Molecular Characterization of a Lysozyme Gene and Its Altered Expression Profile in Crowded Beet Webworm (Loxostege sticticalis)

Abstract

There is growing evidence that insects living in high-density populations exhibit an increase in immune function to counter a higher risk of disease. This phenomenon, known as density-dependent prophylaxis, has been experimentally tested in a number of insect species. Although density-dependent prophylaxis is especially prevalent in insects exhibiting density-dependent phase polyphenism, the molecular mechanism remains unclear. Our previous study demonstrated that the antibacterial activity of lysozyme is important for this process in the beet webworm Loxostege sticticalis. In this study, a lysozyme cDNA from L. sticticalis was cloned and characterized. The full-length cDNA is 1078 bp long and contains an open reading frame of 426 bp that encodes 142 amino acids. The deduced protein possesses structural characteristics of a typical c-type lysozyme and clusters with c-type lysozymes from other Lepidoptera. LsLysozyme was found to be expressed throughout all developmental stages, showing the highest level in pupae. LsLysozyme was also highly expressed in the midgut and fat body. Elevated LsLysozyme expression was observed in L. sticticalis larvae infected by Beauveria bassiana and in larvae reared under crowding conditions. In addition, the expression level of LsLysozyme in infected larvae reared at a density of 10 larvae per jar was significantly higher compared to those reared at a density of l or 30 larvae per jar. These results suggest that larval crowding affects the gene expression profile of this lysozyme. This study provides additional insight into the expression of an immune-associated lysozyme gene and helps us to better understand the immune response of L. sticticalis under crowding conditions.

Fig 1. Nucleotide and deduced amino acid sequences of L. sticticalis lysozyme.

The nucleotides are numbered on the left from the first base of the cDNA. The deduced amino acid sequence is shown below the nucleotide sequence in single-letter code. The codons for initiation, termination, polyadenylation and the poly (A) tail are indicated in bold-italic.

Fig 2. Multiple sequence alignment of beet webworm lysozyme C with other Lepidoptera insect lysozymes.

The star symbols (☆) mark conserved cysteine residues; The 11 residues that compose the lysozyme catalytic cleft on the conserved LYZ1 domain are indicated with triangle (△). The two major residues, Glu⁵³ and Asp⁷¹, responsible for the puptative lysozyme catalytic site on conserved LYZ1 domain are marked with numbersign (#). Asterisks (*) indicate Ca²⁺ binding site on conserved domain LYZ1.

Fig 3. Phylogenetic tree of amino acid sequences of c-type lysozymes from different Insecta orders (Lepidoptera, Hemiptera, Hymenoptera, Coleoptera, Orthoptera and Diptera) was constructed by MEGA 4.0.

Gallus gallus lysozyme was used as the out-group. Bootstrap values are indicated in maximum evolution and minimum likelihood for each root, respectively. L. sticticalis lysozyme is written italic letters.

Fig 4. Expression patterns of LsLysozyme during life stages and in different tissues.

The expression profiles of LsLysozyme during developmental stages are depicted in (A). L1-L5, 1st to 5th-instar larvae. The tissue expression distribution of LsLysozyme is shown in (B). Data are shown as the mean ± SE, n = 3. Different letters indicate significant difference at P < 0.01.

Fig 5. The relative gene expression of LsLysozyme among larvae reared at densities of 1, 10 and 30 larvae per jar and before and after treatment with B. bassiana are shown in A and B, respectively.

The relative gene expression of LsLysozyme in fifth-instar larvae infected with B. bassiana is shown in C. Data are presented as the mean±SE, n = 3. Different characters denote significant difference at P < 0.01.