Genomic analysis of carboxyl/cholinesterase genes in the silkworm Bombyx mori

Abstract

Background: Carboxyl/cholinesterases (CCEs) have pivotal roles in dietary detoxification, pheromone or hormone degradation and neurodevelopment. The recent completion of genome projects in various insect species has led to the identification of multiple CCEs with unknown functions. Here, we analyzed the phylogeny, expression and genomic distribution of 69 putative CCEs in the silkworm, Bombyx mori (Lepidoptera: Bombycidae).

Results: A phylogenetic tree of CCEs in B. mori and other lepidopteran species was constructed. The expression pattern of each B. mori CCE was also investigated by a search of an expressed sequence tag (EST) database, and the relationship between phylogeny and expression was analyzed. A large number of B. mori CCEs were identified from a midgut EST library. CCEs expressed in the midgut formed a cluster in the phylogenetic tree that included not only B. mori genes but also those of other lepidopteran species. The silkworm, and possibly also other lepidopteran species, has a large number of CCEs, and this might be a consequence of the large cluster of midgut CCEs. Investigation of intron-exon organization in B. mori CCEs revealed that their positions and splicing site phases were strongly conserved. Several B. mori CCEs, including juvenile hormone esterase, not only showed clustering in the phylogenetic tree but were also closely located on silkworm chromosomes. We investigated the phylogeny and microsynteny of neuroligins in detail, among many CCEs. Interestingly, we found the evolution of this gene appeared not to be conserved between B. mori and other insect orders.

Conclusions: We analyzed 69 putative CCEs from B. mori. Comparison of these CCEs with other lepidopteran CCEs indicated that they had conserved expression and function in this insect order. The analyses showed that CCEs were unevenly distributed across the genome of B. mori and suggested that neuroligins may have a distinct evolutionary history from other insect order. It is possible that such an uneven genomic distribution and a unique neuroligin evolution are shared with other lepidopteran insects. Our genomic analysis has provided novel information on the CCEs of the silkworm, which will be of value to understanding the biology, physiology and evolution of insect CCEs.

Figure 1

Phylogenetic tree of CCEs. MEGA 4.0 [40] was used to construct the phylogenetic tree using the Minimum Evolution method. Asterisks in the cladogram indicate bootstrap values greater than 50%. The nomenclatures of clades or groups are according to Teese et al [23]. Subclades of CCEs showing 1:1 orthologous relationship are marked red. The CCEs of B. mori are colored light blue, H. armigera blue, S. littoralis red and E. postvittana purple. The tissues in which each CCE is expressed are shown to the right of CCE name. For B. mori CCEs, the number of EST clones identified by the database analysis is shown in parenthesis. Species name abbreviations: Ha, Helicoverpa armigera; Hv, Heliothis virescens; Bm, Bombyx mori; Ep, Epiphyas postvittana; Sl, Spodoptera littoralis; Snon, Sesamia nonagrioides; Apol, Antheraea polyphemus; Mbra, Mamestra brassicae; Ms, Manduca sexta; Cf, Choristoneura fumiferana. Abbreviations for tissues: mg, midgut; sg, silk gland; ov, ovary; he, hemocyte; fb, fat body; ep, epidermis; ant, antenna; phe, pheromone gland; br, brain; wg, wing; tho, thorax; abd, abdomen; ca, corpora allata; mxg, maxillary galea; prob, proboscis; pr, prothoracic gland; ce, compound eye; te, testis; Vg, Verson's gland; Mt, Malpighian tubules.

Figure 2

Phylogenetic tree and intron positions of silkworm CCE genes. Asterisks in the cladogram indicate bootstrap values greater than 50%, and the nomenclatures of clades are according to Teese et al [23]. The intron position of sequences is shown as (|) for a phase 0 intron, ([) for a phase 1 intron and (]) for a phase 2 intron. The arrow indicates the phase 0 intron at position 229 or 230, while the arrowhead shows the phase 2 intron at position 1368. The green brackets indicate the phase 1 introns at positions 792 and 861 shared among CCEs in clades 024-026 and 030, orange brackets the phase 1 intron at position 1022 shared among CCEs in clades 013 and 024-026, purple bars the phase 0 intron at position 1165 shared among CCEs in clade 030 and red brackets the phase 2 introns at position 787 and 865 shared among CCEs in clade 020.

Figure 3

The distribution of the intron length in B. mori CCEs.

Figure 4

Chromosomal locations of silkworm CCE genes. The length of each chromosome is drawn to scale. CCEs that are clustered on chromosomes and the phylogenetic tree (Figures. 1 and 2) are colored purple. CCE019a, 024c and 029a were located in a scaffold whose chromosomal location is unknown and is not shown on this figure.

Figure 5

The organization of the CCE cluster around CCE020 and 016 in the silkworm genome. Six CCEs are clustered around CCE020a on chromosome 25, along with one additional gene (BGIBMGA000789) (A); four CCEs are in a cluster on chromosome 23 (B). The arrows indicate the direction of transcription. CCEs are shown as purple arrows, while other genes are indicated with black arrows.

Figure 6

Phylogenetic and microsynteny analysis of neuroligin genes. (A) The phylogenetic tree of B. mori, D. melanogaster, A. aegypti and A. mellifera neuroligins. Asterisks in the cladogram indicate bootstrap values greater than 50%. For the neuroligins except B. mori, refer to Claudianos et al [2]. Neuroligins of B. mori are colored light blue, D. melanogaster are colored green, A. gambiae are colored brown and A. mellifera are colored orange. (B) The chromosomal locations of B. mori neuroligins. The direction of the arrow indicates the transcription direction. The color of each neuroligin arrow is the same as that for orthologs in Claudianos et al [2].