Postembryonic development of H. illucens.

(A) The overall program of postembryonic development, including the feeding larval stage (L1D0 to L6D0), the non-feeding, wandering 7th instar prepupal stage (PPD0), the pupal stage (PD0), and the adult (AD0). (B) Intra-puparial development of H. illucens, including the cryptocephalic pupa (early PD0), the phanerocephalic pupa (mid-late PD0), the pharate adult (PD1 to PD8), and the newly emerged adult (from PD9 to AD0). L1D0 to PPD0, and AD0 are presented in dorsal view, and the other photos are presented in ventral view. The same individual is exhibited from PD1 to PD9 (AD0).

Expression profiles of key metamorphic genes in H. illucens.

(A) Aligned expression profile of genes encoding four key metamorphic genes in H. illucens: Kr-h1, Chinmo, Br-c and E93. (B-E) Separated expression profiles of (B) Kr-h1, (C) Chinmo, (D) Br-c, and (E) E93. Relative expression level was calculated using the 2-ΔΔCt method, with Actin-5C as the reference gene. All data are shown in mean ± SE (n = 3).

Knockdown of Kr-h1 or Chinmo triggers precocious metamorphosis in H. illucens.

(A) Dorsal view of the control (dsmalE treated) prepupa, the Kr-h1i precocious prepupa, and the Chinmoi precocious prepupa. (B) Body weight comparison of the control (dsmalE treated) prepupa (n = 58), the Kr-h1i precocious prepupa (n = 25), and the Chinmoi precocious prepupa (n = 34). (C) Head capsule width comparison of the control (dsmalE treated) prepupa (n = 58), the Kr-h1i precocious prepupa (n = 25), and the Chinmoi precocious prepupa (n = 34). (D-G) Fold changes of relative expression levels in key metamorphic genes: Kr-h1 (D), Chinmo (E), Br-c (F) and E93 (G) measured 14 days after dsRNA treatment (mean ± SE, n = 6 for each group). The average relative expression level of the control group was normalized to 1 for analysis. Scale bar: 2000 μm. One-way ANOVA with Tukey’s test was performed for (B) and (C); groups labeled with different letters are significantly different (p < 0.05). One-way ANOVA followed by Dunnett’s multiple comparisons test against the control group was performed for (D) to (G); Asterisks denote statistical significance: p < 0.05 (*), and p < 0.01 (**).

Double knockdown of Kr-h1 and Chinmo triggers precocious metamorphosis and results in PP-like L5 larvae in H. illucens.

(A) Dorsal view of the control (dsmalE treated) prepupa and the Kr-Chmi precocious prepupa. (B) Body weight comparison of the control (dsmalE treated) prepupa (n = 58) and the Kr-Chmi precocious prepupa (n = 33). (C) Head capsule width comparison of the control (dsmalE treated) prepupa (n = 58) and the Kr-Chmi precocious prepupa (n = 33). (D) Morphological comparisons among the prepupal-like L5 larva, the control L5 larva and the control prepupa. Dorsal view of the posterior abdominal segments reveals prepupal-like epidermal phenotype in Kr-Chmi L5 larva. (E-H) Fold changes of relative expression levels in key metamorphic genes: Kr-h1 (E), Chinmo (F), Br-c (G) and E93 (H) measured 14 days after dsRNA treatment (mean ± SE, n = 6 for each group). The average relative expression level of the control group was normalized to 1 for analysis. Scale bars: 2000 μm for the whole body, and 1000 μm for body parts. An unpaired Student’s t-test was used to compare the two groups in (B), (C), (E-H). Asterisks denote statistical significance: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Knockdown of Br-c impedes prepupal-pupal metamorphosis in H. illucens.

(A) Phenotypes obtained from dsmalE-injected control individuals and dsBr-c-injected RNAi individuals (Br-ci): phanerocephalic pupa (CPP/PD0), pharate adult (PD2), and Br-ci phase I-V individuals. Phase I: pharate adult wings were expanded but flat, attaching to the 1st and 2nd abdominal segments, and terminal abdominal projections were incompletely developed; Phase II: overall development was arrested at phanerocephalic pupal stage. Eyes were incompletely developed, legs were formed but unextended, wings were partially expanded but flat, attaching to the metathorax and the 1st abdominal segment. Abdominal respiratory tubes were incompletely formed, and the abdominal terminal segment was flat and larval-like. Phase III: overall development was arrested at the cryptocephalic pupal stage. Head and legs were differentiated but undeveloped, wings were partially expanded but small and flat, attaching to the metathorax. Abdominal respiratory tubes were unformed. Phase IV: less differentiated than phase III, the head and legs were almost undifferentiated, and the wing buds were unexpanded. Phase V: overall development was arrested at the phanerocephalic pupal stage, similar to phase II, except that wings failed to reach the 1st abdominal segment. Prepupal-like setae appeared on each abdominal segment. (B-E) Fold changes of relative expression levels in key metamorphic genes: Kr-h1 (B), Chinmo (C), Br-c (D), and E93 (E) measured 7 days after dsRNA treatment (mean ± SE, n = 6 for each group). The average relative expression level of the control group was normalized to 1 for analysis. Important morphological characteristics described are labeled with white arrowheads, and prepupal-like setae are indicated by white arrows. Scale bars: 2000 μm. An unpaired Student’s t-test was used to compare the two groups in (B-E). Asterisks denote statistical significance: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Knockdown of E93 inhibits adult formation in H. illucens.

(A) Phenotypes obtained from dsmalE-injected control individuals (normal pupa at PD8) and dsE93-injected RNAi individuals (E93i phase I-V). Phase I: all tissues normally developed except slightly smaller compound eyes in some individuals. Pupae failed to complete eclosion and were trapped in the puparium. Phase II: all tissues were generally well developed, except that the compound eyes were markedly reduced in size. An ectopic swelling appeared between the antennal bases. Phase III: body pigmentation was restricted to antennae, part of the eyes, and wings. Pigmented range in eyes was significantly small, wings were partially expanded but flat, attaching to the metathorax and the 1st abdominal segment. Phase IV: Body pigmentation was generally absent. Antennae were curved, and wings were partially expanded but flat and transparent. Phase V: Body pigmentation was totally absent. The head was incompletely developed, and the wings were partially expanded but flat and transparent. Legs were incompletely extended, with the posterior end attaching to the apex of the wings. (B-E) Fold changes of relative expression levels in key metamorphic genes: Kr-h1 (B), Chinmo (C), Br-c (D) and E93 (E) measured at PD3 (mean ± SE, n = 6 for each group). Important morphological characteristics described are labeled with white arrowheads. Scale bars: 2000 μm. An unpaired Student’s t-test was used to compare the two groups in (B-E). Asterisks denote statistical significance: p < 0.05 (*), and p < 0.01 (**).

Postembryonic developmental model and schematic diagrams of molecular mechanisms underlying growth and metamorphosis in Hermetia illucens.

(A) The Postembryonic developmental model of H. illucens in comparison with D. melanogaster. Solid lines between stages indicate stage transitions achieved through molting or pupation, whereas dotted lines indicate stage transitions achieved without molting. The model of D. melanogaster is adapted from (Martín et al., 2021). (B-G) Schematic diagrams of the molecular mechanism underlying growth and metamorphosis in H. illucens. (B) Larval stage maintenance: a high JH titer in the blood induces the expression of Kr-h1, and possibly Chinmo as well, as a secondary effect. The Kr-h1 protein suppresses the expression of Br-c and E93, with Chinmo also suppressing Br-c, thereby maintaining the larval status. (C) Prepupa formation: 20E signaling induces gradual expression of Br-c in the final instar larva. The coexistence of both JH and 20E signaling triggers the larval-prepupal metamorphosis. (D) Prepupa maintenance: Br-c expression is maintained at a high level throughout the prepupal stage. The JH signal is low but still present, inducing the low-level Chinmo and Kr-h1 expression. (E) Pupa formation: The loss of Kr-h1 and Chinmo eliminates the prepupal stage maintenance effect, allowing 20E to induce a gradually increased expression of E93. The function of Br-c plays a major role in inducing the prepupal-pupal metamorphosis. (F) Adult formation: the elevated presence of E93 protein during the pupal stage represses Br-c expression and adult tissue development, triggering pupal-adult metamorphosis. (G) Adult development: the JH signal reappears in adults, inducing the upregulation of Kr-h1, whereas Chinmo expression remains strongly repressed. The coexistence of Kr-h1 and E93 together promotes adult development and maturation.

Developmental changes in head capsule width across larval and prepupal stages in H. illucens (mean ± SD, n = 60).

Head morphology of H. illucens final instar larva and prepupa.

(A) Head morphology of the final instar larva (L6D0). (B) Head morphology of the prepupa (PPD0). Scale bars: 200 μm. Abbreviations: e – eye, eph – epipharynx, mdr – mandibular ridges, mxb – maxillary brush, mxp – maxillary palp, mxr maxillary ridge.

Expression profiles of genes encoding JH and ecdysone receptors.

(A) Aligned expression profile of genes encoding JH and ecdysone receptors: Met and EcR. (B) Expression profile of Met. (C) Expression profile of EcR. Relative expression level was calculated using the 2-ΔΔCt method, with Actin-5C as the reference gene. All data are shown in mean ± SE (n = 3).

Expression profiles of genes encoding key degradation enzymes of JH and 20E.

(A) Aligned expression profile of genes encoding key degradation enzymes of JH and 20E: JHE and Cyp18a1. (B) Expression profile of JHE. (C) Expression profile of Cyp18a1. Relative expression level was calculated using the 2-ΔΔCt method, with Actin-5C as the reference gene. All data are shown in mean ± SE (n = 3).

Knockdown efficiency analysis of dsKr-h1 and dsChinmo in H. illucens larvae.

(A) Knockdown efficiency analysis of dsKr-h1. (B) Knockdown efficiency analysis of dsChinmo. L4D0 larvae were injected with 2.0 μL of dsRNA solution, and collected at L4D2 for qRT-PCR analysis (mean ± SE, n = 6 for each group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Different letters indicate significant differences among groups (p < 0.05).

Knockdown efficiency analysis of dsBr-c in H. illucens prepupae.

PPD0 larvae were injected with 5.0 μL of dsRNA solution, and collected at PPD2 for qRT-PCR analysis (mean ± SE, n = 6 for both groups). An unpaired Student’s t-test was used to compare the two groups. Asterisks denote statistical significance: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Morphological comparisons between the Br-ci phase V arrested pupa and the control (dsmalE treated) prepupa’s abdomen.

(A) ventral view of the Br-ci phase V individual’s 1st to 3rd abdominal segments (left), and the enlarged image of the abdominal setae on the Br-ci phase V individual’s 2nd abdominal segment (right). (B) ventral view of the control prepupa’s 1st to 3rd abdominal segments, and the enlarged image of the abdominal setae on the control prepupa’s 2nd abdominal segment (right). Scale bars: 500 μm for abdominal segments (left), and 200 μm for enlarged images (right).

Knockdown efficiency analysis of dsE93 in H. illucens pupae.

PPD7 individuals were injected with 5.0 μL of dsRNA solution, and collected at PPD9 (mean ± SE, n = 6 for both groups). An unpaired Student’s t-test was used to compare the two groups. Asterisks denote statistical significance: p < 0.05 (*), and p < 0.01 (**).

Morphological comparisons between the E93i phase III arrested pupa and the control (dsmalE treated) pupa.

(A) Ventral view of the control pupa at PD2 with amber eyes. (B) Ventral view of the E93i phase III arrested pupa at PD14: only a restricted area of the eyes was pigmented amber color; wings were partially expanded but flat, showing pigmentation in black like mature pupa; black pigmentation was also observed in legs from femora to tibiae. (C) Enlarged image of the head of the E93i phase III arrested pupa. (D) Enlarged image of the thorax and the 1st abdominal segment of the E93i phase III arrested pupa. Scale bars: 2000 μm for the whole body, and 500 μm for body parts.

Intra-puparial developmental stages of black soldier fly.

Statistical results of dsKr-h1 or dsChinmo treatment to L4D0 larvae.

Statistical results of simultaneous dsKr-h1 and dsChinmo treatment to L4D0 larvae.

Statistical results of dsBr-c treatment to PPD0 (or PPD0∼6) prepupae.

Statistical results of dsE93 treatment to PPD7 (or PPD6∼10) prepupae.

Composition of the artificial diet per liter.

Insect body parts and the amount of TRIzol reagent used for total RNA extraction.

Primers used for qRT-PCR in the present study.

Primers used for dsRNA synthesis in the present study.