Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long-standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin-binding protein type 2 (TBT-bp2) domains. In this study, we examined the evolutionary dynamics of TBT-bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR-based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT-bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT-bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT-bp2 genes. In addition, a detailed comparison of Takifugu TBT-bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT-bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes.

The Fanconi anaemia (FA) pathway is responsible for interstrand crosslink (ICL) repair. Among the FA core complex components, FANCM is believed to act as a damage sensor for the ICL-blocked replication fork and also as a molecular platform for FA core complex assembly and interaction with Bloom's syndrome (BS) complex that is thought to play an important role in the processing of DNA structures such as stalled replication forks. In the present study, we found that in silkworms, Bombyx mori, a species lacking the major FA core complex components (FANCA, B, C, E, F, and G), FancM is required for FancD2 monoubiquitination and cell proliferation in the presence of mitomycin C (MMC). Silkworm FancM (BmFancM) was phosphorylated in the middle regions, and the modification was associated with its subcellular localization. In addition, BmFancM interacted with Mhf1, a histone-fold protein, and Rmi1, a subunit of the BS complex, in the different regions. The interaction region containing at least these two protein-binding domains played an essential role in FancM-dependent resistance to MMC. Our results suggest that BmFancM also acts as a platform for recruitment of both the FA protein and the BS protein, although the silkworm genome seems to lose FAAP24, a FancM-binding partner protein in mammals.

Small RNA-mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA-induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double-stranded RNA (dsRNA)-induced RNAi, whereas Ago3 and Siwi are involved in the Piwi-interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)-mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA-decapping enzyme 2 (Dcp2) protein in the Drosophila processing-bodies (P-bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P-bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori.

The AHA1 (activator of Hsp90 ATPase) family of proteins were exclusively conserved from yeast to humans, but little is known about their tissue distribution or biological function. In this study, a cDNA for a Bombyx mori AHA1 homologue, BmAHA1, was isolated from the testes of larvae on day 3 of the fifth instar using an mRNA differential display method. This cDNA encodes a protein with 341 amino acid residues. Gene expression studies revealed that BmAHA1 mRNA occurred prominently in the testes. In situ hybridization and immunostaining showed that the BmAHA1 mRNA signals were strongly detected in spermatogonial cells and primary spermatocytes at the fifth larval instar stage, whereas the BmAha1 protein was abundant in round and elongated spermatids at the pupal stage. The localization pattern of the accumulated protein in the elongated spermatids was reminiscent of that reported previously for microtubules, but the BmAha1 protein showed a decrease in apparent concentration during maturation process. The stage- and cell-specific expression indicated that BmAha1 might play a role in silkworm spermatogenesis, especially in postmeiotic differentiation.

Twenty bacteria were isolated from four ant lion larvae. The isolates were classified into three groups by biological characteristics. Since Group I, Group II and Group III were isolated from individual larvae Kuo1, Kuo3, 4 and Kuo2, respectively, with exception of one isolate Kuo2‐1, each ant lion tested had its own dominant bacterial flora. Groups I and II were closer to Serratia liquefaciens and Enterobacter cloacae, respectively, whereas Group III could not be identified by the test used. The phylogenetic analysis of GroEL amino acid sequences revealed that Group I, II and III were related to those of Serratia spp., E. cloacae and Salmonella spp. –Escherichia/Shigella spp., respectively. Among these groups, Group I was highly virulent against Bombyx mori and Periplaneta americana, and caused 100% mortality within 24 h. The other two groups (Group II and III) were avirulent to these insect species. The culture filtrate of Group I caused killing activity to B. mori larvae and the insecticidal substance was purified from culture filtrate of Group I bacterium. Since the insecticidal activity highly correlated with proteolytic activity in the chromatographies, Group I bacterium may secret insecticidal proteinase in vitro.