Siu F. Lee

Temporal expression of heat shock genes during cold stress and recovery from chill coma in adult Drosophila melanogaster.

A common physiological response of organisms to environmental stresses is the increase in expression of heat shock proteins (Hsps). In insects, this process has been widely examined for heat stress, but the response to cold stress has been far less studied. In the present study, we focused on 11 Drosophila melanogaster Hsp genes during the stress exposure and recovery phases. The temporal gene expression of adults was analyzed during 9 h of cold stress at 0 °C and during 8 h of recovery at 25 °C. Increased expression of some, but not all, Hsp genes was elicited in response to cold stress. The transcriptional activity of Hsp genes was not modulated during the cold stress, and peaks of expression occurred during the recovery phase. On the basis of their response, we consider that Hsp60, Hsp67Ba and Hsc70‐1 are not cold‐inducible, whereas Hsp22, Hsp23, Hsp26, Hsp27, Hsp40, Hsp68, Hsp70Aa and Hsp83 are induced by cold. This study suggests the importance of the recovery phase for repairing chilling injuries, and highlights the need to further investigate the contributions of specific Hsp genes to thermal stress responses. Parallels are drawn between the stress response networks resulting from heat and cold stress.

Rapid sequential spread of two Wolbachia variants in Drosophila simulans.

The maternally inherited intracellular bacteria Wolbachia can manipulate host reproduction in various ways that foster frequency increases within and among host populations. Manipulations involving cytoplasmic incompatibility (CI), where matings between infected males and uninfected females produce non-viable embryos, are common in arthropods and produce a reproductive advantage for infected females. CI was associated with the spread of Wolbachia variant wRi in Californian populations of Drosophila simulans, which was interpreted as a bistable wave, in which local infection frequencies tend to increase only once the infection becomes sufficiently common to offset imperfect maternal transmission and infection costs. However, maternally inherited Wolbachia are expected to evolve towards mutualism, and they are known to increase host fitness by protecting against infectious microbes or increasing fecundity. We describe the sequential spread over approximately 20 years in natural populations of D. simulans on the east coast of Australia of two Wolbachia variants (wAu and wRi), only one of which causes significant CI, with wRi displacing wAu since 2004. Wolbachia and mtDNA frequency data and analyses suggest that these dynamics, as well as the earlier spread in California, are best understood as Fisherian waves of favourable variants, in which local spread tends to occur from arbitrarily low frequencies. We discuss implications for Wolbachia-host dynamics and coevolution and for applications of Wolbachia to disease control.

Effects of small Hsp genes on developmental stability and microenvironmental canalization

BackgroundProgression of development has to be insulated from the damaging impacts of environmental and genetic perturbations to produce highly predictable phenotypes. Molecular chaperones, such as the heat shock proteins (HSPs), are known to buffer various environmental stresses, and are deeply involved in protein homeostasis. These characteristics of HSPs imply that they might affect developmental buffering and canalization.ResultsWe examined the role of nine Hsp genes using the GAL4/UAS-RNAi system on phenotypic variation of various morphological traits in Drosophila melanogaster. The stability of bristle number, wing size and wing shape was characterized through fluctuating asymmetry (FA) and the coefficient of variation (CV), or among-individual variation. Progeny of the GAL4/Hsp-RNAi crosses tended to have reduced trait means for both wing size and wing shape. Transcriptional knockdown of Hsp67Bc and Hsp22 significantly increased FA of bristle number, while knockdown of Hsp67Ba significantly increased FA and among-individual variation of wing shape but only in males. Suppression of Hsp67Bb expression significantly increased among-individual variation of bristle number. The knockdown of gene expression was confirmed for Hsp67Ba, Hsp67Bc, Hsp22, and Hsp67Bb. Correlation between FA and CV or among-individual variation of each trait is weak and not significant except for the case of male wing shape.ConclusionFour small Hsp genes (Hsp22, Hsp67Ba, Hsp67Bb and Hsp67Bc) showed involvement in the processes of morphogenesis and developmental stability. Due to possible different functions in terms of developmental buffering of these small Hsps, phenotypic stability of an organism is probably maintained by multiple mechanisms triggered by different environmental and genetic stresses on different traits. This novel finding may lead to a better understanding of non-Hsp90 molecular mechanisms controlling variability in morphological traits.

Knocking down expression of Hsp22 and Hsp23 by RNA interference affects recovery from chill coma in Drosophila melanogaster.

SUMMARY To protect cells from the damaging effects of environmental stresses, all organisms possess a universal stress response involving upregulation of heat shock proteins (Hsps). The mechanisms underlying chilling injuries and the subsequent recovery phase are only beginning to be understood in insects. Hsp22 and Hsp23 are both upregulated during the recovery from prolonged chill coma in Drosophila melanogaster. This prompted us to investigate the functional significance of these modulations by testing whether expression of these two small Hsps is necessary for recovery after cold stress. We used the GAL4/UAS system to separately knock down expression of Hsp22 and Hsp23, and assayed three aspects of recovery performance in transgenic adults that had undergone 12 h of chill coma at 0°C. The time to recover (short-term recovery) and mobility parameters (medium-term recovery) were significantly impaired in the transgenic flies in which Hsp22 or Hsp23 was suppressed. Our findings show that both Hsp22 and Hsp23 play important roles in the recovery from chill coma in adult males, and suggest that these contribute to adaptive responses to fluctuating thermal conditions.

High-throughput PCR assays to monitor Wolbachia infection in the dengue mosquito (Aedes aegypti) and Drosophila simulans.

ABSTRACT We have developed and validated two new fluorescence-based PCR assays to detect the Wolbachia wMel strain in Aedes aegypti and the wRi and wAu strains in Drosophila simulans. The new assays are accurate, informative, and cost-efficient for large-scale Wolbachia screening.

Functional characterization of the Frost gene in Drosophila melanogaster: importance for recovery from chill coma.

Background Almost all animals, including insects, need to adapt to temperature fluctuations. The molecular basis of thermal adaptation is not well understood, although a number of candidate genes have been proposed. However, a functional link between candidate genes and thermal tolerance has rarely been established. The gene Frost (Fst) was first discovered when Drosophila flies were exposed to cold stress, but the biological function(s) of Fst has so far not been characterized. Because Fst is up-regulated after a cold stress, we tested whether it was essential for chill-coma recovery. Methodology/Principal Findings A marked increase in Fst expression was detected (by RT-PCR) during recovery from cold stress, peaking at 42-fold after 2 h. The GAL4/UAS system was used to knock down expression of Fst and recovery ability was assessed in transgenic adults following 12 h of chill coma at 0°C. The ability to recover from cold stress (short-, medium- and long-term) was significantly altered in the transgenic adults that had Fst silenced. These findings show that Fst plays an essential role in the recovery from chill coma in both males and females. Conclusions/Significance The Frost gene is essential for cold tolerance in Drosophila melanogaster and may play an important role in thermal adaptation.

Lucilia cuprina genome unlocks parasitic fly biology to underpin future interventions

Lucilia cuprina is a parasitic fly of major economic importance worldwide. Larvae of this fly invade their animal host, feed on tissues and excretions and progressively cause severe skin disease (myiasis). Here we report the sequence and annotation of the 458-megabase draft genome of Lucilia cuprina. Analyses of this genome and the 14,544 predicted protein-encoding genes provide unique insights into the flys molecular biology, interactions with the host animal and insecticide resistance. These insights have broad implications for designing new methods for the prevention and control of myiasis.

Molecular Basis of Adaptive Shift in Body Size in Drosophila melanogaster: Functional and Sequence Analyses of the Dca Gene

Latitudinal body size clines in animals conforming to Bergmanns rule occur on many continents but isolating their underlying genetic basis remains a challenge. In Drosophila melanogaster, the gene Dca accounts for approximately 5-10% of the natural wing size variation (McKechnie SW, Blacket MJ, Song SV, Rako L, Carroll X, Johnson TK, Jensen LT, Lee SF, Wee CW, Hoffmann AA. 2010. A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila. Mol Ecol. 19:775-784). We present here functional evidence that Dca is a negative regulator of wing size. A significant negative latitudinal cline of Dca gene expression was detected in synchronized third instar larvae. In addition, we clarified the evolutionary history of the three most common Dca promoter alleles (Dca237-1, Dca237-2, and Dca247) and showed that the insertion allele (Dca247), whose frequency increases with latitude, is associated with larger wing centroid size and higher average cell number in male flies. Finally, we showed that the overall linkage disequilibrium (LD) was low in the Dca promoter and that the insertion/deletion polymorphism that defines the Dca alleles was in strong LD with two other upstream sites. Our results provide strong support that Dca is a candidate for climatic adaptation in D. melanogaster.

Polymorphism in the couch potato gene clines in eastern Australia but is not associated with ovarian dormancy in Drosophila melanogaster

Natural selection can generate parallel latitudinal clines in traits and gene frequencies across continents, but these have rarely been linked. An amino acid (isoleucine to lysine, or I462K) polymorphism of the couch potato (cpo) gene in Drosophila melanogaster is thought to control female reproductive diapause cline in North America ( Schmidt et al. 2008 , Proc Natl Acad Sci USA, 105, 16207–16211). Here, we show that under standard diapause‐inducing conditions (12 °C and short photoperiod) ( Saunders et al. 1989 , Proc Natl Acad Sci USA, 86, 3748–3752), egg maturation in Australian flies is delayed, but not arrested at previtellogenic stages. At 12 °C, the phenotypic distribution in egg development was bimodal at stages 8 and 14 and showed a strong nonlinear pattern on the east coast of Australia, with incidence of egg maturation delay (ovarian dormancy) increasing both toward tropical and temperate climates. Furthermore, we found no evidence for an association between the cpo I462K polymorphism and ovarian dormancy at either 12 or 10 °C (when egg maturation was often delayed at stage 7). Owing to strong linkage disequilibrium, the latitudinal cline in cpo allele frequencies was no longer evident once variation in the In(3R)P inversion polymorphism was taken into account. Our results suggest that the standard diapause‐inducing conditions (12 °C and short photoperiod) were not sufficient to cause the typical previtellogenic developmental arrest in Australian flies and that the cpo I462K polymorphism does not explain the observed delay in egg development. In conclusion, ovarian dormancy does not show a simple latitudinal cline, and the lack of cpo‐dormancy association suggests a different genetic basis to reproductive dormancy in North America and Australia.

Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia

Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations.