Optimization of a Diet for the Greater Wax Moth (Lepidoptera: Pyralidae) Using Full Factorial and Mixture Design.

Diet optimization is an important process to increase the efficiency of rearing insects and can be used to develop high-quality insects with specific fitness and life-history traits. Galleria mellonella (L.), the greater wax moth, is widely used in research, microbiology assays, as pet food, and host for biological control agents. Although artificial diets for G. mellonella have been researched and optimized for decades, preliminary tests indicated that the predominantly utilized G. mellonella diet could be improved to yield larger larvae with a short development time. We used a design of experiments (DOE) approach that incorporated multiple full factorial designs and a final mixture design to test the qualitative and quantitative effects of ingredients and their interactions on larval mass and survival. Analysis of 17 ingredient variations in 35 diet formulations yielded an optimized diet that supported high survival and 2.4-fold greater larval body mass than the standard rearing diet. This study demonstrates the importance and efficiency of statistical DOE in guiding the optimization of insect diets to improve traits that represent the quality and fitness of the reared insects.

Effect of antifungal agents on biological fitness of Lygus hesperus (Heteroptera: Miridae).

Artificial diets have become important components of rearing systems for insects that are used for research purposes and in commercial production. Because the rearing conditions for insects also provide ideal settings for mold growth, antifungal additives are often used to reduce diet contamination. However, the antifungal agents must not only be effective in mold suppression, they must also be safe to the target insects of the rearing programs. The toxicity of five commonly used antifungal agents (benzoic acid, formalin, methyl paraben, propionic acid, and sorbic acid) was tested using diet bioassays on Lygus hesperus Knight, and the effect on biological fitness was measured. Biological fitness was defined as total number of survivors, mean biomass (dry weight) accumulated per cage over the total treatment period, egg production, time to adult emergence, and time to start of egg laying. Methyl paraben and formalin were found to have significant negative effects on these measurements of biological fitness. Challenge tests to determine the ability of the antifungal agents to suppress mold growth when inoculated into the diet medium are currently in progress.

Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae).

Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.