Insights Into the Immune Response of the Black Soldier Fly Larvae to Bacteria

Abstract

In insects, a complex and effective immune system that can be rapidly activated by a plethora of stimuli has evolved. Although the main cellular and humoral mechanisms and their activation pathways are highly conserved across insects, the timing and the efficacy of triggered immune responses can differ among different species. In this scenario, an insect deserving particular attention is the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae). Indeed, BSF larvae can be reared on a wide range of decaying organic substrates and, thanks to their high protein and lipid content, they represent a valuable source of macromolecules useful for different applications (e.g., production of feedstuff, bioplastics, and biodiesel), thus contributing to the development of circular economy supply chains for waste valorization. However, decaying substrates bring the larvae into contact with different potential pathogens that can challenge their health status and growth. Although these life strategies have presumably contributed to shape the evolution of a sophisticated and efficient immune system in this dipteran, knowledge about its functional features is still fragmentary. In the present study, we investigated the processes underpinning the immune response to bacteria in H. illucens larvae and characterized their reaction times. Our data demonstrate that the cellular and humoral responses in this insect show different kinetics: phagocytosis and encapsulation are rapidly triggered after the immune challenge, while the humoral components intervene later. Moreover, although both Gram-positive and Gram-negative bacteria are completely removed from the insect body within a few hours after injection, Gram-positive bacteria persist in the hemolymph longer than do Gram-negative bacteria. Finally, the activity of two key actors of the humoral response, i.e., lysozyme and phenoloxidase, show unusual dynamics as compared to other insects. This study represents the first detailed characterization of the immune response to bacteria of H. illucens larvae, expanding knowledge on the defense mechanisms of this insect among Diptera. This information is a prerequisite to manipulating the larval immune response by nutritional and environmental factors to increase resistance to pathogens and optimize health status during mass rearing.

Keywords: Hermetia illucens; cellular response; hemocytes; humoral response; immune system.

Figure 1

Analysis of the antimicrobial activity. (A, B) Humoral and cellular activity against Escherichia coli (A) and Micrococcus luteus (B). (C, D) Humoral antimicrobial activity against E. coli (C) and M. luteus (D). Values represent mean ± s.e.m. Different letters indicate statistically significant differences among treatments (One-Way ANOVA: A) F _4-10 = 91.65, p < 0.0001; B) F _4-10 = 74.62, p < 0.0001; C) F _4-10 = 52.36, p < 0.0001; D) F _4-10 = 4.694, p < 0.0216).

Figure 2

Hemocyte counts. Hemocyte counts of naïve larvae (Naïve) and larvae infected with 10⁵ CFU/ml E. coli/M. luteus mix (Injected) at different time points (6, 14, 24, and 48 hours after infection). Values represent mean ± s.e.m. Different letters indicate statistically significant differences among naïve and injected larvae (Unpaired Student’s t-test: for 6h t = 3.289, df = 5, p = 0.0217; for 14h t = 7.864, df = 5, p = 0.0005; for 24h t = 4.661, df = 4, p = 0.0096; for 48h t = 1.595, df = 4, p = 0.1859).

Figure 3

Analysis of phagocytosis. Hemocytes undergoing phagocytosis 5 minutes (A, B), 60 minutes (C, D), 14 hours (E, F), and 24 hours (G, H) after injection of Staphylococcus aureus (A, C, E, G) and Escherichia coli (B, D, F, H) conjugated with pHrodo. Arrows indicate phagocytosed bacteria. Bars: 10 µm.

Figure 4

Analysis of encapsulation. Effect of B-Agarose (A, E, I, M), B-Sephadex (B, F, J, N), CM-Sephadex (C, G, K, O), and DEAE-Sephadex (D, H, L, P) beads on encapsulation and melanization. Hemolymph was analyzed 2 (A–D), 4 (E–H), 14 (I–L), and 24 (M-P) hours after injecting the beads in the larvae. Arrows, adherent hemocytes; arrowheads, melanin deposition; B, beads. Bars: 40 µm.

Figure 5

Analysis of PO system activation. ΔAbs at 490 nm of phenoloxidase in (A) hemolymph of naïve larvae (Naïve) and larvae infected for 7, 30 and 60 minutes with 10⁵ CFU/ml of E. coli/M. luteus mix; (B) hemolymph isolated from naïve larvae (Naïve + Zym) and larvae infected for 7, 30, and 60 minutes with 10⁵ CFU/ml of E. coli/M. luteus, treated with Zymosan; (C) hemolymph isolated from larvae infected for 7 minutes with different concentrations of E. coli/M. luteus mix. Values represent mean ± s.e.m. Different letters indicate statistically significant differences among treatments (One Way ANOVA: A) F _3-8 = 41.54, p < 0.0001; B) F _3-8 = 22.95, p = 0.0003; C) F _3-8 = 122.7, p < 0.0001).

Figure 6

Lysozyme activity and HiLysozyme expression. (A) Lysozyme relative activity in the hemolymph of naïve and infected larvae at different time points. (B) HiLysozyme mRNA levels in hemocytes of naïve and infected larvae at different time points. Naïve larvae starved for 6 and 3 hours were used as controls for the activity and the expression of lysozyme, respectively. Values represent mean ± s.e.m. Different letters indicate statistically significant differences among treatments (One-Way ANOVA: A) F _4-10 = 128.1, p < 0.0001; B) F _4-10 = 107.2, p < 0.0001).

Figure 7

qRT-PCR analysis of antimicrobial peptides. (A, C) mRNA levels of HiDiptericin in the fat body (A) and hemocytes (C) of naïve and infected larvae at different time points; (B, D) mRNA levels of HiDefensin in the fat body (B) and hemocytes (D) of naïve and infected larvae at different time points. Naïve larvae starved for 3 hours were used as controls. Values represent mean ± s.e.m. Different letters indicate statistically significant differences among treatments (One-Way ANOVA: A) F _5-12 = 178.8, p < 0.0001; B) F _5-12 = 127, p < 0.0001; C) F _4-10 = 532.6, p < 0.0001; D) F _4-10 = 658.3, p < 0.0001).

Figure 8

Schematic representation of cellular and humoral responses in H. illucens larvae and activation timing.