Molecular mechanisms underlying the prepupal-instar-forming metamorphosis in black soldier fly (Hermetia illucens)

Joint Public Review

Summary:

In this study, the authors investigated the developmental and molecular basis of the unusual metamorphic program of the black soldier fly, Hermetia illucens, which differs from the canonical holometabolous life cycle by inserting a distinct, non-feeding prepupal instar between the final larval stage and pupation. Most insects that undergo complete metamorphosis molt to the final instar and then develop into the prepupal stage without molting. H. illucens, however, undergoes a molt before entering a non-feeding prepupal stage. Thus, it is an unusual, novel developmental strategy, and its regulation has remained a mystery. Through an integrated approach combining detailed morphological characterization, developmental gene expression profiling, and RNAi-mediated functional analyses of the core components of the Metamorphic Gene Network (MGN), the authors examine the developmental identity of this prepupal stage and how the temporal deployment of conserved metamorphic regulators has been reorganized to accommodate this atypical developmental program. In particular, they show that the prepupal stage expresses a distinct combination of the key genes known to regulate life history transitions, including unusually high levels of Br-C expression.

Strengths:

The study represents a valuable contribution to insect developmental biology. A major strength is the comprehensive characterization of postembryonic development, which establishes a robust developmental framework for H. illucens. This is complemented by detailed expression profiling and RNAi-based functional analyses of the Metamorphic Gene Network (MGN), comprising the temporal specifier factors, Kr-h1, chinmo, Br-C, and E93. The results show that these conserved regulators are deployed in a modified temporal sequence that accommodates the distinctive prepupal stage while largely preserving their canonical developmental functions. Together, the morphological, molecular, and functional data support the conclusion that the prepupal stage of H. illucens is a distinct developmental transition associated with a characteristic configuration of the metamorphic gene network. The results are supported by solid methodology and approaches and will serve as valuable resources for future investigations into insect development, the evolution of metamorphosis, and the diversification of insect life-history strategies.

Weaknesses:

While the study successfully establishes the developmental identity of the prepupal stage and its association with a modified temporal deployment of the MGN, some aspects of the proposed regulatory model are less directly supported by the experimental evidence.

(1) Several regulatory interactions within the MGN remain inferential rather than experimentally demonstrated in H. illucens. In particular, the proposed relationship between juvenile hormone (JH), Kr-h1, and chinmo is based primarily on expression dynamics and RNAi-induced transcriptional changes. Although these observations are consistent with the proposed model, they do not directly demonstrate that JH induces chinmo expression or establish the regulatory relationship between Kr-h1 and chinmo in this species. As a result, the corresponding regulatory interactions presented in the final model should be regarded as plausible hypotheses rather than experimentally validated mechanisms.

(2) A second limitation concerns the developmental role assigned to Br-C and E93 during the larval-to-prepupal transition. The authors conclude that sustained Br-C expression is a defining molecular feature of the prepupal stage and discuss its potential role in prepupal specification. However, the functional analyses of both Br-C and E93 were initiated only after larvae had already entered the prepupal stage. Consequently, while the RNAi experiments convincingly demonstrate essential roles for Br-C during the prepupal-to-pupal transition and for E93 during adult differentiation, they do not directly address whether either factor is required to trigger the formation of the prepupal stage itself. Therefore, the molecular mechanisms governing the initiation of this distinctive developmental transition remain unresolved. In particular, the proposed lack of repression of E93 by Br-C is only weakly supported, yet may be an essential feature of the prepupal stage of Hermetia illucens.

(3) Although knockdowns of Kr-h1 and chinmo knockdowns look superficially similar, it would be good to confirm this with higher-magnification views of the cuticles for all three treatments (control, Kr-h1 RNAi, and chinmo RNAi). In other species, Kr-h1 knockdown leads to premature adult cuticle development, whereas chinmo knockdown typically leads to premature appearance of pupal characteristics. Similarly, in Fig. 4A and 4D, higher-magnification images of the cuticle would be helpful.

(4) (Relating to Line 336 and Figure 7): "This low but persistent prepupal Kr-h1 expression, together with modest chinmo expression from PPD0 to PPD8, may be correlated to a JH-dependent antimetamorphic effect that maintains the prepupal stage." However, we are not aware of a function of JH in extending the prepupal stage. In addition, in most insects, the prepupal stage expresses high Kr-h1 expression; this peak likely prevents the animal from turning into an adult instead of the pupa. We presume the same holds true for H. illucens (although the lower expression of Kr-h1 during that stage is curious). As a result, we suggest that Fig. 7D be revised as it may be difficult to distinguish between pupal formation and prepupal maintenance given the experimental set-up. Fig. 7E may also need to be modified since the development of the pupa may require Kr-h1. It is worth noting that at the prepupal stage, JH and Br-C are co-expressed in many insects. If the authors think that Kr-h1 expression needs to be low at this time, this would imply a novel interaction between Kr-h1 and Br-C, and should be discussed.

Author response:

We thank the editors and reviewers for their careful evaluation and constructive suggestions. During the review process, we identified and corrected several presentation, terminology, citation, and figure-legend errors, and these corrections have been incorporated into the current version of preprint. Following the reviewers’ comment, we have also revised the title of the manuscript. We are now preparing a substantive revision that will distinguish more clearly between experimentally supported conclusions and hypothetical regulatory relationships. We plan to examine gene expression at an earlier time point after Br-c knockdown, further investigate the relationships among Kr-h1, chinmo, and E93 using additional RNAi experiments, characterize the cuticular phenotypes of precocious prepupae at higher magnification, and determine whether severe E93-knockdown individuals exhibit evidence of a repeated pupal developmental program. After completing these experiments, we will cautiously revise the proposed regulatory model and moderate conclusions that are not directly supported by the current evidence.