Comparative proteome profiling of egg, larva and adult Hermetia illucens (Diptera: Stratiomyidae)

Comparative proteome profiling of egg, larva and adult Hermetia illucens (Diptera: Stratiomyidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparative proteome profiling of egg, larva and adult Hermetia illucens (Diptera: Stratiomyidae) Pablo Saldivia, Paul Amouroux, Guillermo Nourdin, Cristian Emhart, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6908850/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract The black soldier fly (BSF), Hermetia illucens , is the insect with the greatest potential to contribute to sustainable human development because of its nutritional characteristics, low environmental impact, and bioremediation ability. In this study, we characterized and quantified the proteome of three BSF stages: egg, larva, and adult, using bottom-up proteomics. A total of 6,116 proteins were identified across all BSF developmental stages. Processes related to information processing, such as chromatin structure, replication, transcription, translation, and the cell cycle, were notably more abundant in eggs than in larvae and adults. Nevertheless, metabolic processes, such as amino acid, carbohydrate, lipid, and nucleotide metabolism, were more abundant in larvae and adults than in eggs. The quantitative analysis revealed four expression clusters of proteins involved in amino acid transport and metabolism, carbohydrate metabolism, coenzyme transport and metabolism, energy production, inorganic ion transport and metabolism, lipid metabolism, posttranscriptional modification and translation. Furthermore, the detection of xenobiotic detoxification proteins, including P450s, esterases, and GSTs, suggests that BSF can metabolize xenobiotics. This overview of the BSF proteome provided information for further investigations as biotechnological or practical applications based on newly identified detoxification enzymes, such as enhancing rearing practices with new diet formulations or bioremediation wastes. Biological sciences/Biological techniques/Proteomic analysis Biological sciences/Biotechnology Biological sciences/Developmental biology Biological sciences/Molecular biology Biological sciences/Systems biology Biological sciences/Zoology/Entomology shotgun proteomics development stage detoxification bioremediation metabolic processes insect rearing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The sustainable exploitation of natural resources can be further enhanced by integrating nature-based solutions to address the problems of overproduction, solid organic waste disposal, and the growing demand for natural raw materials [ 1 ]. Natural bioactive components are found in various natural sources, such as plants, animals, and microorganisms [ 2 ]. The exploitation of insect mass culture as a promising alternative source of proteins and lipids has increased in recent years. Insects are highly efficient bioconverters, generating a low environmental impact since they emit fewer greenhouse gases, consume less water and reduce the risk of zoonoses [ 3 , 4 ]. The black soldier fly (BSF), Hermetia illucens (L., 1758) (Diptera: Stratiomyidae), can contribute to sustainable human development because of its nutritional characteristics, low environmental impact, and bioremediation ability, among other advantages. Many studies have described promising farmed insects that provide multiple services and uses for humans [ 5 , 6 ]. Saprophagous larvae are highly polyphagous and consume vegetal and animal substrates such as food waste, manure, animal corpses, and other organic materials [ 7 , 8 ]. They represent one of the most efficient and quick fly species that consumes raw waste [ 9 ]. Consequently, BSF has a potential role in human waste management and could participate in a sustainable circular economy through waste bioconversion [ 10 – 12 ]. Moreover, the activity of BSF larvae reduces the number of noxious bacteria and insect pests, such as the common fly or Drosophila flies, in waste. The biomass generated by BSF larvae is rich in proteins and lipids and is successfully used to feed fish such as rainbow trout [ 13 ] or Atlantic salmon [ 14 ]. The high fat content of larvae could also be a source for biodiesel production [ 15 , 16 ]. In addition, various products of high biological value can be extracted from BSF larvae as chymotrypsin-like proteases, serine endopeptidases [ 17 ] or antimicrobial peptides with activity against bacteria, fungi, and viruses [ 18 – 20 ]. Finally, insects have a high protein content and are rich in essential amino acids. Consequently, BSF, which is currently used in animal feed, represents a potential source of nutrients for humans [ 21 ]. The polyphagy of BSF larvae is a central feature of interest in this species. First, BSF can modulate nutrient digestion and absorption, which affect diet quality [ 22 ]. Then, digestive enzymes, which are found in the gut as amylases, lipases, and proteases, ensure that the first stage of food digestion [ 9 ] is associated with the gut microbiota [ 23 , 24 ]. Finally, different biochemical mechanisms transform the digested elements into valuable ones, such as proteins or lipids. Nevertheless, their accumulation can be influenced by diet quality [ 25 ] and larval growth performance [ 11 ]. The food conversion rate can also be affected by the diet composition and the presence of secondary metabolites or toxins (pesticides, fungal agents), negatively affecting BSF production. Metabolic enzymes such as cytochrome P450 monooxygenases (P450s), esterases, and glutathione-S-transferases (GSTs) can support the detoxification process [ 26 , 27 ]. The mechanisms involved in these processes in BSF larvae have rarely been studied. Previous biological, genomic, and proteomic studies on BSF have focused on the larval instars used for biotechnology applications. Nevertheless, studies examining other development stages are controversial. Until recently, adults were considered unable to eat [ 5 ]. However, a recent morphological and functional study formally demonstrated that BSF adults possess an operative digestive system [ 28 ]. To date, no studies have characterized the BSF egg proteome. In other insect species, this characterization has been fundamental in developmental biology studies and for identifying enzymes with high biotechnological value [ 29 – 32 ]. Proteomic studies of BSF eggs and adults could lead to the characterization of new cellular processes and future applications. Here, we characterized and quantified the proteomes of three developmental stages of BSF, egg, larva, and adult, via a bottom-up proteomics strategy. The analyses focused on metabolic processes related to carbohydrates, lipids and amino acids and xenobiotic detoxification processes involving the metabolic enzymes P450s, esterases, and GSTs. As a result, we provide valuable information about the whole life cycle of BSF, revealing potential functions related to regulatory mechanisms of development, evolution, and interaction with the surrounding environment (bacteria, diet, etc.). This knowledge will help provide relevant information for optimizing the breeding conditions of BSF, developing new biotechnology based on three BSF development stages, and producing quality proteins as required by industry. 2. Results 2.1 Protein identification Overview and Functional annotation by KOG A total of 6,116 proteins were identified across the three BSF developmental stages. By stage, 4,344 proteins were identified in the egg, 3,543 in the larva, and 4,301 in the adult (Table 1 ). The proteomes shared by the three individuals at each stage were 2,261, 2,149, and 2,360 proteins for eggs, larvae, and adults, respectively (Table 1 ). A comparison of the proteomes between the BSF stages revealed that 1,768 proteins were shared by all three stages: 1,718 unique to the egg, 911 unique to the larva, and 949 unique to the adult (Fig. 1 A). Notably, the highest number of proteins was detected in the egg stage. On the basis of the complete dataset, the PCA clustered each stage separately, with low variation among the three individuals within each stage (Fig. 1 B). Table 1 Number of proteins identified in each individual per stage, total number of unique proteins per stage (panproteome), and number of proteins shared among the three individuals at each stage. Biological replicates Egg Larva Adult R1 3,181 2,837 2,986 R2 3,521 2,853 3,095 R3 2,961 2,721 3,369 Panproteome 4,344 3,543 4,031 Shared Proteins 2,261 2,149 2,360 Each protein dataset was annotated, achieving an annotation yield of 90–92% using EggNOG Mapper, 89–91% using eukaryotic orthologous groups (KOG), and 88–99% using protein families (PFAM) (Table 2 ). To analyze the processes in which the proteins participate and to assess the distribution of unique proteins at each developmental stage, the percentage of proteins associated with different pathways was determined through KOG analysis (Fig. 1 C). Table 2 Functional annotation of each stage’s panproteome using EggNOG (Evolutionary Genealogy of Genes: Nonsupervised Orthologous Groups), KOG (Clusters of Orthologous Groups of proteins), and PFAM (protein families) databases. Annotation yields are shown in parentheses. Stage Number of identified proteins EggNog KOG PFAM Egg 4,344 4,006 (92.2%) 3,947 (90.9%) 3,887 (89.5%) Larva 3,543 3,198 (90.3%) 3,157 (89.1%) 3,119 (88.0%) Adult 4,031 3,689 (91.5%) 3,604 (89.4%) 3,557 (88.2%) Compared with the larval and adult stages, the egg stage presented the highest percentage of assigned proteins in each category. The most abundant processes in the egg were replication, recombination, and repair (100%); cell cycle control, cell division, and chromosome partitioning (88%); transcription (83%); chromatin structure and dynamics (75%); translation and ribosomal structure (72%); RNA biogenesis, processing, and modification (67%); nuclear structure (60%); and intracellular trafficking, secretion, and vesicular transport (59%). In the larva, the most abundant processes were secondary metabolite biosynthesis, transport, and catabolism (64%); cell wall/membrane/envelope biogenesis (58%); carbohydrate transport and metabolism (52%); defense mechanisms (50%); posttranslational modification, protein turnover, and chaperones (41%); and amino acid transport and metabolism (39%). In adults, the most abundant processes were extracellular structures (52%), energy production and conversion (49%), lipid transport and metabolism (42%), and nucleotide transport and metabolism (39%). In summary, processes related to information processing were significantly more common in the egg stage than in the larva and adult stages, whereas metabolic processes were more common in the larva and adult stages than in the egg stage. 2.2 Analysis of protein clustering at different BSF stages To determine which proteins significantly varied in expression (i.e. DEPs), we compared the three developmental stages of BSF. First, we identified 1,688, 2,042 and 2,269 quantifiable proteins (Fig. 2 B) and 802,839 and 1,136 DEPs for larva vs. egg, adult vs. egg and adult vs. larva, respectively (Fig. 2 A). A greater number of quantifiable proteins and DEPs were found between adults and larvae. Next, 1,091 proteins were significantly differentially expressed across the three BSF stages and were divided into eleven clusters (Supplementary Fig. 2). Clusters 02, 04, 05, and 06 were composed of 198, 43, 11 and 15 proteins, respectively, and presented consistent expression between development stages (Fig. 3 A). The proteins in clusters 02, 04, and 05 were highly expressed in eggs but weakly expressed in larvae and adults. In cluster 06, the expression of these proteins was higher in larvae but lower in eggs and adults. Additionally, out of 21 pathways, we highlighted six KOG pathways: carbohydrate metabolism, coenzyme transport and metabolism, energy production, inorganic ion transport and metabolism, lipid transport and metabolism and amino acid transport and metabolism. These genes were relevant within the three development stages (Fig. 3 B). To synthesize and analyze the protein interactions of these four clusters, the mean relative abundance scaled as the Z score of the proteins was plotted (Fig. 4 ). The heatmap revealed that the expression profile across the clusters was consistent with the trajectories shown in the profile plots. Figure 4 shows the protein distribution in the KOG pathways of amino acid transport and metabolism, carbohydrate metabolism, coenzyme transport and metabolism, energy production, inorganic ion transport and metabolism, lipid metabolism, posttranscriptional modification and translation, and protein‒protein interactions in the STRING database mapping against Drosophila melanogaster . As shown in the profile plots (Fig. 3 A), the analysis of these clusters revealed that the proteins in clusters 02, 04, and 05 presented a higher mean abundance in egg and a lower mean abundance in larvae and adults. In contrast, the proteins in cluster 06 had a higher abundance in larvae and a lower abundance in eggs and adults. In cluster 02, the largest cluster, proteins belonged predominantly to posttranscriptional modification, with numerous interactions within the proteins involved in this process, and with proteins belonging to the processes of translation and lipid metabolism. For cluster 04, the most relevant biological process was post modification with 15 of 24 proteins, but with only one and four interactions with proteins from Translation and Amino acid transport and metabolism, respectively. In cluster 05, three processes were represented Post Transcriptional modification, Aminoacid transport, metabolism, and Translation. The proteins of the first process presented successive interactions. Finally, the cluster 06 was composed of proteins that mainly participated in Translation, and in interacting processes such as Energy Production, Carbohydrate metabolism and Coenzyme Transport and Metabolism. 2.3 Bioremediation- and detoxification-associated protein families (PFAMs). The protein annotation performed on the PFAM database identified 15 proteins belonging to the COesterase, cytochrome and GST protein families. These proteins were plotted on a chord plot showing their relative abundances for the larva vs. egg, adult vs. egg and adult vs. larva comparisons (Fig. 5 ). In general, proteins such as cyc, cyc1, levy, and COX5A of the “cytochrome family” and Gst1 and Fax of the “GST family” were upregulated between larva or adult vs. egg and between the adult and larva. The unique protein in the “COesterase family” was downregulated in egg vs. larva and adult comparisons but upregulated in adult vs larva comparisons. 3. Discussion Using a bottom-up approach, we performed the first proteomic description, which included three BSF development stages: Egg, L5 Larva and Adult. A total of 6,116 proteins were identified, of which 28.9% were shared by the three stages, 28.1% were unique to egg, 14.8% were unique to larvae, and 15.5% were unique to adults. More than 88% of these proteins were annotated in the databases EggNog, KOG, and PFAM. A total of 1,091 differentially expressed proteins were identified and divided into 11 clusters. Hierarchical clustering separated eggs from the other two stages, but the variation in protein expression between stages was dependent on each cluster. The proteins involved in bioremediation belonging to the Cytochrome and GST families were generally more highly expressed in larvae and adults than in eggs. This valuable information from egg to adult BSFs revealed potential functions related to regulatory mechanisms of development, evolution, and interactions with the surrounding environment. This knowledge will be helpful for enhancing rearing methods and providing potential new biotechnology uses for the less studied stages: egg and adult. Previous investigations have focused on the BSF larva [ 43 ] because this is the main stage used as the final product, e.g., for feeding breeding animals and in the cosmetics industry. In our study, we obtained 6,116 identifications among the three stages and 3,543 for the sole L5 larva. Other studies identified approximately 500 proteins from imprecise instars of BSF larvae, whereas others characterized approximately 5,000 proteins from the five larval instars [ 44 , 45 ]. Previously, these authors annotated between 26 and 66% of the larva proteins [ 45 ]. In our study, each dataset of proteins was annotated to obtain an annotation yield of 90 to 92% using Eggnog Mapper, 89 to 91% using EuKaryotic Orthologous Groups (KOG) and 88 to 99% using protein families (PFAM). This enhancement mainly resulted from the increasing amount of information available in proteomics databases. This knowledge will provide a better understanding of the pathways and functions involved in BSF development. One of the insights acquired in the present study is the description of the egg proteome. During this critical stage, the embryo fully develops into a larva. Approximately 1.8-fold more unique proteins were found in the egg stage than in the other two stages. Specifically, the number of egg proteins was greater in 15 of 24 pathways noted in the KOG, corresponding to 62.5% of the total number of proteins, highlighting pathways related to metabolic processes and storage information and processing. These results confirmed that active embryogenesis required specific biological processes for embryo development. Cluster 2 included numerous proteins and interactions involved in these processes (Fig. 4 ). The same relevant changes in development processes from egg to larva were observed in Bactrocera latifrons (Hendel) (Diptera: Tephritidae) [ 46 ] and Apis mellifera L. (Hymenoptera: Apidae). Gala and collaborators (2012) reported that proteins related to energy production, metabolism development and amino acid metabolism were strongly expressed in honeybee embryos [ 47 ]. In the case of BSF egg, we discovered a high percentage of unique proteins that participate in processes related to transport together with storage information and processing. Interestingly, when the DEPs in the KOG category were analyzed, we observed that the relative activity of the abovementioned processes was increased mainly in larvae and that processes related to amino acid metabolism and transport were favored in egg. Moreover, analyses of unique proteins related to the transport of amino acids and nucleotides revealed an increase in the larval stage. However, when comparing DEPs, the relative activity of this pathway, such as carbohydrate metabolism and organic ion transport, was increased mainly in egg. This phenomenon may be related to the fact that, in L5, the larva has a higher food intake; therefore, pathways related to metabolic processes are increased regardless of the number of proteins [ 48 , 49 ]. The second insight of this study is the description of the adult proteome. Previous studies on BSF adults have focused mainly on reproductive aspects via biological [ 50 – 52 ], ecological [ 53 ] and morphological [ 28 ] experiments. However, the potential of BSF adults as providers of the final product has rarely been considered. Indeed, metamorphosis highly modifies an individual’s composition and weight, reducing interest in adult-based products. In the adult stage, a significant number of unique proteins related to energy production and conversion were identified according to the KOG annotation. However, for DEPs, most of the processes evaluated increased with respect to egg but not larva, which may be related to the above phenomenon. Our results could favor new investigations for applications in industrial processes, such as those performed for larva, e.g., antibacterial substances [ 20 ] or digestive enzymes [ 17 , 23 ]. These findings are consistent with proteomic studies in different larval stages [ 54 ]. Understanding the digestive system is essential for improving applications and BSF mass rearing [ 43 ]. By exhibiting more cytochromes, COesterases, and glutathione S-transferases than adults, larvae can efficiently digest food and organic waste [ 9 , 55 ]. Indeed, P450s play essential roles in developmental processes, xenobiotic metabolism, phytochemical detoxification, and insecticide resistance [ 56 , 57 ]. In Drosophila melanogaster (Diptera: Drosophilidae), CYP303a1 is essential for wing extension and likely plays a role in the metabolism of an ecdysteroid-like molecule [ 57 ], whereas CYP6a9 is related to DDT resistance [ 58 ]. The characterization of these enzymes can be used to understand the growth performance of larvae and to formulate an optimized diet that suppresses noxious byproducts or organic waste used to feed the larvae. For this last objective, the role of the gut microbiota also needs to be considered [ 59 , 60 ]. The presence of eight proteins belonging to the cytochrome family in BSF adults suggests that these proteins can also detoxify exogenous molecules and could be considered in the development of future adult diets. In this way, nutriproteomics (how diet affects protein synthesis) has great potential for optimizing artificial diets for mass rearing, as is the case for Arma chinensis (Fallou) (Hemiptera: Pentatomidae), a biocontrol agent [ 61 ]. Overall, OMICS approaches will be helpful for improving selection practices for identifying specific lineages with the desired traits [ 62 ], taking advantage of phenotypic plasticity in the development and waste conversion of BSF [ 63 ]. Another result could be the enhancement of the BSF larva meal based on metabolic processes. Interdisciplinary approaches are needed to understand the interactions between BSF and its controlled environment and to optimize the final BSF product (meal composition, lipid extraction). To date, this study presents the most complete description of the BSF proteome, including all life stages: egg, larva, and adult. The overview of the BSF proteome provided information for further investigations such as biotechnological applications based on newly identified enzymes or practical applications to enhance rearing practices such as the dietary formulation. 4. Methods 4.1 Insect rearing and sampling Each stage of BSF was provided by F4F (Food 4 Future, Talca - Chile). From the third instar, the larvae were reared on vegetable and fruit wastes in a chamber with 80% humidity and 18°C. Individuals were collected via principal rearing: 12-h-old eggs, fifth-instar larvae, and two-day-old adults were collected and immediately processed. 4.2 Protein extraction and digestion and sample preparation for nLC-MS/MS Protein extraction was carried out as previously described [ 33 ]. Three individuals at each development stage (egg, larva, and adult) were selected and individually processed. The BSF samples were lyophilized overnight, ground in a mortar with a pestle in liquid nitrogen until obtaining a fine powder and resuspended in lysis buffer composed of 10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1% SDS, 25 mM DTT and 1 mM EDTA. Proteins were subjected to precipitation using 5:1 (v/v) cold 100% acetone and incubated overnight at -80°C. Then, the mixture was centrifuged at 15,000 × g for 10 min, the supernatant was discarded, and the pellet was washed three times with acetone at 90%, dried in a rotary concentrator at 4°C, and finally resuspended in 8 M urea with 25 mM ammonium bicarbonate (pH 8.0). The proteins were quantified with a Qubit protein assay, where 100 µg was reduced with 20 mM DTT for one hour, alkylated with 20 mM iodoacetamide in the dark for one hour, diluted ten times with 25 mM ammonium bicarbonate (pH 8.0) and digested with trypsin/LyC (Promega) at a 1:50 ratio overnight at 37°C. Peptides were cleaned using Pierce C-18 Spin Columns (Thermo Scientific, USA) according to the protocol suggested by the manufacturer. The eluted peptides were dried via a rotary concentrator at 4°C, resuspended in 2% acetonitrile (MERCK Germany) with 0.1% v/v formic acid (MERCK Germany) and quantified via direct detection (MERCK Millipore). 4.3 Mass spectrometry analysis A NanoElute (Bruker Daltonics) liquid chromatography system was used. Two hundred nanograms of tryptic peptides were separated within 90 min at a flow rate of 400 nL/min on an Aurora Series CSI reversed-phase column (25 cm × 75 µm i.d. C18 1.6 µm) (IonOpticks, Australia) at 50°C. Mobile phases A and B were water and acetonitrile with 0.1 vol% formic acid, respectively. The B percentage linearly increased from 2 to 17% within 57 min, followed by an increase to 25% B within 21 min and further to 35% within 13 min, followed by a washing step at 85% B and re-equilibration. All samples were analyzed on a timsTOF Pro (Bruker Daltonics) hybrid trapped ion mobility quadrupole time‒of-flight mass spectrometer via a CaptiveSpray nanoelectrospray ion source. The mass spectrometer was operated in data-dependent acquisition (DDA) mode for ion mobility-enhanced spectral library generation. The accumulation and ramp times were 100 ms each, and mass spectra were recorded in the range from m/z 100–1,700 in positive electrospray mode. The ion mobility was scanned from 0.6 to 1.6 Vs/cm². The overall acquisition cycle of 1.16 s comprised one full TIMS-MS scan and 10 parallel accumulation-serial fragmentation (PASEF) MS/MS scans. 4.4 Database searching Tandem mass spectra were extracted using Tims Control version 2.0 software. All MS/MS samples were analyzed using PEAKS Studio X+ (Bioinformatics Solutions, Waterloo, ON Canada; version 10.5 [2019-11-20]), which was set up to search the Hermetia illucens protein database, available in NCBI (24,794 entries, [2022-01-02]), with trypsin assumed to be a digestion enzyme. PEAKS studio X + was configured with fragment ion mass tolerance of 0.05 Da. and parent ion tolerance of 50 ppm. The carbamidomethyl of cysteine was specified as a fixed modification. Deamination of asparagine and glutamine, oxidation of methionine and acetylation of the N-terminus were specified as variable modifications. FDR estimation was included using a decoy database. FDR < 0.01 and 1 minimal unique peptide per protein were used for identification. 4.5 Bioinformatics analysis Perseus (v1.6.15.0) was used for bioinformatics and statistical analysis using the intensity matrix from PEAKS Studio X + protein identification [ 34 ]. The label-free quantification (LFQ) intensity data were normalized to the Z score. Only proteins with 100% valid values were considered for analysis. To determine the variability of each biological replicate and development stage, a principal component analysis (PCA) was performed. To compare multiple groups, one-way ANOVA, controlled by a permutation-based FDR threshold of 0.05, was used to identify significant differences in protein abundance during the BSF development stages. Proteins with p values less than 0.05 were considered significant. Protein clusters were determined via hierarchical clustering analysis through Euclidean distances. Panproteomes were annotated through eggNOG-mapper [ 35 , 36 ] using the following databases: Clusters of Orthologous Groups of proteins [ 37 ] and the protein families PFAM database [ 38 ]. Protein‒protein interactions were determined by mapping protein sequences against Drosophila melanogaster STRING database [ 39 ]. The resulting network was imported and processed in Cytoscape v.3.91 [ 40 ]. The charts were created via R v.3.6.0 with ComplexHeatmap v.2.0.0 [ 41 ], GOplot [ 42 ] and base packages. 4.6 Data Availability The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository under the dataset identifier PXD042131. Declarations Acknowledgments: We are grateful to Food4Future (Talca, Chile) for providing us with the biological material and their technical support. Author Contributions: Conceptualization: C.V.; Methodology: M.H., G.N., P.S.; Software: G.N., P.S.; Validation: P.S., P.A.; Formal analysis: G.N., P.S.; Investigation: P.A.; Resources: C.E.; Data curation: P.S., P.A., G.N.; Writing – original draft: P.S., P.A.; Writing – review & editing: P.A., C.V.; Visualization: P.S.; Supervision: C.V., M.H.; Project administration: C.V., M.C.A., R.D.; Funding acquisition: C.V. All authors have read and approved the final version of the manuscript. Data Availability. 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Bertinetti, C., Samayoa, A. C. & Hwang, S-Y. Effects of feeding adults of Hermetia illucens (diptera: stratiomyidae) on longevity, oviposition, and egg hatchability: insights into optimizing egg production. J. Insect Sci. 19 10.1093/jisesa/iez001 (2019). Liu, Z., Najar-Rodriguez, A. J., Minor, M. A., Hedderley, D. I. & Morel, P. C. H. Mating success of the black soldier fly, Hermetia illucens (Diptera: Stratiomyidae), under four artificial light sources. J. Photochem. Photobiol B . 205 , 111815. 10.1016/j.jphotobiol.2020.111815 (2020). Xu, Q., Wu, Z., Zeng, X. & An, X. Identification and expression profiling of chemosensory genes in Hermetia illucens via a transcriptomic analysis. Front. Physiol. 11 , 720. 10.3389/fphys.2020.00720 (2020). Almeida, C., Rijo, P. & Rosado, C. Bioactive compounds from Hermetia illucens larvae as natural ingredients for cosmetic application. Biomolecules 10 10.3390/biom10070976 (2020). Rane, R. V. et al. Detoxifying enzyme complements and host use phenotypes in 160 insect species. Curr Opin Insect Sci . 31, 131–138;10.1016/j.cois.12.008 (2019). (2018). Nauen, R., Bass, C., Feyereisen, R. & Vontas, J. The role of cytochrome P450s in insect toxicology and resistance. Annu. Rev. Entomol. 67 , 105–124. 10.1146/annurev-ento-070621-061328 (2022). Wu, L. et al. CYP303A1 has a conserved function in adult eclosion in Locusta migratoria and Drosophila melanogaster . Insect Biochem. Mol. Biol. 113 , 103210. 10.1016/j.ibmb.2019.103210 (2019). Chen, S. & Li, X. Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes. BMC Evol. Biol. 7 , 46. 10.1186/1471-2148-7-46 (2007). Li, X., Zhou, S., Zhang, J., Zhou, Z. & Xiong, Q. Directional changes in the intestinal bacterial community in black soldier fly ( Hermetia illucens ) larvae. Anim. Basel . 11 10.3390/ani11123475 (2021). Jiang, C. L. et al. Black soldier fly larvae ( Hermetia illucen s) strengthen the metabolic function of food waste biodegradation by gut microbiome. Microb. Biotechnol. 12 , 528–543. 10.1111/1751-7915.13393 (2019). Zou, D. et al. Differential proteomics analysis unraveled mechanisms of Arma chinensis responding to improved artificial diet. Insects 13 10.3390/insects13070605 (2022). Leung, K. et al. Next-generation biological control: the need for integrating genetics and genomics. Biol. Rev. Camb. Philos. Soc. 95 , 1838–1854. 10.1111/brv.12641 (2020). Zhou, F., Tomberlin, J. K., Zheng, L., Yu, Z. & Zhang, J. Developmental and waste reduction plasticity of three black soldier fly strains (Diptera: Stratiomyidae) raised on different livestock manures. J. Med. Entomol. 50 , 1224–1230. 10.1603/me13021 (2013). Additional Declarations No competing interests reported. Supplementary Files BSFsupplementary.pdf Cite Share Download PDF Status: Published Journal Publication published 24 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 30 Jun, 2025 Reviews received at journal 28 Jun, 2025 Reviews received at journal 27 Jun, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviewers invited by journal 24 Jun, 2025 Editor invited by journal 19 Jun, 2025 Editor assigned by journal 18 Jun, 2025 Submission checks completed at journal 17 Jun, 2025 First submitted to journal 16 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6908850","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":476493048,"identity":"51e1a94c-bdb0-4817-b79f-55564a4ba959","order_by":0,"name":"Pablo Saldivia","email":"","orcid":"","institution":"Fundación de Investigación San Ramón (FISAR)","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Saldivia","suffix":""},{"id":476493049,"identity":"fc5acfa8-1250-4eb3-a38f-25a95d6d92b7","order_by":1,"name":"Paul Amouroux","email":"","orcid":"","institution":"Universidad Mayor","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Amouroux","suffix":""},{"id":476493050,"identity":"2b4f0e5b-fafc-4028-966a-7a2260b7fc15","order_by":2,"name":"Guillermo Nourdin","email":"","orcid":"","institution":"MELISA Institute","correspondingAuthor":false,"prefix":"","firstName":"Guillermo","middleName":"","lastName":"Nourdin","suffix":""},{"id":476493051,"identity":"60f67583-5afc-49db-ac6d-b0ed9a5fd679","order_by":3,"name":"Cristian Emhart","email":"","orcid":"","institution":"Food4Future - F4F","correspondingAuthor":false,"prefix":"","firstName":"Cristian","middleName":"","lastName":"Emhart","suffix":""},{"id":476493052,"identity":"0e6a4893-0d41-4a55-bcd7-2f9c6729d6fa","order_by":4,"name":"María Cristina Au","email":"","orcid":"","institution":"Food4Future - F4F","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Cristina","lastName":"Au","suffix":""},{"id":476493053,"identity":"829ea8fd-24b9-439c-a38d-68ef64aafdd2","order_by":5,"name":"Rocío Delgado","email":"","orcid":"","institution":"Food4Future - F4F","correspondingAuthor":false,"prefix":"","firstName":"Rocío","middleName":"","lastName":"Delgado","suffix":""},{"id":476493055,"identity":"27ee61cd-f0d1-49df-a45d-1bb6539e6f10","order_by":6,"name":"Mauricio Hernández","email":"","orcid":"","institution":"MELISA Institute","correspondingAuthor":false,"prefix":"","firstName":"Mauricio","middleName":"","lastName":"Hernández","suffix":""},{"id":476493056,"identity":"c27692cf-cf5c-41ef-9679-62c93ca74608","order_by":7,"name":"Cristian Vargas","email":"data:image/png;base64,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","orcid":"","institution":"MELISA Institute","correspondingAuthor":true,"prefix":"","firstName":"Cristian","middleName":"","lastName":"Vargas","suffix":""}],"badges":[],"createdAt":"2025-06-16 23:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6908850/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6908850/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-28130-2","type":"published","date":"2025-11-24T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85479830,"identity":"1c3fe121-e447-44b4-a2c8-8b79abaddadb","added_by":"auto","created_at":"2025-06-26 10:45:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":532037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative proteomic overview across BSF developmental stages. \u003c/strong\u003e(A) Venn diagram showing shared and unique proteins identified in eggs, larvae, and adults. (B) Principal component analysis (PCA) of protein profiles from three biological replicates per stage. (C) Functional classification of identified proteins according to KOG categories, showing the number of proteins assigned to each function per stage.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6908850/v1/e87931803042d39121aa2625.png"},{"id":85479840,"identity":"928d9556-2c70-49e6-9616-8e344644c7be","added_by":"auto","created_at":"2025-06-26 10:45:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":368250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential proteomic analysis across BSF developmental stages. Bar plot showing the number of quantified proteins (orange) and differentially expressed proteins (DEPs) (sky blue) for each pairwise comparison: Larva vs Egg, Adult vs Egg, and Adult vs Larva. \u003c/strong\u003eVolcano plots display quantified proteins for each comparison. Vertical dashed lines indicate the 0.5-fold change threshold; the horizontal line marks the p-value = 0.05 cutoff. Red and blue dots represent significantly upregulated and downregulated proteins, respectively\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6908850/v1/099a4d569db5888209b73205.png"},{"id":85479832,"identity":"d6c5f3ec-e894-441c-91a9-ff9c2a12e43e","added_by":"auto","created_at":"2025-06-26 10:45:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":387544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein expression clusters and relative activity of key KOG categories across BSF developmental stages.\u003c/strong\u003e (A) Scaled expression trajectories (Z-score) of protein clusters showing consistent patterns across egg, larva, and adult stages. Each plot indicates the cluster name and number of proteins included. (B) Line plots of the relative activity (Z-score) of selected KOG categories across pairwise comparisons: larva vs egg, adult vs egg, and adult vs larva. Categories with noticeable activity shifts are highlighted.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6908850/v1/53c7483a901f936a7499b8aa.png"},{"id":85480300,"identity":"0f82f28a-c0ad-4910-9d59-81c004d4d593","added_by":"auto","created_at":"2025-06-26 10:53:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1024251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelevant biological processes represented in the most consistent protein expression clusters across BSF developmental stages.\u003c/strong\u003e From left to right: heatmap showing the scaled protein abundance (Z-score) for each developmental stage (Egg, Larva, and Adult); KOG annotation indicating the functional classification of proteins within each cluster; and STRING protein–protein interaction network highlighting the main biological processes enriched in each expression pattern. Clusters were selected based on their consistency across stages and biological relevance.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6908850/v1/46897d03a7b1c985bad85fbe.png"},{"id":85481160,"identity":"d48124b7-998e-4338-be03-766b4acc0f2b","added_by":"auto","created_at":"2025-06-26 11:01:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":533576,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteins involved in bioremediation-associated PFAM terms across BSF developmental stages.\u003c/strong\u003e Chord plots showing bipartite networks between proteins (left semicircle) and their associated PFAM domains (right semicircle), including GST (purple), Cytochrome P450 (green), and Carboxylesterase (yellow). Each panel represents one pairwise comparison: Larva vs Egg, Adult vs Egg, and Adult vs Larva. The outer colored bars next to each protein node indicate differential expression status: red for upregulated and blue for downregulated proteins.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6908850/v1/50ae1e9c02c6e834c2a59b1c.png"},{"id":97179323,"identity":"cd7dd955-74dd-4aa6-b57f-0ed596a75267","added_by":"auto","created_at":"2025-12-01 16:14:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3966473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6908850/v1/340ddb0f-eac6-4b43-b5b6-ff46528193d9.pdf"},{"id":85479834,"identity":"4531c49a-5abd-4ead-ad82-6b79583e88ad","added_by":"auto","created_at":"2025-06-26 10:45:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":330254,"visible":true,"origin":"","legend":"","description":"","filename":"BSFsupplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6908850/v1/a2a9d3ab4f609ff1992aadf0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative proteome profiling of egg, larva and adult Hermetia illucens (Diptera: Stratiomyidae)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe sustainable exploitation of natural resources can be further enhanced by integrating nature-based solutions to address the problems of overproduction, solid organic waste disposal, and the growing demand for natural raw materials [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Natural bioactive components are found in various natural sources, such as plants, animals, and microorganisms [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The exploitation of insect mass culture as a promising alternative source of proteins and lipids has increased in recent years. Insects are highly efficient bioconverters, generating a low environmental impact since they emit fewer greenhouse gases, consume less water and reduce the risk of zoonoses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The black soldier fly (BSF), \u003cem\u003eHermetia illucens\u003c/em\u003e (L., 1758) (Diptera: Stratiomyidae), can contribute to sustainable human development because of its nutritional characteristics, low environmental impact, and bioremediation ability, among other advantages. Many studies have described promising farmed insects that provide multiple services and uses for humans [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Saprophagous larvae are highly polyphagous and consume vegetal and animal substrates such as food waste, manure, animal corpses, and other organic materials [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. They represent one of the most efficient and quick fly species that consumes raw waste [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, BSF has a potential role in human waste management and could participate in a sustainable circular economy through waste bioconversion [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, the activity of BSF larvae reduces the number of noxious bacteria and insect pests, such as the common fly or \u003cem\u003eDrosophila\u003c/em\u003e flies, in waste. The biomass generated by BSF larvae is rich in proteins and lipids and is successfully used to feed fish such as rainbow trout [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] or Atlantic salmon [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The high fat content of larvae could also be a source for biodiesel production [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, various products of high biological value can be extracted from BSF larvae as chymotrypsin-like proteases, serine endopeptidases [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] or antimicrobial peptides with activity against bacteria, fungi, and viruses [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Finally, insects have a high protein content and are rich in essential amino acids. Consequently, BSF, which is currently used in animal feed, represents a potential source of nutrients for humans [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe polyphagy of BSF larvae is a central feature of interest in this species. First, BSF can modulate nutrient digestion and absorption, which affect diet quality [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Then, digestive enzymes, which are found in the gut as amylases, lipases, and proteases, ensure that the first stage of food digestion [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] is associated with the gut microbiota [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Finally, different biochemical mechanisms transform the digested elements into valuable ones, such as proteins or lipids. Nevertheless, their accumulation can be influenced by diet quality [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and larval growth performance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The food conversion rate can also be affected by the diet composition and the presence of secondary metabolites or toxins (pesticides, fungal agents), negatively affecting BSF production. Metabolic enzymes such as cytochrome P450 monooxygenases (P450s), esterases, and glutathione-S-transferases (GSTs) can support the detoxification process [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The mechanisms involved in these processes in BSF larvae have rarely been studied.\u003c/p\u003e \u003cp\u003ePrevious biological, genomic, and proteomic studies on BSF have focused on the larval instars used for biotechnology applications. Nevertheless, studies examining other development stages are controversial. Until recently, adults were considered unable to eat [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, a recent morphological and functional study formally demonstrated that BSF adults possess an operative digestive system [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To date, no studies have characterized the BSF egg proteome. In other insect species, this characterization has been fundamental in developmental biology studies and for identifying enzymes with high biotechnological value [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Proteomic studies of BSF eggs and adults could lead to the characterization of new cellular processes and future applications.\u003c/p\u003e \u003cp\u003eHere, we characterized and quantified the proteomes of three developmental stages of BSF, egg, larva, and adult, via a bottom-up proteomics strategy. The analyses focused on metabolic processes related to carbohydrates, lipids and amino acids and xenobiotic detoxification processes involving the metabolic enzymes P450s, esterases, and GSTs. As a result, we provide valuable information about the whole life cycle of BSF, revealing potential functions related to regulatory mechanisms of development, evolution, and interaction with the surrounding environment (bacteria, diet, etc.). This knowledge will help provide relevant information for optimizing the breeding conditions of BSF, developing new biotechnology based on three BSF development stages, and producing quality proteins as required by industry.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Protein identification Overview and Functional annotation by KOG\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA total of 6,116 proteins were identified across the three BSF developmental stages. By stage, 4,344 proteins were identified in the egg, 3,543 in the larva, and 4,301 in the adult (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The proteomes shared by the three individuals at each stage were 2,261, 2,149, and 2,360 proteins for eggs, larvae, and adults, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A comparison of the proteomes between the BSF stages revealed that 1,768 proteins were shared by all three stages: 1,718 unique to the egg, 911 unique to the larva, and 949 unique to the adult (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Notably, the highest number of proteins was detected in the egg stage. On the basis of the complete dataset, the PCA clustered each stage separately, with low variation among the three individuals within each stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of proteins identified in each individual per stage, total number of unique proteins per stage (panproteome), and number of proteins shared among the three individuals at each stage.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiological replicates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEgg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLarva\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3,181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2,837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2,986\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3,521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2,853\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3,095\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2,961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2,721\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3,369\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePanproteome\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4,344\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e3,543\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4,031\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShared Proteins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2,261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2,149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2,360\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEach protein dataset was annotated, achieving an annotation yield of 90\u0026ndash;92% using EggNOG Mapper, 89\u0026ndash;91% using eukaryotic orthologous groups (KOG), and 88\u0026ndash;99% using protein families (PFAM) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To analyze the processes in which the proteins participate and to assess the distribution of unique proteins at each developmental stage, the percentage of proteins associated with different pathways was determined through KOG analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFunctional annotation of each stage\u0026rsquo;s panproteome using EggNOG (Evolutionary Genealogy of Genes: Nonsupervised Orthologous Groups), KOG (Clusters of Orthologous Groups of proteins), and PFAM (protein families) databases. Annotation yields are shown in parentheses.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of identified proteins\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEggNog\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKOG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePFAM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEgg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4,344\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4,006 (92.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3,947 (90.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3,887 (89.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3,543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3,198 (90.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3,157 (89.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3,119 (88.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdult\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4,031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3,689 (91.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3,604 (89.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3,557 (88.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCompared with the larval and adult stages, the egg stage presented the highest percentage of assigned proteins in each category. The most abundant processes in the egg were replication, recombination, and repair (100%); cell cycle control, cell division, and chromosome partitioning (88%); transcription (83%); chromatin structure and dynamics (75%); translation and ribosomal structure (72%); RNA biogenesis, processing, and modification (67%); nuclear structure (60%); and intracellular trafficking, secretion, and vesicular transport (59%). In the larva, the most abundant processes were secondary metabolite biosynthesis, transport, and catabolism (64%); cell wall/membrane/envelope biogenesis (58%); carbohydrate transport and metabolism (52%); defense mechanisms (50%); posttranslational modification, protein turnover, and chaperones (41%); and amino acid transport and metabolism (39%). In adults, the most abundant processes were extracellular structures (52%), energy production and conversion (49%), lipid transport and metabolism (42%), and nucleotide transport and metabolism (39%).\u003c/p\u003e \u003cp\u003eIn summary, processes related to information processing were significantly more common in the egg stage than in the larva and adult stages, whereas metabolic processes were more common in the larva and adult stages than in the egg stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Analysis of protein clustering at different BSF stages\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo determine which proteins significantly varied in expression (i.e. DEPs), we compared the three developmental stages of BSF. First, we identified 1,688, 2,042 and 2,269 quantifiable proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and 802,839 and 1,136 DEPs for larva vs. egg, adult vs. egg and adult vs. larva, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A greater number of quantifiable proteins and DEPs were found between adults and larvae. Next, 1,091 proteins were significantly differentially expressed across the three BSF stages and were divided into eleven clusters (Supplementary Fig.\u0026nbsp;2). Clusters 02, 04, 05, and 06 were composed of 198, 43, 11 and 15 proteins, respectively, and presented consistent expression between development stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The proteins in clusters 02, 04, and 05 were highly expressed in eggs but weakly expressed in larvae and adults. In cluster 06, the expression of these proteins was higher in larvae but lower in eggs and adults. Additionally, out of 21 pathways, we highlighted six KOG pathways: carbohydrate metabolism, coenzyme transport and metabolism, energy production, inorganic ion transport and metabolism, lipid transport and metabolism and amino acid transport and metabolism. These genes were relevant within the three development stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo synthesize and analyze the protein interactions of these four clusters, the mean relative abundance scaled as the Z score of the proteins was plotted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The heatmap revealed that the expression profile across the clusters was consistent with the trajectories shown in the profile plots. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the protein distribution in the KOG pathways of amino acid transport and metabolism, carbohydrate metabolism, coenzyme transport and metabolism, energy production, inorganic ion transport and metabolism, lipid metabolism, posttranscriptional modification and translation, and protein‒protein interactions in the STRING database mapping against \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. As shown in the profile plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), the analysis of these clusters revealed that the proteins in clusters 02, 04, and 05 presented a higher mean abundance in egg and a lower mean abundance in larvae and adults. In contrast, the proteins in cluster 06 had a higher abundance in larvae and a lower abundance in eggs and adults. In cluster 02, the largest cluster, proteins belonged predominantly to posttranscriptional modification, with numerous interactions within the proteins involved in this process, and with proteins belonging to the processes of translation and lipid metabolism. For cluster 04, the most relevant biological process was post modification with 15 of 24 proteins, but with only one and four interactions with proteins from Translation and Amino acid transport and metabolism, respectively. In cluster 05, three processes were represented Post Transcriptional modification, Aminoacid transport, metabolism, and Translation. The proteins of the first process presented successive interactions. Finally, the cluster 06 was composed of proteins that mainly participated in Translation, and in interacting processes such as Energy Production, Carbohydrate metabolism and Coenzyme Transport and Metabolism.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Bioremediation- and detoxification-associated protein families (PFAMs).\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe protein annotation performed on the PFAM database identified 15 proteins belonging to the COesterase, cytochrome and GST protein families. These proteins were plotted on a chord plot showing their relative abundances for the larva vs. egg, adult vs. egg and adult vs. larva comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In general, proteins such as cyc, cyc1, levy, and COX5A of the \u0026ldquo;cytochrome family\u0026rdquo; and Gst1 and Fax of the \u0026ldquo;GST family\u0026rdquo; were upregulated between larva or adult vs. egg and between the adult and larva. The unique protein in the \u0026ldquo;COesterase family\u0026rdquo; was downregulated in egg vs. larva and adult comparisons but upregulated in adult vs larva comparisons.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eUsing a bottom-up approach, we performed the first proteomic description, which included three BSF development stages: Egg, L5 Larva and Adult. A total of 6,116 proteins were identified, of which 28.9% were shared by the three stages, 28.1% were unique to egg, 14.8% were unique to larvae, and 15.5% were unique to adults. More than 88% of these proteins were annotated in the databases EggNog, KOG, and PFAM. A total of 1,091 differentially expressed proteins were identified and divided into 11 clusters. Hierarchical clustering separated eggs from the other two stages, but the variation in protein expression between stages was dependent on each cluster. The proteins involved in bioremediation belonging to the Cytochrome and GST families were generally more highly expressed in larvae and adults than in eggs. This valuable information from egg to adult BSFs revealed potential functions related to regulatory mechanisms of development, evolution, and interactions with the surrounding environment. This knowledge will be helpful for enhancing rearing methods and providing potential new biotechnology uses for the less studied stages: egg and adult.\u003c/p\u003e \u003cp\u003ePrevious investigations have focused on the BSF larva [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] because this is the main stage used as the final product, e.g., for feeding breeding animals and in the cosmetics industry. In our study, we obtained 6,116 identifications among the three stages and 3,543 for the sole L5 larva. Other studies identified approximately 500 proteins from imprecise instars of BSF larvae, whereas others characterized approximately 5,000 proteins from the five larval instars [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Previously, these authors annotated between 26 and 66% of the larva proteins [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In our study, each dataset of proteins was annotated to obtain an annotation yield of 90 to 92% using Eggnog Mapper, 89 to 91% using EuKaryotic Orthologous Groups (KOG) and 88 to 99% using protein families (PFAM). This enhancement mainly resulted from the increasing amount of information available in proteomics databases. This knowledge will provide a better understanding of the pathways and functions involved in BSF development.\u003c/p\u003e \u003cp\u003eOne of the insights acquired in the present study is the description of the egg proteome. During this critical stage, the embryo fully develops into a larva. Approximately 1.8-fold more unique proteins were found in the egg stage than in the other two stages. Specifically, the number of egg proteins was greater in 15 of 24 pathways noted in the KOG, corresponding to 62.5% of the total number of proteins, highlighting pathways related to metabolic processes and storage information and processing. These results confirmed that active embryogenesis required specific biological processes for embryo development. Cluster 2 included numerous proteins and interactions involved in these processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The same relevant changes in development processes from egg to larva were observed in \u003cem\u003eBactrocera latifrons\u003c/em\u003e (Hendel) (Diptera: Tephritidae) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and \u003cem\u003eApis mellifera\u003c/em\u003e L. (Hymenoptera: Apidae). Gala and collaborators (2012) reported that proteins related to energy production, metabolism development and amino acid metabolism were strongly expressed in honeybee embryos [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In the case of BSF egg, we discovered a high percentage of unique proteins that participate in processes related to transport together with storage information and processing. Interestingly, when the DEPs in the KOG category were analyzed, we observed that the relative activity of the abovementioned processes was increased mainly in larvae and that processes related to amino acid metabolism and transport were favored in egg. Moreover, analyses of unique proteins related to the transport of amino acids and nucleotides revealed an increase in the larval stage. However, when comparing DEPs, the relative activity of this pathway, such as carbohydrate metabolism and organic ion transport, was increased mainly in egg. This phenomenon may be related to the fact that, in L5, the larva has a higher food intake; therefore, pathways related to metabolic processes are increased regardless of the number of proteins [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe second insight of this study is the description of the adult proteome. Previous studies on BSF adults have focused mainly on reproductive aspects via biological [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], ecological [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and morphological [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] experiments. However, the potential of BSF adults as providers of the final product has rarely been considered. Indeed, metamorphosis highly modifies an individual\u0026rsquo;s composition and weight, reducing interest in adult-based products. In the adult stage, a significant number of unique proteins related to energy production and conversion were identified according to the KOG annotation. However, for DEPs, most of the processes evaluated increased with respect to egg but not larva, which may be related to the above phenomenon. Our results could favor new investigations for applications in industrial processes, such as those performed for larva, e.g., antibacterial substances [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] or digestive enzymes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings are consistent with proteomic studies in different larval stages [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnderstanding the digestive system is essential for improving applications and BSF mass rearing [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. By exhibiting more cytochromes, COesterases, and glutathione S-transferases than adults, larvae can efficiently digest food and organic waste [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Indeed, P450s play essential roles in developmental processes, xenobiotic metabolism, phytochemical detoxification, and insecticide resistance [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (Diptera: Drosophilidae), CYP303a1 is essential for wing extension and likely plays a role in the metabolism of an ecdysteroid-like molecule [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], whereas CYP6a9 is related to DDT resistance [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The characterization of these enzymes can be used to understand the growth performance of larvae and to formulate an optimized diet that suppresses noxious byproducts or organic waste used to feed the larvae. For this last objective, the role of the gut microbiota also needs to be considered [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The presence of eight proteins belonging to the cytochrome family in BSF adults suggests that these proteins can also detoxify exogenous molecules and could be considered in the development of future adult diets.\u003c/p\u003e \u003cp\u003eIn this way, nutriproteomics (how diet affects protein synthesis) has great potential for optimizing artificial diets for mass rearing, as is the case for \u003cem\u003eArma chinensis\u003c/em\u003e (Fallou) (Hemiptera: Pentatomidae), a biocontrol agent [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Overall, OMICS approaches will be helpful for improving selection practices for identifying specific lineages with the desired traits [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], taking advantage of phenotypic plasticity in the development and waste conversion of BSF [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Another result could be the enhancement of the BSF larva meal based on metabolic processes. Interdisciplinary approaches are needed to understand the interactions between BSF and its controlled environment and to optimize the final BSF product (meal composition, lipid extraction).\u003c/p\u003e \u003cp\u003eTo date, this study presents the most complete description of the BSF proteome, including all life stages: egg, larva, and adult. The overview of the BSF proteome provided information for further investigations such as biotechnological applications based on newly identified enzymes or practical applications to enhance rearing practices such as the dietary formulation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Insect rearing and sampling\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEach stage of BSF was provided by F4F (Food 4 Future, Talca - Chile). From the third instar, the larvae were reared on vegetable and fruit wastes in a chamber with 80% humidity and 18\u0026deg;C. Individuals were collected via principal rearing: 12-h-old eggs, fifth-instar larvae, and two-day-old adults were collected and immediately processed.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Protein extraction and digestion and sample preparation for nLC-MS/MS\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProtein extraction was carried out as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Three individuals at each development stage (egg, larva, and adult) were selected and individually processed. The BSF samples were lyophilized overnight, ground in a mortar with a pestle in liquid nitrogen until obtaining a fine powder and resuspended in lysis buffer composed of 10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1% SDS, 25 mM DTT and 1 mM EDTA. Proteins were subjected to precipitation using 5:1 (v/v) cold 100% acetone and incubated overnight at -80\u0026deg;C. Then, the mixture was centrifuged at 15,000 \u0026times; g for 10 min, the supernatant was discarded, and the pellet was washed three times with acetone at 90%, dried in a rotary concentrator at 4\u0026deg;C, and finally resuspended in 8 M urea with 25 mM ammonium bicarbonate (pH 8.0). The proteins were quantified with a Qubit protein assay, where 100 \u0026micro;g was reduced with 20 mM DTT for one hour, alkylated with 20 mM iodoacetamide in the dark for one hour, diluted ten times with 25 mM ammonium bicarbonate (pH 8.0) and digested with trypsin/LyC (Promega) at a 1:50 ratio overnight at 37\u0026deg;C. Peptides were cleaned using Pierce C-18 Spin Columns (Thermo Scientific, USA) according to the protocol suggested by the manufacturer. The eluted peptides were dried via a rotary concentrator at 4\u0026deg;C, resuspended in 2% acetonitrile (MERCK Germany) with 0.1% v/v formic acid (MERCK Germany) and quantified via direct detection (MERCK Millipore).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Mass spectrometry analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA NanoElute (Bruker Daltonics) liquid chromatography system was used. Two hundred nanograms of tryptic peptides were separated within 90 min at a flow rate of 400 nL/min on an Aurora Series CSI reversed-phase column (25 cm \u0026times; 75 \u0026micro;m i.d. C18 1.6 \u0026micro;m) (IonOpticks, Australia) at 50\u0026deg;C. Mobile phases A and B were water and acetonitrile with 0.1 vol% formic acid, respectively. The B percentage linearly increased from 2 to 17% within 57 min, followed by an increase to 25% B within 21 min and further to 35% within 13 min, followed by a washing step at 85% B and re-equilibration. All samples were analyzed on a timsTOF Pro (Bruker Daltonics) hybrid trapped ion mobility quadrupole time‒of-flight mass spectrometer via a CaptiveSpray nanoelectrospray ion source. The mass spectrometer was operated in data-dependent acquisition (DDA) mode for ion mobility-enhanced spectral library generation. The accumulation and ramp times were 100 ms each, and mass spectra were recorded in the range from m/z 100\u0026ndash;1,700 in positive electrospray mode. The ion mobility was scanned from 0.6 to 1.6 Vs/cm\u0026sup2;. The overall acquisition cycle of 1.16 s comprised one full TIMS-MS scan and 10 parallel accumulation-serial fragmentation (PASEF) MS/MS scans.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Database searching\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTandem mass spectra were extracted using Tims Control version 2.0 software. All MS/MS samples were analyzed using PEAKS Studio X+ (Bioinformatics Solutions, Waterloo, ON Canada; version 10.5 [2019-11-20]), which was set up to search the \u003cem\u003eHermetia illucens\u003c/em\u003e protein database, available in NCBI (24,794 entries, [2022-01-02]), with trypsin assumed to be a digestion enzyme. PEAKS studio X\u0026thinsp;+\u0026thinsp;was configured with fragment ion mass tolerance of 0.05 Da. and parent ion tolerance of 50 ppm. The carbamidomethyl of cysteine was specified as a fixed modification. Deamination of asparagine and glutamine, oxidation of methionine and acetylation of the N-terminus were specified as variable modifications. FDR estimation was included using a decoy database. FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and 1 minimal unique peptide per protein were used for identification.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Bioinformatics analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePerseus (v1.6.15.0) was used for bioinformatics and statistical analysis using the intensity matrix from PEAKS Studio X\u0026thinsp;+\u0026thinsp;protein identification [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The label-free quantification (LFQ) intensity data were normalized to the Z score. Only proteins with 100% valid values were considered for analysis. To determine the variability of each biological replicate and development stage, a principal component analysis (PCA) was performed. To compare multiple groups, one-way ANOVA, controlled by a permutation-based FDR threshold of 0.05, was used to identify significant differences in protein abundance during the BSF development stages. Proteins with p values less than 0.05 were considered significant. Protein clusters were determined via hierarchical clustering analysis through Euclidean distances. Panproteomes were annotated through eggNOG-mapper [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] using the following databases: Clusters of Orthologous Groups of proteins [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and the protein families PFAM database [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Protein‒protein interactions were determined by mapping protein sequences against \u003cem\u003eDrosophila melanogaster\u003c/em\u003e STRING database [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The resulting network was imported and processed in Cytoscape v.3.91 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The charts were created via R v.3.6.0 with ComplexHeatmap v.2.0.0 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], GOplot [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and base packages.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Data Availability\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository under the dataset identifier PXD042131.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u0026nbsp;We are grateful to Food4Future (Talca, Chile) for providing us with the biological material and their technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u0026nbsp;Conceptualization: C.V.; Methodology: M.H., G.N., P.S.; Software: G.N., P.S.; Validation: P.S., P.A.; Formal analysis: G.N., P.S.; Investigation: P.A.; Resources: C.E.; Data curation: P.S., P.A., G.N.; Writing – original draft: P.S., P.A.; Writing – review \u0026amp; editing: P.A., C.V.; Visualization: P.S.; Supervision: C.V., M.H.; Project administration: C.V., M.C.A., R.D.; Funding acquisition: C.V. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability. \u003c/strong\u003eThe mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository under the dataset identifier PXD042131.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u003c/strong\u003e\u0026nbsp;The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u0026nbsp;This research was supported by the FISAR Foundation, grant number CLA042024-01, and the APC was funded by F4F - Food4Future.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKuan, Z. J., Chan, B. K. N. \u0026amp; Gan, S. K. E. Worming the Circular Economy for Biowaste and Plastics: \u003cem\u003eHermetia illucens, Tenebrio molitor, and Zophobas morio\u003c/em\u003e. \u003cem\u003eSustain. Sci. Pract. 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Entomol.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 1224\u0026ndash;1230. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1603/me13021\u003c/span\u003e\u003cspan address=\"10.1603/me13021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"shotgun proteomics, development stage, detoxification, bioremediation, metabolic processes, insect rearing","lastPublishedDoi":"10.21203/rs.3.rs-6908850/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6908850/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe black soldier fly (BSF), \u003cem\u003eHermetia illucens\u003c/em\u003e, is the insect with the greatest potential to contribute to sustainable human development because of its nutritional characteristics, low environmental impact, and bioremediation ability. In this study, we characterized and quantified the proteome of three BSF stages: egg, larva, and adult, using bottom-up proteomics. A total of 6,116 proteins were identified across all BSF developmental stages. Processes related to information processing, such as chromatin structure, replication, transcription, translation, and the cell cycle, were notably more abundant in eggs than in larvae and adults. Nevertheless, metabolic processes, such as amino acid, carbohydrate, lipid, and nucleotide metabolism, were more abundant in larvae and adults than in eggs. The quantitative analysis revealed four expression clusters of proteins involved in amino acid transport and metabolism, carbohydrate metabolism, coenzyme transport and metabolism, energy production, inorganic ion transport and metabolism, lipid metabolism, posttranscriptional modification and translation. Furthermore, the detection of xenobiotic detoxification proteins, including P450s, esterases, and GSTs, suggests that BSF can metabolize xenobiotics. This overview of the BSF proteome provided information for further investigations as biotechnological or practical applications based on newly identified detoxification enzymes, such as enhancing rearing practices with new diet formulations or bioremediation wastes.\u003c/p\u003e","manuscriptTitle":"Comparative proteome profiling of egg, larva and adult Hermetia illucens (Diptera: Stratiomyidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 10:45:00","doi":"10.21203/rs.3.rs-6908850/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-30T15:19:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-28T06:55:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-27T04:06:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"195614986789880507747061568066265883038","date":"2025-06-24T13:29:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3048750524651290668987151430549980654","date":"2025-06-24T11:29:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60907771473782194459772453596132122055","date":"2025-06-24T09:42:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104915806875062051849245066833200506460","date":"2025-06-24T06:39:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-24T06:22:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-19T12:25:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-18T06:46:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-17T13:04:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-16T23:02:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a8edc5b-b6b6-45e9-905c-2bb9eb4c63ac","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50599580,"name":"Biological sciences/Biological techniques/Proteomic analysis"},{"id":50599581,"name":"Biological sciences/Biotechnology"},{"id":50599582,"name":"Biological sciences/Developmental biology"},{"id":50599583,"name":"Biological sciences/Molecular biology"},{"id":50599584,"name":"Biological sciences/Systems biology"},{"id":50599585,"name":"Biological sciences/Zoology/Entomology"}],"tags":[],"updatedAt":"2025-12-01T16:08:59+00:00","versionOfRecord":{"articleIdentity":"rs-6908850","link":"https://doi.org/10.1038/s41598-025-28130-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-24 15:57:39","publishedOnDateReadable":"November 24th, 2025"},"versionCreatedAt":"2025-06-26 10:45:00","video":"","vorDoi":"10.1038/s41598-025-28130-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-28130-2","workflowStages":[]},"version":"v1","identity":"rs-6908850","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6908850","identity":"rs-6908850","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}