The armoured cuticle of the black soldier fly Hermetia illucens

Abstract

We characterise in detail the larval and pupal cuticle of the black soldier fly Hermetia illucens L. (Diptera: Stratiomyidae), a key insect species in circular economy. In particular, we focus on ultrastructure using scanning and transmission electron microscopy, material characterization and composition (elements and minerals) with confocal laser scanning microscope, energy dispersive X-ray microanalysis, powder X-ray diffraction and mechanical properties with nanoindentation measurements. Calcium carbonate crystallizes on the epicuticle as blocks of calcite in the pupal cuticle. Calcium carbonate granules are stored in two specialised Malpighian tubules. CaCO₃ is already present in the cuticle of young larval instars, but it is mainly in the form of amorphous calcium carbonate while the amount of calcite increases during larval development. The presence of calcite leads to cuticle hardening. Larval and pupal cuticles contain large amounts of resilin which guarantee cuticle flexibility.

Figure 1

H. illucens larval instars in light microscope (a) and in SEM ((b–e), back scattered electrons, brighter areas indicate presence of CaCO₃ owing to its higher atomic number composition material compared to cuticle; (f), secondary electrons). (a) from the left to the right: larva with a size of 2–3 mm, larva with a size of 5 mm, clear larva with a size of 10–15 mm, dark larva with a size of 10–15 mm (prepupa); (b) body surface of larvae of 2–3 mm, whose cuticle is still not divided in hexagonal tiles and the CaCO₃ does not cover completely the cuticle but forms small aggregates scattered on the cuticle; (c) body surface of the larvae of 5 mm with a cuticle separated in hexagonal tiles. CaCO₃ does not cover completely the cuticle but is localised at the centre of each tile forming three polygonal platelets in the centre; (d) prepupa body surface divided in hexagonal tiles fully covered by blocks of CaCO_3, which in their centre are crystallised in numerous juxtaposed polygonal platelets forming rosettes (arrows); (e) Puparium surface showing the same features of the prepupa; (f) prepupa body surface treated with HCl dissolving the blocks of CaCO₃. Note the cuticle with its hexagonal tiles with a depression in their centre (arrow head).

Figure 2

Cross sections of the cuticle of the larvae of 2–3 mm (a,b) and of the prepupa of H. illucens (c,d) at SEM (backscattered images) showing the CaCO₃ blocks (arrows) in longitudinal section. In the young larvae the blocks are not developed and CaCO₃ forms thin aggregates scattered on the cuticular surface while in the prepupa the CaCO₃ blocks covers the hexagonal cuticular tile filling its central depression (arrow heads). A thick cuticle (C) underlies the CaCO₃ blocks. H, hairs-like setae.

Figure 3

SEM image (a), spatial distribution of the most abundant elements represented by Ca (b), P (c) and Mg (d) and EDX analysis (e,f) on the body surface of the prepupa of H. illucens. Typical EDX spectrum (e,f) shows the presence of high amount of Ca (atomic percentage) both on the crystallised platelets forming rosettes on the blocks (e) and in the lateral portion of the blocks (f).

Figure 4

SEM image (a), spatial distribution of the most abundant elements represented by Ca (b), P (c) and Mg (d) and EDX analysis (e,f) on the prepupa blocks of H. illucens in longitudinal section. Typical EDX spectrum (e,f) shows that the amount of Ca (atomic percentage) is high compared with that of P and Mg both in the inner (e) and in the outer (f) portion of the block. A higher percentage of P and Mg is visible on the external portion of the block compared with its inner portion (e,f).

Figure 5

PXRD patterns collected on the young larvae (a), and the pupal exuviae (b) of BSF, the calculated one of calcite is reported in grey. In the young larvae the calcite typical reflections present a low intensity and broad peaks, due to the low crystallinity of the CaCO₃ also observed in the SEM analysis. In the pupal exuviae of BSF sample, the PXRD pattern shows the presence of crystalline calcite.

Figure 6

Sections of the cuticle of the prepupa of H. illucens in TEM. (a) Fine structure of a CaCO₃ blocks with their crystallised platelets (P) and of the underlining cuticle. G, groove separating the tiles in the cuticle. Ep, epicuticle; Pr, procuticle. The procuticle is darker (melanised) in correspondence of the depression of each tile forming electron dense cones just under each CaCO₃ block (arrow head). (b–d) Details of the blocks wrapped by a multi-layered membrane constituted of the most external epicuticular layers (arrow).

Figure 7

Confocal laser scanning micrographs of H. illucens cuticle. Prepupa cuticle, visualized from dorsal surface (a), ventral surface (b), section of the dorsal side (c) and section of the ventral side (d). The section of the dorsal side (d) was visualized by light microscopy (e), and increased in signal strength (f). Dorsal surface (g) and section of the dorsal side (h) of larva cuticle. Arrows point out the wide canals in the multilayered procuticle allowing the passage of CaCO₃ granules (see Fig. 8).

Figure 8

H. illucens prepupa after the moult in SEM (backscattered images). (a) just moulted larval body surface lacking the blocks of CaCO₃; (b) In some hours after the moults, in correspondence of the central depression, ACC spherical aggregates appear (arrow); (c,d) the ACC spherical aggregates (arrows) increase gradually their size and number; (e) Encrustations of CaCO₃ (arrow) covering the tiles; (f) Crystallised CaCO₃ polygonal platelets (arrow) forming rosettes appear after about 48 h from the moult.

Figure 9

Sections of the just secreted cuticle of the fifth larval instar of H. illucens about to moult to the prepupa stage (the old cuticle has been removed) in TEM. (a) New multi-layered procuticle (Pr) under the central portion of each tile interrupted by wide canals (arrows) allowing the passage of dark granules (arrow head) from the epithelial cells (EC) to the epicuticular surface (E); (b) Detail of a); (c,d) Pore canals with dark secretions visible just under the epicuticular layer.

Figure 10

H. illucens (second instar larva) Malpighian tubules in light microscopy (a) and in SEM ((b–e), back scattered electrons. (a) Four Malpighian tubules emerging from a common trunk (T), two of them represent the real Malpighian tubules (arrow head) while the other two (white in colour) represent two storage organs of CaCO₃ (arrows). Asterisk points their content made of lime; (b,d) Cross (b) and longitudinal (c) sections of dry larvae showing lime glands brighter compared with the surrounding structures, owing to presence of CaCO₃ granules; (e,f) Details of (b) showing the CaCO₃ spherical microgranules.

Figure 11

Mechanical properties of the cuticle of H. illucens. (a) Elastic modulus and (b) Hardness measured from the dorsal side of prepupa (PD, n = 5), the ventral side of prepupa (PV, n = 5), the dorsal side of larva (LD, n = 5) and the ventral side of the larva (LV, n = 5). Capital letters indicate significant differences (P < 0.05, Holm-Sidak one-way ANOVA).