Insect Antimicrobial Peptides: Advancements, Enhancements and New Challenges.

Several insects are known as vectors of a wide range of animal and human pathogens causing various diseases. However, they are also a source of different substances, such as the Antimicrobial Peptides (AMPs), which can be employed in the development of natural bioactive compounds for medical, veterinary and agricultural applications. It is well known that AMP activity, in contrast to most classical antibiotics, does not lead to the development of natural bacterial resistance, or at least the frequency of resistance is considered to be low. Therefore, there is a strong interest in assessing the efficacy of the various peptides known to date, identifying new compounds and evaluating possible solutions in order to increase their production. Moreover, implementing AMP modulation in insect rearing could preserve insect health in large-scale production. This review describes the current knowledge on insect AMPs, presenting the validated ones for the different insect orders. A brief description of their mechanism of action is reported with focus on proposed applications. The possible effects of insect diet on AMP translation and synthesis have been discussed.

Impact of the Diet on the Mortality and on Gene Expression of the Antimicrobial Peptide Tenecin 3 in Tenebrio molitor Larvae Infected by Beauveria bassiana.

Large-scale insect rearing can be subjected to microbial infections, leading to serious economic losses. When possible, the use of antibiotics is to be avoided for insects intended as feed or food and new strategies to preserve the health of the farmed insects are required. The effectiveness of insect immune system depends on several factors, including the nutritional composition of the diet. The possibility to modulate immune responses through the diet is currently a topic of great interest from an application point of view. We evaluated the effect of two different diets on the survival rate and gene expression of the antimicrobial peptide Tenecin 3 in uninfected and Beauveria bassiana-infected Tenebrio molitor L. larvae. A wheat bran diet, mixed 50% with brewers' spent grains, could positively influence the expression of Tenecin 3 gene when uninfected T. molitor is allowed to develop on such a substrate from early larval stages. Even if, in our trial, the diet with added brewers' spent grains could not reduce the mortality of the larvae infected with B. bassiana, higher transcriptional levels of the antifungal peptide were observed in insects fed this diet, depending on the timing of diet administration.

Immune Responses of the Black Soldier Fly Hermetia illucens (L.) (Diptera: Stratiomyidae) Reared on Catering Waste.

The black soldier fly (BSF), Hermetia illucens L. (Diptera: Stratiomyidae), has a great bioconversion potential and ability to develop on diverse substrates. Although the use of catering waste and food by-products containing meat and fish would reduce the footprint of the insect sector, to date, in Europe, their use is still facing legal obstacles for insects as food and feed. Since a major request from the EU insect sector is to diversify the spectrum of allowed substrates, and considering that variations in diet composition could influence insect immune responses, we evaluated the impact of different catering wastes on BSF prepupae immunity. Insects were reared on five diets: one based on feed for laying hens and four based on catering waste containing (i) fruits and vegetables; (ii) fruits, vegetables and bread; (iii) fruit, vegetables, bread and dairy products; (iv) fruits, vegetables, bread, meat and fish. The gene expression of two antimicrobial peptides (AMPs), one defensin and one cecropin, was assessed. Moreover, the hemolymph inhibitory activity against Escherichia coli DH5α and Micrococcus yunnanensis HI55 was evaluated using diffusion assays in solid media. The up-regulation of both AMPs' encoding genes was observed in insects fed a bread-added and dairy product-added diet. All hemolymph samples showed inhibitory activity against both bacteria, affecting the colony size and number. The obtained results show how catering waste positively influences the H. illucens immune system. The possibility of modulating AMP expression levels through the diet opens up new perspectives in the management of insect health in mass rearings.

Effect of the rearing diet on gene expression of antimicrobial peptides in Hermetia illucens (Diptera: Stratiomyidae).

Insect proteins have been proposed for human and animal food production. Safeguarding the health status of insects in mass rearing allows to obtain high-quality products and to avoid severe economic losses due to entomopathogens. Therefore, new strategies for preserving insect health must be implemented. Modulation of the insect immune system through the diet is one such strategy. We evaluated gene expression of two antimicrobial peptides (one defensin and one cecropin) in Hermetia illucens (L.) (Diptera: Stratiomyidae) reared on different diets. Analyses were performed on prepupae and 10-day-old larvae reared on cereal- and municipal organic waste-based diets and on only prepupae reared on a cereal-based diet supplemented with sunflower, corn, or soybean oil. The inclusion of sunflower oil at different points in the cereal-based diet was also evaluated. Moreover, diet-driven differences in the inhibitory activity of the hemolymph were tested against Escherichia coli DH5α and Micrococcus yunnanensis HI55 using diffusion assays in solid media. Results showed that a municipal organic waste-based diet produced a significant overexpression of antimicrobial peptides only in prepupae. Inclusion of vegetable oils caused an upregulation of at least one peptide, except for the corn oil. Higher expression of both genes was observed when sunflower oil was added 5 days before pupation. All hemolymph samples showed an inhibitory activity against bacteria colonies. Our results suggest that municipal organic waste-based diet and vegetable oil-added diet may successfully impact the immune system of H. illucens. Such alternatives may also exist for other species of economic interest.

In Vitro Effects of Leaf Extracts from Brassica rapa on the Growth of Two Entomopathogenic Fungi.

This study aimed to determine the inhibitive or stimulatory effects of leaf extracts from two Brassica rapa subspecies on the hyphal growth of two well-known entomopathogenic fungi, Cordyceps fumosorosea and Beauveria bassiana. Extract concentrations of 50, 25, and 10% w/v based on leaf fresh weight were prepared from turnip (B. rapa subspecies rapa) and bok choy (B. rapa subspecies chinensis) leaves. Each concentration was individually incorporated into potato dextrose agar plates for in vitro bioassays. The center of each plate was inoculated with 20 µL of a fungal suspension that was allowed 24 h to soak into the agar before sealing the plates and incubating them at 25 °C under a 14-h photophase. The fungal colony perimeter was marked 5 days after inoculation on two perpendicular lines drawn on the bottom of each plate. Radial colony growth was measured from 4 marks per plate 5, 10, and 15 days later. Radial growth rates for both fungi were 1.3-2.0 and 0.9-1.4 times faster with bok choy and turnip extracts, respectively, at the 25% and 50% concentrations compared to the no-extract control treatment. Therefore, bok choy and turnip leaf extracts can stimulate entomopathogenic fungus growth within 15 days. Biochemical compounds in the extracts include sesquiterpenes, α-copaene, ß-selinene, γ-gurjunene, calamenene, cubenene, and α-calacorene.

First Multi-Target Application of Exclusion Net in Nectarine Orchards: Effectiveness against Pests and Impact on Beneficial Arthropods, Postharvest Rots and Fruit Quality.

Over the past few years, there has been an increasing interest in the development of alternative pest control strategies to reduce environmental impact. In this contest, exclusion nets have been evaluated as a sustainable alternative to pesticides. In this study, the use of a photoselective exclusion net was investigated in semi-field conditions as a potential strategy to protect nectarine orchards from different pests (i.e., fruit moths, Halyomorpha halys and Drosophila suzukii) in NW Italy. The presence and abundance of pest populations inside and outside the net, as well as the damage they caused on fruits, were evaluated. Moreover, any possible effects of the net on beneficial arthropods, postharvest rots and fruit quality and nutraceutical parameters were considered. The exclusion net significantly reduced pest populations. At harvest, fruit damage caused by Grapholita molesta and H. halys in netted plots was reduced up to 90% and to 78%, respectively, compared with insecticide-treated plots. The exclusion net allowed the production of healthier fruits with a strong reduction of insecticide treatments (up to seven less) and of their related costs without any negative impact on postharvest rots, neither fruit quality nor nutraceutical properties.

Temporal Dynamics of 'Ca. Phytoplasma mali' Load in the Insect Vector Cacopsylla melanoneura.

The transmission of phytoplasmas is the result of an intricate interplay involving pathogens, insect vectors and host plants. Knowledge of the vector's competence during its lifespan allows us to define more sustainable well-timed control strategies targeted towards the most worrisome life stages. We investigated the temporal dynamics of 'Candidatus Phytoplasma mali' load in Cacopsylla melanoneura in the different developmental stages in Northwest Italy. The phytoplasma load in the vector was evaluated in overwintering adults, nymphs and newly emerged adults after different acquisition access periods. Moreover, we followed the multiplication of the phytoplasma during the aestivation and the overwintering period on conifers. Our results confirmed the ability of remigrants to retain the phytoplasma until the end of winter. We also highlighted the high acquisition efficiency and vector competence, based on phytoplasma load, of nymphs and newly emerged adults. Therefore, particular attention should be paid to the management of overwintered C. melanoneura as soon as they return to the orchards, but also to newly emerged adults, particularly in orchards with a high infection rate and when the migration to conifers is delayed.

Photoselective exclusion netting in apple orchards: effectiveness against pests and impact on beneficial arthropods, fungal diseases and fruit quality.

BACKGROUND: Frequent pesticide treatments in fruit orchards increase hazards for workers, consumers and the environment. Moreover, the indiscriminate and excessive use of pesticides often induces resistance in pests. In the past few years, physical exclusion strategies have been proposed as an alternative for the control of insect pests. The goal of this study was to evaluate the effectiveness of anti-hail photoselective netting in protecting apples against key and emerging pests, as well as the impact on beneficial arthropods, fungal diseases and fruit quality. RESULTS: In netted plots, a significant reduction in pest populations, i.e. fruit moths, Halyomorpha halys (Stål) and Drosophila suzukii (Matsumura), was recorded in comparison with un-netted controls. Moreover, the damage on fruits caused by H. halys was reduced up to 62% compared with insecticidal treatments. The net did not negatively affect the abundance of predators and the incidence of post-harvest rot. In addition, the incidence of bitter pit on apple was reduced up to 52%. Furthermore, fruit quality was unaffected by the net coverage (both at harvest and after 4 months of storage). CONCLUSION: Anti-hail photoselective pearl netting proved a promising exclusion system that can prevent attack by more than one insect pest at a time, allowing for a strong reduction in insecticide treatments and relative costs. At the same time, the netting did not negatively influence the presence of predators, the incidence of fungal disease or fruit quality. © 2019 Society of Chemical Industry.