Characterization of silk genes in Ephestia kuehniella and Galleria mellonella revealed duplication of sericin genes and highly divergent sequences encoding fibroin heavy chains.

Silk is a secretory product of numerous arthropods with remarkable mechanical properties. In this work, we present the complete sequences of the putative major silk proteins of E. kuehniella and compare them with those of G. mellonella, which belongs to the same moth family Pyralidae. To identify the silk genes of both species, we combined proteomic analysis of cocoon silk with a homology search in transcriptomes and genomic sequences to complement the information on both species. We analyzed structure of the candidate genes obtained, their expression specificity and their evolutionary relationships. We demonstrate that the silks of E. kuehniella and G. mellonella differ in their hydrophobicity and that the silk of E. kuehniella is highly hygroscopic. In our experiments, we show that the number of genes encoding sericins is higher in G. mellonella than in E. kuehniella. By analyzing the synteny of the chromosomal segment encoding sericin genes in both moth species, we found that the region encoding sericins is duplicated in G. mellonella. Finally, we present the complete primary structures of nine fibH genes and proteins from both families of the suborder Pyraloidea and discuss their specific and conserved features. This study provides a foundation for future research on the evolution of silk proteins and lays the groundwork for future detailed functional studies.

Functional Analysis of Adipokinetic Hormone Signaling in Bombyx mori.

Insect adipokinetic hormones (AKHs) are short peptides produced in the corpora cardiaca and are responsible for mobilizing energy stores from the fat body to the hemolymph. Three related peptides, AKH1, AKH2, and AKH/corazonin-related peptide (ACP) as well as three AKH receptors have been reported in Bombyx mori. AKH1 and AKH2 are specific for the AKHR1 receptor, whereas ACP interacts with the other two AKHRs. To assess the effect of the two silkworm AKHs and ACP in the regulation of energy homeostasis we examined the expression pattern of the three peptides and their receptors as well as their effect on the level of carbohydrates and lipids in the hemolymph. Our results support the hypothesis that only AKH1 and AKH2 peptides together with the AKHR1 receptor are involved in the maintenance of energy homeostasis. Because Bombyx AKHR1 (BmAKHR1) seems to be a true AKHR we generated its mutation. The BmAKHR1 mutant larvae display significantly lower carbohydrate and lipid levels in the hemolymph and reduced sensitivity to starvation. Our study clarifies the role of BmAKHR1 in energy homeostasis.

The expansion of genes encoding soluble silk components in the greater wax moth, Galleria mellonella.

Lepidopteran silk is a complex assembly of proteins produced by a pair of highly specialized labial glands called silk glands. Silk composition has been examined only in a handful of species. Here we report on the analysis of silk gland-specific transcriptomes from three developmental stages of the greater wax moth, Galleria mellonella, combined with proteomics, Edman microsequencing and northern blot analysis. In addition to the genes known earlier, we identified twenty seven candidate cDNAs predicted to encode secretory proteins, which may represent novel silk components. Eight were verified by proteomic analysis or microsequencing, and several others were confirmed by similarity with known silk genes and their expression patterns. Our results revealed that most candidates encode abundant secreted proteins produced by middle silk glands including ten sericins, two seroins, one or more mucins, and several sequences without apparent similarity to known proteins. We did not detect any novel PSG-specific protein, confirming that there are only three fibroin subunits. Our data not only show that the number of sericin genes in the greater wax moth is higher than in other species thus far examined, but also the total content of soluble proteins in silk is twice as high in G. mellonella than in B. mori or A. yamamai. Our data will serve as a foundation for future identification and evolutionary analysis of silk proteins in the Lepidoptera.

Targeted mutagenesis and functional analysis of adipokinetic hormone-encoding gene in Drosophila.

Adipokinetic hormones (Akhs) are small peptides (8-10 amino acid [aa] residues long) found in insects that regulate metabolic responses to stress by stimulating catabolic reactions and mobilizing energy stores. We employed Transcription activator-like effector nuclease (TALEN) mutagenesis and isolated an Akh(1) mutant carrying a small deletion in the gene that resulted in a truncated peptide; the second aa (Leu) was missing from the functional octapeptide. This null Dmel/Akh mutant is suitable to study Akh function without any effect on the C-terminal associated peptide encoded by the same gene. The mutant flies were fully viable and compared to the control flies, had significantly low levels of hemolymph saccharides including trehalose and were resistant to starvation. These characteristics are similar to those obtained from the flies carrying targeted ablation of Akh-expressing neurons (reported earlier). We also found that the Akh(1) mutants are slightly heavy and had a slow metabolic rate. Furthermore, we showed that the ectopic expression of Dmel∖Akh reverses the Akh(1) phenotype and restores the wild-type characteristics. Our results confirmed that Akh is an important regulator of metabolic homeostasis in Drosophila.