. Therefore, we hypothesized that, in temperate regions, the infection frequencies may decrease in cool seasons but increase in the summer season. To clarify the temporal population dynamics of the spiroplasma infection, D. hydei were collected from two Japanese populations in 2006–2008 from May to early August, representing the only period when a number of D. hydei are collectable in Japan, and examined for spiroplasma infection. Within each year, the frequency of spiroplasma infection fluctuated considerably in both populations. Consistent with our hypothesis, the infection frequency showed an increasing trend in both populations in 2007. However, the data in 2006 and 2008 did not show consistent patterns of increase. The population dynamics of spiroplasma infection may be affected but not critically determined by temperature. Moreover, despite the fluctuation within each year, the infection frequencies seemed to be stable across the years. The frequencies of spiroplasma infection in D. hydei populations may be stabilized by multiple factors. One of these factors may involve a context-dependent positive effect of spiroplasma on the fitness of D. hydei, as was recently observed in laboratory experiments.

Taxonomic study of the Drosophila auraria species complex (Diptera: Drosophilidae) with description of a new species

The Drosophila auraria species complex, especially from the Ryukyu archipelago and Taiwan, was reviewed. A new species, D. neoasahinai Watada and Kondo, sp. nov., was described from Okinawa‐jima and surrounding islands. Two synonymies were proposed on the basis of the present and previous morphological comparisons and cross experiments: (i) D. yuwanensis Kim and Okada, 1988 as a junior synonym of D. asahinai Okada, 1964; and (ii) D. quadraria Bock and Wheeler, 1972 as a junior synonym of D. triauraria Bock and Wheeler, 1972. A laboratory stock (no. 14020–0011.01) maintained in the Drosophila Species Stock Center at the University of California, San Diego and so far designated as D. rufa Kikkawa and Peng, 1938 was identified as D. tani Cheng and Okada, 1985, based on morphology.

Maternally transmitted non-bacterial male killer in Drosophila biauraria

A maternally inherited, all-female trait is widely found among arthropods, which is caused by bacterial endosymbionts such as Wolbachia, Rickettsia, Spiroplasma and Cardinium. We discovered a single female of Drosophila biauraria, collected from Tomakomai, Hokkaido, Japan, that produced all-female offspring. This all-female trait was maternally inherited in the iso-female line (SP12F) by backcrossing with males of a normal line (SP11-20) with a 1 : 1 sex ratio derived from the same population. The all-female trait was not affected by tetracycline treatment performed for two consecutive generations. However, the microinjection of filter-sterilized homogenate of SP12F females into SP11-20 females established all-female matrilines. Our data suggest the role of transmissible agents, most likely viruses, but not bacteria or protists, as the possible cause of the all-female phenotype, which is likely to be achieved by killing of male embryos because egg hatch rates of SP12F were nearly half those of SP11-20. This is the first report in Diptera to demonstrate a maternally inherited virus-like element as the cause of the male-killing phenotype in D. biauraria.