Transcriptomic survey of the midgut of Anthonomus grandis (Coleoptera: Curculionidae)

Abstract

Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase, and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information.

Keywords: 454 pyrosequencing; cotton pest; midgut expressed gene.

Fig. 1.

Gene ontology (GO) assignments for A. grandis larval midgut transcriptome. GO terms distribution by (A) molecular functions at level 2, (B) biological processes at level 2 and (C) cellular component at level 2. The number shows the percentage of GO terms included in each. One sequence could be associated with more than one GO term at the same time.

Fig. 2.

Diversity and relationships of A. grandis cathepsins. Homologous nucleotide sequences (>300 pb) to Cathepsins obtained from A. grandis midgut were aligned and used to analyze a phylogenetic tree in order to know the diversity within each class and between classes.

Fig. 3.

Multiple alignments of A. grandis cathepsins and dendrogram. Cathepsin-L from A. grandis (Contig 230, accession number JR948171.1) and representatives organisms were individually aligned. Phylogenetic tree was inferred with MEGA 5 program. (A) The C1A domain is underlined and the catalytic sites are marked with an asterisk. Propeptide inhibitor domain is double underlined. Identical residues are boxed with dark shading. (B) Phylogenetic tree showing relationships between Cathepsins L. from organisms of different orders included in Insecta Class: Tribolium castaneum (NP_001164088.1), N. vitripennis (XP_001605879.1), Bombus terrestris (XP_003402785.1), Periplaneta americana (BAA86911.1), Camponotus floridanus (EFN68284.1), Drosophila melanogaster (NP_620470.1), Drosophila mojavensis (XP_002008774.1), Anopheles gambiae str. PEST (XP_307325.4), Glossina morsitans morsitans (ABC48937.1), Sarcophaga peregrina (BAA76272.1), Solenopsis invicta (EFZ13575.1), Aedesaegypti (XP_001657758.1), Culex quinquefasciatus (XP_001867470.1), Harpegnathos saltator (EFN82144.1), Plautia stali (BAF94153.1), Pediculus humanus corporis (XP_002425065.1), and Manduca sexta (CAX16636.1). Node support is indicated by bootstrap values.

Fig. 4.

Carbohydrate metabolism. Major metabolic pathways associated with sugars were analyzed indicating the enzymes found in the A. grandis intestine and their relative abundance. (A) and (B) were used as reference the Keggs terms of each metabolic pathway. (C) variety of cellulases found and their relative abundances.

Fig. 5.

Microsatellite. Distribution of most abundant microsatellite motifs indicating the number of scorings.