Nobuyuki Inomata

Protein crosslinking by transglutaminase controls cuticle morphogenesis in Drosophila.

Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins—two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin—as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.

EVOLUTION OF THE RESPONSE PATTERNS TO DIETARY CARBOHYDRATES AND THE DEVELOPMENTAL DIFFERENTIATION OF GENE EXPRESSION OF ALPHA -AMYLASE IN DROSOPHILA

Intraspecific variation of α-amylase activity in D. melanogaster and D. immigrans, which is distantly related to D. melanogaster, and interspecific variation of α-amylase activity in 18 Drosophila species were examined. The amount of intraspecific variation of α-amylase activities measured in terms of coefficient of variation in D. melanogaster and D. immigrans was one-half and one-tenth or less, respectively, of the interspecific variation in 18 Drosophila species. We also surveyed the response patterns of α-amylase activity to dietary carbohydrates at the larval and adult stages. The levels of α-amylase activity depended on both repression by dietary glucose (glucose repression) and induction by dietary starch (starch induction). In general, our data suggest that glucose repression was conserved among species at both stages while starch induction was mainly observed in larvae, although the degree of the response depended on species. In D. lebanonensis lebanonensis and D. serrata, larvae expressed electrophoretically different α-amylase variants (isozymes) from those of adult flies. These results may suggest that the regulatory systems responsible both for the response to environment and developmental expression are different among species in Drosophila.

Evolutionary History and Mode of the amylase Multigene Family in Drosophila

Previous studies indicate that the tandemly repeated members of the amylase (Amy) gene family evolved in a concerted manner in the melanogaster subgroup and in some other species. In this paper, we analyzed all of the 49 active and complete Amy gene sequences in Drosophila, mostly from subgenus Sophophora. Phylogenetic analysis indicated that the two types of diverged Amy genes in the Drosophilamontium subgroup and Drosophilaananassae, which are located in distant chromosomal regions from each other, originated independently in different evolutionary lineages of the melanogaster group after the split of the obscura and melanogaster groups. One of the two clusters was lost after duplication in the melanogaster subgroup. Given the time, 24.9 mya, of divergence between the obscura and the melanogaster groups (Russo et al. 1995), the two duplication events were estimated to occur at about 13.96 ± 1.93 and 12.38 ± 1.76 mya in the montium subgroup and D. ananassae, respectively. An accelerated rate of amino acid changes was not observed in either lineage after these gene duplications. However, the G+C contents at the third codon positions (GC3) decreased significantly along one of the two Amy clusters both in the montium subgroup and in D.ananassae right after gene duplication. Furthermore, one of the two types of the Amy genes with a lower GC3 content has lost a specific regulatory element within the montium subgroup species and D.ananassae. While the tandemly repeated members evolved in a concerted manner, the two types of diverged Amy genes in Drosophila experienced frequent gene duplication, gene loss, and divergent evolution following the model of a birth-and-death process.

Phylogeny and the Evolution of the Amylase Multigenes in the Drosophila montium Species Subgroup

To investigate the phylogenetic relationships and molecular evolution of α-amylase (Amy) genes in the Drosophila montium species subgroup, we constructed the phylogenetic tree of the Amy genes from 40 species from the montium subgroup. On our tree the sequences of the auraria, kikkawai, and jambulina complexes formed distinct tight clusters. However, there were a few inconsistencies between the clustering pattern of the sequences and taxonomic classification in the kikkawai and jambulina complexes. Sequences of species from other complexes (bocqueti, bakoue, nikananu, and serrata) often did not cluster with their respective taxonomic groups. This suggests that relationships among the Amy genes may be different from those among species due to their particular evolution. Alternatively, the current taxonomy of the investigated species is unreliable. Two types of divergent paralogous Amy genes, the so-called Amy1- and Amy3-type genes, previously identified in the D. kikkawai complex, were common in the montium subgroup, suggesting that the duplication event from which these genes originate is as ancient as the subgroup or it could even predate its differentiation. Thc Amy1-type genes were closer to the Amy genes of D. melanogaster and D. pseudoobscura than to the Amy3-type genes. In the Amy1-type genes, the loss of the ancestral intron occurred independently in the auraria complex and in several Afrotropical species. The GC content at synonymous third codon positions (GC3s) of the Amy1-type genes was higher than that of the Amy3-type genes. Furthermore, the Amy1-type genes had more biased codon usage than the Amy3-type genes. The correlations between GC3s and GC content in the introns (GCi) differed between these two Amy-type genes. These findings suggest that the evolutionary forces that have affected silent sites of the two Amy-type genes in the montium species subgroup may differ.

Inferences of evolutionary history of a widely distributed mangrove species, Bruguiera gymnorrhiza, in the Indo-West Pacific region

Inference of genetic structure and demographic history is fundamental issue in evolutionary biology. We examined the levels and patterns of genetic variation of a widespread mangrove species in the Indo-West Pacific region, Bruguiera gymnorrhiza, using ten nuclear gene regions. Genetic variation of individual populations covering its distribution range was low, but as the entire species it was comparable to other plant species. Genetic differentiation among the investigated populations was high. They could be divided into two genetic clusters: the West and East clusters of the Malay Peninsula. Our results indicated that these two genetic clusters derived from their ancestral population whose effective size of which was much larger compared to the two extant clusters. The point estimate of speciation time between B. gymnorrhiza and Bruguiera sexangula was two times older than that of divergence time between the two clusters. Migration from the West cluster to the East cluster was much higher than the opposite direction but both estimated migration rates were low. The past Sundaland and/or the present Malay Peninsula are likely to prevent gene flow between the West and East clusters and function as a geographical or land barrier.

Population structure and demographic history of a tropical lowland rainforest tree species Shorea parvifolia (Dipterocarpaceae) from Southeastern Asia

Distribution of tropical rainforests in Southeastern Asia has changed over geo-logical time scale, due to movement of tectonic plates and/or global climatic changes. Shorea parvifolia is one of the most common tropical lowland rainforest tree species in Southeastern Asia. To infer population structure and demographic history of S. parvifolia, as indicators of temporal changes in the distribution and extent of tropical rainforest in this region, we studied levels and patterns of nucleotide polymorphism in the following five nuclear gene regions: GapC, GBSSI, PgiC, SBE2, and SODH. Seven populations from peninsular Malaysia, Sumatra, and eastern Borneo were included in the analyses. STRUCTURE analysis revealed that the investigated populations are divided into two groups: Sumatra-Malay and Borneo. Furthermore, each group contained one admixed population. Under isolation with migration model, divergence of the two groups was estimated to occur between late Pliocene (2.6 MYA) and middle Pleistocene (0.7 MYA). The log-likelihood ratio tests of several demographic models strongly supported model with population expansion and low level of migration after divergence of the Sumatra-Malay and Borneo groups. The inferred demographic history of S. parvifolia suggested the presence of a scarcely forested land bridge on the Sunda Shelf during glacial periods in the Pleistocene and predominance of tropical lowland rainforest at least in Sumatra and eastern Borneo.

Demographic history and interspecific hybridization of four Shorea species (Dipterocarpaceae) from Peninsular Malaysia inferred from nucleotide polymorphism in nuclear gene regions

Shorea acuminata Dyer, Shorea curtisii Dyer ex King, Shorea leprosula Miq., and Shorea parvifolia Dyer are dominant tree species in the tropical rainforest of Peninsular Malaysia, which experienced several climatic changes during Pleistocene. To investigate the current population structure and demographic history of these species, we analyzed levels and patterns of nucleotide polymorphism of the nuclear gene region PgiC. We also used sequence data of the GapC gene region obtained in our previous study. Negative Tajimas D values observed in both investigated gene regions for S. curti- sii, S. leprosula, and S. parvifolia implied that all three species have experienced population expansion events. Little to moderate levels of population differentiation in S. acuminata and S. curtisii suggested recent divergence of the investigated populations after postglacial colonization of the Peninsular Malaysia. In addition, some haplotypes were similar or identi- cal to haplotypes of the other species. The existence of such haplotypes could be partially explained by interspecific hy- bridization. Indeed, we found some putative hybrid individuals. Interspecific hybridization among closely related species might have contributed to the polymorphism of the investigated species.

Seasonal Changes in the Long-Distance Linkage Disequilibrium in Drosophila melanogaster

Seasonal environmental changes have the potential to influence the genetic structure of species with a short generation time, such as Drosophila. We previously found the seasonal change in linkage disequilibrium (LD) between the chemoreceptor (Cr) genes in a local Japanese population (Kyoto [KY]). This could be caused by fluctuation in the population size or selection in temporally heterogeneous environments or both. Here, we analyzed the scale of LD between 51 X-linked polymorphisms (10 Cr and 41 non-Cr gene markers) in the 2 seasonal samples from the KY population and an autumn sample from 106 localities in and around Japan (Ja03au). Many of the non-Cr genes have receptor function but fewer functional connections to each other. The magnitude of LD in Ja03au did not significantly differ from that in the KY autumn sample. The lack of local differentiation was confirmed in an autumn sample from another local Japanese population. On the other hand, the magnitude of LD was significantly larger in spring than in autumn in the 2 independent KY samples. This suggests that reduction in the population size during winter increased the magnitude of LD in spring in the mainland population in Japan. Long-distance LD could be a useful measure for assessing seasonal fluctuation in effective population size.

Evolution of the amylase isozymes in the Drosophila melanogaster species subgroup.

The relationship between the net charge ofmolecules and their mobility on electrophoresis wasanalyzed for Drosophila alpha-amylases. Most ofthe differences in electrophoretic mobility, 98.2%, canbe explained by the charge state. Therefore fivereference amino acid sites, which are informativeresidues for charge differences among amylase isozymes,were considered for the evolution of the isozymes in Drosophila melanogaster. Theamylase isozymes in D. melanogaster can beclassified into three groups, I (AMY1,AMY2, and AMY3-A), II(AMY3-B and AMY4), and III(AMY5, AMY6-A, andAMY6-B), based on the differences in the reference sites. The mostprimitive amylase in D. melanogaster was foundto belong to Group I, most likely the AMY2isozyme. Groups II and III could have been derived fromGroup I. These results were confirmed by the analysis of 38amino acid sites with charge differences inDrosophila.

Effect of nucleotide polymorphism in cis-regulatory and coding regions on amylase activity and fitness in Drosophila melanogaster

In natural populations of Drosophila melanogaster, there are many amylase (AMY) isozymes encoded by the duplicated genes, but their adaptive significance remains unclear. One approach to elucidate this issue is to understand the molecular basis of functional differences between the allelic classes. In this study, the effects of nucleotide polymorphism in 5′-flanking (cis-regulatory) and coding regions on AMY activity were examined, both on glucose and starch food media and in larvae and adults, using three chimeric Amylase (Amy) genes, Amyc111, Amyc161 and Amyfc661. In this notation, the first number in the superscript indicates the sequence of the 5′-flnaking region (similar to Amy1 or Amy6), the second number refers to the coding region and the third number to the 3′-flanking region. We found that effect of nucleotide polymorphism in the coding region differed between larvae and adults. In larvae, the coding sequence of the Amy6 allele resulted in higher AMY activity than that of Amy1 allele, indicating the post-transcriptional differences between them. The cis-regulatory region derived from the Amy6 allele resulted in higher AMY activity in both larvae and adults. Thus, two fitness components, developmental time and productivity, were measured to examine whether polymorphism in the cis-regulatory region between the two alleles has an effect on them, but no significant difference was detected. We raise the implications for the evolution of subfunctionalization in multigene families.