The alarming escalation of infectious diseases resistant to conventional antibiotics requires urgent global actions, including the development of new therapeutics. Antimicrobial peptides (AMPs) represent potential alternatives in the treatment of multi-drug resistant (MDR) infections. Here, we focus on Cecropins (Cecs), a group of naturally occurring AMPs in insects, and on synthetic Cec-analogs. We describe their action mechanisms and antimicrobial activity against MDR bacteria and other pathogens. We report several data suggesting that Cec and Cec-analog peptides are promising antibacterial therapeutic candidates, including their low toxicity against mammalian cells, and anti-inflammatory activity. We highlight limitations linked to the use of peptides as therapeutics and discuss methods overcoming these constraints, particularly regarding the introduction of nanotechnologies. New formulations based on natural Cecs would allow the development of drugs active against Gram-negative bacteria, and those based on Cec-analogs would give rise to therapeutics effective against both Gram-positive and Gram-negative pathogens. Cecs and Cec-analogs might be also employed to coat biomaterials for medical devices as an approach to prevent biomaterial-associated infections. The cost of large-scale production is discussed in comparison with the economic and social burden resulting from the progressive diffusion of MDR infectious diseases.

With approximately 160,000 identified species of butterflies and moths, Lepidoptera are among the most species-rich and diverse insect orders. Lepidopteran insects have fundamental ecosystem functions as pollinators and valuable food sources for countless animals. Furthermore, Lepidoptera have a significant impact on the economy and global food security because many species in their larval stage are harmful pests of staple food crops. Moreover, domesticated species such as the silkworm Bombyx mori produce silk and silk byproducts that are utilized by the luxury textile, biomedical, and cosmetics sectors. Several Lepidoptera have been fundamental as model organisms for basic biological research, from formal genetics to evolutionary studies. Regarding chronobiology, in the 1970s, Truman’s seminal transplantation experiments on different lepidopteran species were the first to show that the circadian clock resides in the brain. With the implementation of molecular genetics, subsequent studies identified key differences in core components of the molecular circadian clock of Lepidoptera compared to the dipteran Drosophila melanogaster, the dominant insect species in chronobiological research. More recently, studies on the butterfly Danaus plexippus have been fundamental in characterizing the interplay between the circadian clock and navigation during the seasonal migration of this species. Moreover, the advent of Next Generation Omic technologies has resulted in the production of many publicly available datasets regarding circadian clocks in pest and beneficial Lepidoptera. This review presents an updated overview of the molecular and anatomical organization of the circadian clock in Lepidoptera. We report different behavioral circadian rhythms currently identified, focusing on the importance of the circadian clock in controlling developmental, mating and migration phenotypes. We then describe the ecological importance of circadian clocks detailing the complex interplay between the feeding behavior of these organisms and plants. Finally, we discuss how the characterization of these features could be useful in both pest control, and in optimizing rearing of beneficial Lepidoptera.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen causing severe infections in hospitalized and immunosuppressed patients, particularly individuals affected by cystic fibrosis. Several clinically isolated P. aeruginosa strains were found to be resistant to three or more antimicrobial classes indicating the importance of identifying new antimicrobials active against this pathogen. Here, we characterized the antimicrobial activity and the action mechanisms against P. aeruginosa of two natural isoforms of the antimicrobial peptide cecropin B, both isolated from the silkworm Bombyx mori. These cecropin B isoforms differ in a single amino acid substitution within the active portion of the peptide, so that the glutamic acid of the E53 CecB variant is replaced by a glutamine in the Q53 CecB isoform. Both peptides showed a high antimicrobial and membranolytic activity against P. aeruginosa, with Q53 CecB displaying greater activity compared with the E53 CecB isoform. Biophysical analyses, live-cell NMR, and molecular-dynamic-simulation studies indicated that both peptides might act as membrane-interacting elements, which can disrupt outer-membrane organization, facilitating their translocation toward the inner membrane of the bacterial cell. Our data also suggest that the amino acid variation of the Q53 CecB isoform represents a critical factor in stabilizing the hydrophobic segment that interacts with the bacterial membrane, determining the highest antimicrobial activity of the whole peptide. Its high stability to pH and temperature variations, tolerance to high salt concentrations, and low toxicity against human cells make Q53 CecB a promising candidate in the development of CecB-derived compounds against P. aeruginosa.

The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. mori strains significantly enhances basic and applied silkworm research. In recent years, CRISPR/Cas9 technology is being rapidly adopted as the most efficient molecular tool for generating silkworm lines carrying mutations in target genes. Here we illustrate a complete and efficient workflow to screen, characterize rapidly and follow mutations through generations, allowing the generation of B. mori lines, stably inheriting single CRISPR/Cas9-induced mutations. This approach relies on the use of different molecular methods, the heteroduplex assay, cloning followed by Sanger sequencing, and the amplification refractory mutation system PCR. The use of these methodologies in a sequential combination allows the identification of CRISPR/Cas9-induced mutations in genes mapping on both autosomes and sex chromosomes, and the selection of appropriate individuals to found stable mutant B. mori lines. This protocol could be further applied to screen CRISPR/Cas9 mutations in haploid insects.

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