Truncated Higiant enhances the bioconversion ability of Hermetia illucens

Abstract

The black soldier fly (BSF), Hermetia illucens, has garnered attention for its proficiency in converting organic waste into valuable biomass. In the context of rapid population growth and urbanization, BSF with higher bioconversion efficiency is in urgent need. One promising approach to improve the efficiency is generating new lines by molecular breeding, which has succeeded in plants and various livestock. Here, we developed a BSF strain with an enhanced bioconversion efficiency using CRISPR/Cas9–mediated genome editing. By knocking out the gene giant (gt) in BSF, we obtained individuals with larger size and higher reproductive capacity. Notably, the mutant strain increased the bioconversion efficiency of food waste by 13.10%, and exhibited consistent performance across diverse organic waste substrates. In addition, our study elucidates multifaceted roles of gt in larval growth and developmental plasticity. Moreover, mutants showed reduced reproductive competitiveness against WT in mixed populations. In conclusion, our study provides a superior and safe insect chassis for biomanufacturing and underscores the potential of molecular breeding to enhance efficiency in BSF farming for sustainable organic waste recycling.

By knocking out the gene giant in Hermetia illucens, we obtained the strain with higher biconversion and reproductive capacity, providing a superior and safe insect chassis for biomanufacturing.

Introduction

Organic waste accounts for approximately a third of the global waste stream¹. Untreated organic waste leads to escalating environmental pollution^2,3. While traditional methods such as landfilling, burning, and composting may result in secondary pollution^4,5, economically viable and environmentally friendly methods to optimize organic waste treatment are urgently needed.

The black soldier fly (BSF), Hermetia illucens L. (Diptera: Stratiomyidae), has been recognized as an excellent candidate for organic waste recycling, aligning with the circular economy principle^6,7. BSF larvae (BSFL) effectively convert various organic waste (e.g., food waste (FW), and animal feces) into insect biomass rich in edible protein, fat, and bioactive compounds (e.g., antimicrobial peptides, chitin, and lauric acid)^6,7. Bioconversion of organic waste by BSFL reduces pollutants such as pathogenic bacteria, antibiotics, and mycotoxin^6,8,9. These features make BSFL biomass an alternative protein supplement for human and livestock^10–12. Another advantage of BSFL is its remarkably low carbon footprint (CO₂ production, 130.18 ± 13.21 g/kg, pH = 9), compared to livestock such as beef cattle (2835 g/kg)^13,14. The carbon footprint of traditional farming meat for human consumption is estimated to be higher than 7.1 billion tones CO₂ equivalent, which represents 14.5% of all anthropogenic greenhouse gas emissions¹⁰. Thus, the application of BSF provides a sustainable solution to the global environmental issue of organic waste and animal husbandry pollution. However, the current production scale and capabilities of BFSL is insufficient to meet the increasing demand¹⁵. In the next decade, the world edible insect market is expected to grow at an annual rate of 20 to 30%¹⁰. Therefore, obtaining BSFL with higher bioconversion efficiency and biomass is key to the application of BSF.

Organism improvement can be achieved through artificial selection and molecular breeding. While artificial selection has been effective in advancing animal breeding, the long breeding cycles and challenges of meeting demands of diverse environment limits its applications^16,17. Molecular breeding refers to the application of gene manipulation to plant or animal breeding; with its short cycle and high operability, this approach has been utilized with several economic organisms for improved characteristics^18–20. For example, genetically engineered corn and cotton have been planted worldwide to manage insect pests²¹. The yeast and tobacco served as platforms for the heterologous expression to obtain high–value products^22,23. In the insect, Bombyx mori and BSF have been employed as bioreactors, such as producing spider silk and carotenoids^24,25. Notably, our group has established an efficient platform for the CRISPR/Cas9–mediated genome editing and piggyBac–based gene over–expression in BSF^12,26. By knocking out the gene vestigial, we obtained wingless BSF adults, which provide a candidate approach to address the challenge of BSF escaping in large–scale production¹². Molecular breeding in BSF has shown feasibility in varied conditions, making it possible to generate a superior line with increased conversion efficiency and resource production²⁷.

Here, to improve bioconversion ability of BSFL, we carried out molecular breeding screening based on homologous genes in model species Drosophila melanogaster, Meigen (Diptera: Drosophilidae), and identified the gene giant (gt) in BSF. The gt knockout of D. melanogaster exhibited significantly enhanced body size while maintaining fertility²⁸, suggesting the gt ortholog may serve as a promising candidate for trait optimization in BSF breeding. The H. illucens gt loss–of–function mutants obtained using the CRISPR/Cas9 technique exhibited a significant increase in growth rate and weight gain, with a 13.10% increase in conversion efficiency of FW. The content of fat and protein was not affected. In addition, the conversion capacity for various organic wastes has steadily increased. The reproductive assays showed that the increase in the size of the mutant individuals did not affect the mating, and egg counts (i.e., amount generated) were significantly increased (i.e., 55.53%). The gt mutation promoted growth of hatchling larvae by elevating ilps expression, resulting in a larger basal body size and weight. RNA–seq and histological information indicated that thinner cuticle structure of gt mutants could be an adaptation to the larger body. Crucially, the strain showed a competitive disadvantage in reproduction compared to wild type (WT). Overall, our molecular breeding approach exhibits a superior chassis for biomanufacturing and provides a basis for future high–throughput breeding of resource insects, benefiting for both synthetic biology and environment management field.

Results

Identification and analyses of gt

BSFL goes through five instar stages and can be utilized to convert organic waste after 2nd instar stage (Fig. 1a). To obtain BSFL with higher conversion efficiency with molecular breeding method, we referred to the studies in the model species D. melanogaster^28–31, which also belongs to the order Diptera. Among the genes affecting body size in fruit fly, knockouts show larger size but with higher mortality rate²⁹ or significant developmental delay³¹ were excluded. Consequently, we noticed the gene gt, which result in mutants with larger size and slight developmental delay with a normal percentage of adults²⁸. To ascertain the presence of homologous genes in BSF, we employed the D. melanogaster Gt protein (FBgn0001150) as the query for BLAST analysis against the genomic database of BSF. The homologous gene gt (LOC119655181) was found in BSF. Subsequently, the cDNA sequence of the putative gt gene with 1065 base pairs was amplified and cloned. Analysis of multiple sequence alignments revealed a high degree of conservation among Gt protein sequences, with the protein featuring a basic leucine zipper (bZIP) domain (Fig. 1b, Supplementary Fig. 1). These results strongly suggest that the function of gt in BSF might be analogous to that of its homologs in other insect species.

Fig. 1. Ablation of gt results in larger body size but not affect development.

a Schematic diagram of the life history of Hermetia illucens. b Maximum likelihood based phylogenetic tree of Giant proteins from Hermetia illucens (XP_037916866.1), Drosophila melanogaster (NP_525049.1), Culex quinquefasciatus (XP_001849886.2), Aedes aegypti (XP_001653816.1), Musca domestica (XP_005177400.2), and Apis dorsata (XP_006612918.1). Numbers are fragment lengths in amino acids. c PCR analyses of the gt homozygotes with mutation checking primers. WT: wild type; M1: line 1; M2: line 2. d The phenotype of the D12 larvae of WT and Δgt strain. Scale bar: 1 cm. e The average weight of WT and the strains at D12 (n = 3 biological independent experiments with 40 individuals from 4 replicates). f Percent pupation of late fifth instar stage for WT and Δgt strains (n = 4 replicates with 20 individuals). g The average weight of WT and Δgt strains at adult stage (n = 3 biological independent experiments with 20 individuals from 4 replicates). Statistical comparisons between WT and Δgt strains were examined using one–way ANOVA with Tukey’s multiple comparison test. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

CRISPR/Cas9 induced gt mutation and phenotypic changes

Based on the high similarity between Gt protein sequences in BSF and other Diptera species (Fig. 1b, Supplementary Fig. 1), we propose that gt may also regulate development in BSFL. To validate the role of gt in the growth of BSFL, we utilized the CRISPR/Cas9 system. Two specific target sites within exon 2 of the gt locus were selected for mutagenesis (Fig. 1b). After two generations of crossing, the homozygous strains of gt were generated, and PCR analysis and sequencing confirmed the deletion of large fragments with frame shift mutation in both strains (lines 1 (–316 + 4 bp) and 2 (–308 + 6 bp); Fig. 1c, Supplementary Fig. 2a). The size of Gt protein in WT was 39.3 kDa, and the remaining proteins of both mutated strains were 19.8 kDa.

We first focused on the weight of larvae and the growth rate. Under standardized rearing conditions, the gt mutant strains exhibited a notable increase in weight compared to WT from 4 days after hatching (DAH) onward (Fig. 1d, Supplementary Fig. 2b). The average weight of 40 freshly harvested mutant larvae (line 1: 0.284 g, line 2: 0.273 g) were significantly higher than WT (0.243 g) at 12 DAH (Fig. 1e). This translated to a growth enhancement of 16.8% (line 1) and 12.3% (line 2) in individual mutants. Importantly, despite the difference in weight, there was no impact on the development rate of the mutants (Fig. 1f, Supplementary Fig. 2c), which indicated an accelerated growth rate in the mutants. Furthermore, in contrast to WT counterparts, the mutant pupae and adults consistently exhibited larger body sizes and higher weights (Fig. 1g, Supplementary Fig. 2d–f). To investigate the regulation mechanism of gt in growth, we analyzed mutant growth traits. Notably, Δgt larvae exhibited significantly larger body size than WT from the neonatal stage (Supplementary Fig. 3a, b). Given insulin–like peptides (ILPs) mediated growth–nutrition coupling in insects³², we hypothesized that gt mutants exhibited upregulated ilps expression to increase neonatal weight, promoting larger size. Indeed, ilp2, ilp3 and ilp5 (associated with growth and development³³) expression was significantly elevated in mutants at 24 and 36 h of the first instar compared with WT (Supplementary Fig. 3c–e), demonstrating knockout of gt activated ilps at the neonatal larvae stage to drive growth in BSF. Furthermore, in D. melanogaster, the gt mutation has been associated with the stochastic loss of prothoracicotropic hormone (PTTH) production in PG neurons, resulting in the development of giant larvae, pupae, and adults²⁸. Thus, we evaluated the expression of ptth in the brain of gt homozygous mutants using RT–PCR and qRT–PCR, which showed a significant decrease compared to WT individuals, as observed in D. melanogaster (Supplementary Fig. 4).

Disruption of gt enhances larval conversion efficiency

To evaluate the conversion ability of the mutant strains, we conducted experiments through the disposal of wheat bran (WB). Mass reduction serves as a visual indicator of waste digestion efficacy, a crucial measure of degradation efficiency, and a key determinant of insect bio–transformation rates. The total BSFL yield is a direct outcome of insect rearing. Analysis of WB disposal revealed that the engineered strain exhibited a slightly higher mass reduction rate compared to WT (Fig. 2a). However, the BSFL yield of the strain was noteworthy, reaching 4.78 g (wet weight, WW) and 1.60 g (dry weight, DW). This represents a substantial increase of 15.46 and 12.68%, respectively, compared to the WT value (WW: 4.14 g and DW: 1.42 g) (Fig. 2b). The bioconversion efficiency in WW of the mutant (WW: 4.63% and DW: 5.33%) significantly exceeded that of the WT (WW: 4.27% and DW: 5.06%) (Fig. 2c). In addition, we assessed the fat and protein content of the mutants. The strain exhibited fat (33.58%) and protein (35.01%) contents were similar to that of WT (33.54 and 35.94%) (Fig. 2d, e). Furthermore, there were no significant differences in the relative content of fatty acids (FA) in BSFL between WT and the strain during disposing of WB, with lauric acid (C12:0) being the predominant FA, consistent with previous findings (Fig. 2f)^34,35. These results show that the mutant strain achieves an elevation in total nutrient yield along with enhanced weight gain compared to the WT.

Fig. 2. The Δgt strain has an excellent conversion ability.

a The mass reduction rate of the wheat bran treated with WT and Δgt strain. b The larval yield after disposal of wheat bran. c The bioconversion efficiency of treating with wheat bran (a–c: n = 4 replicates with 20 individuals). WW: wet weight, DW: dry weight. d–f The nutrient content of WT and Δgt strain treated with wheat bran. d The fat content; e the protein content; f the proportion of fatty acids (d–f: n = 3 replicates with 5–7 individuals). Statistical comparisons between WT and Δgt strain were examined using two–tailed, unpaired t–tests. Data are shown as mean ± SD. *P < 0.05, **P < 0.01. ns, not significant.

The Δgt strain exhibits higher organic waste disposal capabilities

In addition to the disposal of WB, to approach the application of BSF in different scenarios, we evaluated the FW disposal capabilities of this strain and compared nutrient compositions between mutants and WT. Table 1 illustrates the capacity of the mutants in conversion of FW. First, the survival rate was higher in the strain population (95.17%) than in the WT (90.50%), suggesting the strain has a stronger ability to adapt to the environment. Remarkably, the engineered strain achieved mass reduction rates of 67.82% (WW) and 52.51% (DW) in FW treatment, surpassing the rates observed in WT (WW: 55.82% and DW: 46.82%). And the BSFL yield of the strain (WW: 127.81 g; DW: 44.99 g) was significantly heavier than WT (WW: 104.65 g; DW: 35.47 g). Strikingly, the increased rate of BSFL yield of the strain was significantly higher when treating with FW compared to WB (Fig. 3a). To determine whether the elevated yield of the strain was entirely due to a high survival rate, the BSFL average weight was measured. The results showed that the mutant BSFL (WW: 0.27 g; DW: 0.09 g) was significantly heavier than WT (WW: 0.23 g; DW: 0.07 g) (Table 1, Fig. 3b). While the conversion efficiency in WW remained relatively stable, the DW treated by the mutant exhibited a significant increase (32.55%) compared to WT (28.78%) in DW (Table 1). After 6 days of treatment, the moisture content in the mutant–treated group (61.18%) was significantly lower than WT (68.31%). Furthermore, compared to WT, the mutant strain achieved more thorough waste disposal, leading to lesser and drier waste residue (Fig. 3c), and concurrently, a higher larval yield (Fig. 3d, e). Furthermore, the nutrient analysis showed no difference in fat and protein contents between WT and the strain (Fig. 3f, g). However, the mutant exhibited a significantly higher relative content of long–chain FA (oleic acid and linoleic acid) along with a decrease in short–chain lauric acid content compared to WT (Fig. 3h), which suggests that the mutants differed from WT in the way they store and metabolize nutrients. In addition, in order to investigate the stability and universality of the strain, we conducted two consecutive generations of conversion capacity measurements using FW, distiller’s grain (DG), and vegetable waste (VW). The results showed that the bioconversion efficiency for FW (1st: 12.16%, 2rd: 13.46%), DG (1st: 13.65%, 2rd: 21.31%), and VW (1st: 18.73%; 2rd: 16.31%) increased by over 10% when using this strain (Supplementary Fig. 5). In summary, the gt mutant strains enhance body weight with increased growth rates and exhibit a higher conversion efficiency for different organic wastes.

Table 1.

The bioconversion ability of WT and the Δgt strain fed with food waste

	Wet weight		Dry weight
Index	WT	Δgt	WT	Δgt
Waste reduction rate (%)	55.82 ± 3.29	67.82 ± 1.41**	46.82 ± 1.55	52.51 ± 3.75ns
BSF larval yield (g)	104.65 ± 5.77	127.81 ± 4.04**	35.47 ± 1.29	44.99 ± 1.79**
Average larval weight (g)	0.234 ± 0.011	0.271 ± 0.005*	0.073 ± 0.002	0.087 ± 0.002**
Bioconversion efficiency (%)	18.75 ± 0.29	18.85 ± 0.11ns	28.78 ± 0.69	32.55 ± 1.69*

Statistical comparisons between WT and Δgt groups were examined using two–tailed, unpaired t–tests. Data are shown as mean ± SD. *P < 0.05, **P < 0.01. ns not significant.

Fig. 3. The bioconversion ability of the Δgt strain is significantly higher than WT upon food waste treatment.

a The yield increase rate of the strain treated with wheat bran and food waste (wheat bran group: n = 4 replicates with 20 individuals; food waste group: n = 3 replicates with 600 individuals). b The average weight of WT and Δgt strain, and the increase rate of Δgt strain after 6 days of food waste disposal (n = 3 replicates with 600 individuals). WW: wet weight, DW: dry weight. c Comparison pictures of the same quality of food waste before and after being treated with the same amount of WT and Δgt strains for 6 days. Scale bar: 10 cm. d,e The larvae of WT and Δgt strain harvested from waste disposal. Scale bar: 5 cm (d), 1 cm (e). f–h The nutrient content of WT and Δgt strain treated with food waste. f The fat content; g the protein content; h the proportion of fatty acids (n = 3 replicates with 5–7 individuals). Statistical comparisons between samples were examined using two–tailed, unpaired t–tests. Data are shown as mean ± SD. *P < 0.05, **P < 0.01. ns, not significant.

The Δgt strain exhibits enhanced fertility

Apart from the bioconversion ability, the reproductive ability of the mutants is also an essential trait of a strain for industrial production. The mating proficiency of the mutants was evaluated in 2 h. The mating rate of line 2 showed no significant difference compared to the WT (Fig. 4a) while the heavier line 1 exhibited a lower mating rate, which may be attributed to the impaired physical agility associated with increased body weight (Fig. 4a). However, with the increase of time, line 1 demonstrated comparable mating frequency to WT individuals, indicating normal mating ability of the mutants (Fig. 4a). What is more, oviposition capacity and fertilization rate remain unaffected in the mutant strain, both remaining consistently around 80.00% (Fig. 4b, c). Intriguingly, both strains produced heavier egg mass than WT, with increases of approximately 55.53% (line 1) and 53.57% (line 2) (Fig. 4d, e). The weight and size of the embryo showed no difference with WT, which pointed to a greater offspring yield (Supplementary Fig. 6). In summary, the mutants exhibit superior reproductive capabilities compared to WT.

Fig. 4. The reproductive ability of Δgt strains is comparable with WT.

a The mating rate of WT and Δgt strains within 2 h. b The egg laying rate of WT and Δgt strains. c The fertilization rate of WT and Δgt strains b,c: n = 3 replicates with 15 pairs). d The phenotype of egg mass of WT and the strains. Scale bar: 1 cm. e The average weight of the egg mass of WT and Δgt strains (n = 3 biological independent experiments with 25 individuals from 3 replicates). Statistical comparisons between WT and Δgt strains were examined using one–way ANOVA with Tukey’s multiple comparison test. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

The Δgt strain leads to alterations in the cuticle

Furthermore, we explored the gene expression profile of mutants and WT at the third larval stage, during which BSFL digest organic waste and convert to biomass. PCA analysis shows only minor differences between mutants and WT individuals (Supplementary Fig. 7a). Genes showing significant differences between the two groups were selected for the heatmap, among which most are related to invertebrate chitin–binding proteins and trypsin–like serine proteases superfamilies (Supplementary Fig. 7b, Fig. 5a, Supplementary Table 3). More genes are down–regulated in Δgt strain and enriched in serine–type endopeptidase activity, chitin binding and extracellular region (Fig. 5b, Supplementary Fig. 7c). Accordingly, thinner cuticles could be observed in the mutants comparing with WT individuals (Fig. 5c, Supplementary Fig. 8). Meanwhile, we found DCXR (Dicarbonyl/L–xylulose reductase) was highly expressed in mutants (Supplementary Fig. 7b, Supplementary Table 3), which played a dual role in carbohydrate metabolism and detoxification in other organisms³⁶.

Fig. 5. Transcriptome analysis between Δgt strain and WT.

a The heatmap of differentially expressed genes between WT and mutants. b GO enrichment analysis of down–regulated genes in the Δgt strain. c Cross–section of cuticle in the WT and Δgt strain at day one in the third larval stage using hematoxylin and eosin staining. Scale bar: 100 μm. The black line indicates the thickness of cuticle. And the thickness of cuticle of WT and Δgt strain was recorded (n = 6 individuals). Statistical comparison was examined using a Mann–Whitney U–test. **P < 0.01.

Since Gt protein was identified as a transcription factor³⁷, and given the conservation of bZIP transcription factors (Fig. 1b, Supplementary Fig. 1), we explored the binding sites using putative sites obtained from D. melanogaster³⁷. However, only 52 hits in BSF were obtained from 1861 putative binding sites of fruit fly, suggesting highly variable regulatory sequences between species. We further extracted genes that overlapped with these putative binding sites to get a closer view of the regulation network in BSF. Notably, 18 out of 31 genes are transcribed into small nuclear RNAs (snRNAs), which are involved in the splicing of precursor messenger RNA (Supplementary Table 4)³⁸. As the expression of gt peaks in the early embryonic stage (Supplementary Fig. 9), we propose that Gt protein may affect the transcription process as a whole during this period. In summary, bioinformatic analysis reveals a relative stable developmental process of mutants, further supporting the application of Δgt strain.

The Δgt strain demonstrates inferior competitive reproductive capacity

To evaluate the ecological safety of the gene–edited BSF, we conducted competitive fitness assays (Fig. 6a). The larva group was designed to simulate the scenario where mutants leak into the wild and compete with wild-type individuals. The adult group aims to compare reproductive capacity directly. The results revealed distinct patterns between the two cohorts. In the larva group, offspring mutation frequency was low (<10%) at day 2 (D2) post–eclosion then increased at D4 and D6 before stabilizing by D8, with overall mutation rates not exceeding 30% (Fig. 6b). The first homozygous offspring was not observed until D8 post–eclosion (Supplementary Fig. 10). In the adult group, offspring mutation frequency showed less variation across time points, consistently stabilizing at approximately 30% (Fig. 6c, Supplementary Fig. 10). A large proportion of mutants were heterozygote (D2: 11/12, D4: 14/15, D6: 15/17; D8: 9/12; Supplementary Fig. 10). This indicates that in mixed WT–mutant populations, homozygous mutants rarely achieve successful biparental mating, suggesting their competitive reproductive disadvantage. Furthermore, the chi–square tests revealed that the genotype ratio of offspring showed significant deviations (P < 0.001) compared with the expected values across all experimental groups (Supplementary Data 2), showing a significantly lower proportion of the mutant than expected. Overall, these results demonstrate a low invasive potential of this strain, thereby supporting its application.

Fig. 6. Disadvantaged competitive reproductive capacity in Δgt strain.

a Schematic diagram of the competitive mating experiments of WT and Δgt strain. The mutation rate in the offspring of the larvae group (b) and adults group (c). Mutation rate indicates the proportion of offspring carrying the gt mutation (heterozygote plus homozygote). Statistical comparisons between WT and Δgt strain were examined using one–way ANOVA with Tukey’s multiple comparison test. Data are shown as mean ± SD. ns, not significant.

Discussion

The bioconversion of organic wastes is becoming an environmentally sound and economically attractive practice due to the rapidly increasing demand for waste disposal and energy supply, as well as the growing public concern about environmental quality³⁹. BSFL are a viable solution for mitigating the environmental impact of FW, as they can provide a sustainable protein source to support the increasing global population^4,12. The main limitation of the large–scale application of BSF is the variable efficiency and yields when treated with different kinds of organic waste, and there is an urgent need to improve the efficiency of larval transformation¹⁵. Current research in BSF multi–condition applications mainly focuses on waste pre–treatment with functional probiotics (e.g., Lactobacillus buchneri, Bacillus subtilis) instead of BSF breeding⁴⁰. To the best of our knowledge, the study proposed and implemented molecular breeding in BSF using the CRISPR/Cas9 system for the first time. The gt mutant exhibited several positive traits, such as increased body weight with more biomass production, improved bioconversion efficiency of diiferent waste types, and enhanced fertility. In short, through molecular breeding, we obtained superior lines with the potential to be used in actual production.

In comparison with the phenotypes observed in D. melanogaster²⁸, gt mutants of BSF show no developmental delay (Fig. 1f, Supplementary Fig. 2c), suggesting their divergence in the regulation of the gt gene networks. Based on the neonate larval phenotype, we demonstrated that the gt mutation regulated early ilps expression, resulting in increased initial body weight in mutants (Supplementary Fig. 3). However, the underlying mechanisms require further investigation. To elucidate the molecular basis of gt mutant phenotypes in BSF, species–specific data are necessary. Given that the expression of gt peaked in the early embryonic stage (Supplementary Fig. 9), ChIP–seq of Gt protein at the corresponding time, for example, could provide more information about the regulatory targets of this protein.

The transcriptome analysis revealed little variation between gt mutants and WT (Fig. 5a, b), which suggests the development of mutants are relatively stable, supporting potential application in the industry. Based on these findings, coupled with the severe phenotypic alterations observed in early–stage mutant larvae (Supplementary Fig. 3), we hypothesized that GT exerted its critical regulatory role in growth and bioconversion as early as the first instar stage. As for the observation of thinner cuticle structure, given that chitin was rigid (Fig. 5c), we hypothesized that it could be an adaptation to the rapid growth of mutants. At the same time, the reduction in the synthesis of cuticle may could be converted to the addition of body weight. In addition, a significant decrease in the content of lauric acid was observed in the mutants during the treatment of FW (Fig. 3h). In insects, FA derivatives, wax esters, fatty alcohols and hydrocarbons are important components of the integument⁴¹. The different diet such as WB and FW would influence FA profiles. Therefore, this decrease might be related to the changes in cuticle phenotype and diet composition, which could be offset by the increase in biomass, maintaining its overall production yield.

Improving conversion efficiency has always been a goal that researchers pursued. Previous studies have shown that the gut microbiota plays an important role in the conversion of organic waste in BSF. For example, when BSFL are fed with cellulose–degrading bacteria, an increase in bioconversion rate could be observed⁴². In another study, the introduction of Bacillus cereus and Bacterium YC–LK–LKJ45 to food waste led to a significant increase of 53.08% in total larval yield and a 60.78% rise in the food waste conversion rate of BSFL¹⁵. Besides, artificial selective breeding has been proven to be a reliable approach. In automated production settings, a consistent 39% augmentation in larval weight was achieved in the selected line compared to the control line⁴³. However, some problems hinder the wide application of these methods. For example, the screened bacteria are often applicable only in specific scenarios and lack versatility across various settings⁴², and selective breeding can be time–consuming^43,44. In contrast, molecular breeding optimizes strains through intrinsic genome modification, reducing the breeding cycle and leading to a more stable strain. Therefore, molecular breeding holds promise for widespread application.

In addition to improving the yield and conversion efficiency of BSF, characteristics such as fertility, nutritional content, and stress tolerance (e.g., heat and pathogens) could be enhanced through genetic manipulation. In the BSF industry, the limited reproductive ability is a bottleneck that hinders large–scale rearing⁴⁵. Simultaneously, the cost–effective, high–quality protein resources offered by BSF can serve as a viable alternative to soybeans and other crops in livestock feed processing⁴⁶. Furthermore, as the pathogen that results in a high mortality rate of BSF larvae has been identified⁴⁷, resistant strains with edited genomes are needed to halt the spread in production. Despite leveraging the qualities of BSF itself, employing BSF as a bioreactor with molecular modification is also a promising development direction, as efficient synthesis of human extracellular superoxide dismutase in transgenic B. mori has been achieved⁴⁸. One interesting aspect of molecular breeding is that it can generate strains with a combination of desirable traits through simple crossing, thereby further reducing the breeding time. And Δgt strain can serve as an excellent chassis to cross with various transgenic BSF strains that synthesize high–value biomolecules to increase production capacity.

Although we have already implemented several genetic modification approaches in BSF, limitations still exist in precise editing for desirable traits. Firstly, unlike traditional livestock, the genetic diversity of farmed insects like BSF remains insufficiently characterized. Although a prior study has established the global population genetic structure of BSF⁴⁹, there is still a need for a systematic collection of genetic resources and comprehensive statistics, and further pan–genomic analysis is required to enhance functional genomic research and germplasm innovation, such as identifying loci controlling quantitative genetic traits⁵⁰. Moreover, addressing the diverse and urgent demand for BSF germplasm resources in the global market requires immediate attention to the research and development of large–scale functional gene studies and high–throughput genetic breeding techniques for BSF. Notably, large–scale mutant libraries constructed using CRISPR and mixed–pool technology in B. mori enable the swift screening of target phenotypes and functional genes⁵¹. Lastly, in response to societal demands for transparency, organizations engaged in research on insect genetics must embrace ethical guidelines and comply with best practices for insect breeding and reproduction⁵². In this study, through competitive fecundity assays, we demonstrated that the gt mutant exhibits negligible transmission risk.

Managing and disposing of organic waste pose serious challenges³. Global waste management statistics indicated that the worldwide generation of organic waste reached 2.01 billion tons in 2016⁵³. The Food and Agriculture Organization of the United Nations (FAO) reports that approximately 1.3 billion tons, one–third of global food production for human consumption have been lost or wasted per year⁵⁴. By 2050, global urban municipal solid waste is expected to reach 9.5 billion tons per year, with FW constituting 25–70% of this amount^55,56. In addition to the large scale, the heterogenous composition and dynamic variability of organic waste increase the difficulty of management^57,58. Thus, cost–effective and environment–friendly approaches, represented by BSFL should be paid more attention and adapted to the practical application scenarios.

As a good source of insect–derived protein, BSF–derived products also meet the demand for pet food and aquaculture feed, which is expected to grow 50 times 2030⁴³. To date, the investments in the insect farming industry for pet diet have exceeded €1 billion and the industry may generate 25,000 jobs by 2030 in Europe⁵⁹. The trend of commercial BSF rearing cannot be halted. From the biomanufacturing perspective, the improved bioconversion efficiency of Δgt strain may generate significant promotion in the industry.

In summary, our research provided compelling evidence supporting the improved genetic strains achieved through molecular breeding in the resource insect BSF, emphasizing the potential for genetically edited chassis to enhance biomanufacturing industry. These strains would undergo further investigation to assess their suitability for industrial–scale production. Moreover, our findings could also be extendedto the entire resource insect industry, and provide interdisciplinary experience to solve environmental problems.

Methods

Insect rearing and waste biomass

The BSF strain (Wuhan, China) was kept in the CAS Center for Excellence in Molecular Plant Sciences (CEMPS, Shanghai, China). The larvae were reared at 28 ± 1 °C and 55 ± 5% relative humidity with a 12 h light/12 h dark photoperiod and were fed an artificial diet comprising 40% WB and 60% water. The adults were kept in the mesh cages with water daily supplied to drink. FW and VW were collected from the student cafeteria of CEMPS, and homogenized into small pieces. DG was from the distillery in Fenyang, Shanxi.

Gene cloning and sequence analysis

Total RNA was extracted from embryos or other tissues at different developmental stages using the TRIzol reagent (Transgen, Beijing, China). First–strand cDNA was synthesized using the PrimeScript^TM RT reagent with a gDNA Eraser kit (TaKaRa, Dalian, China) using 500 ng total RNA. The putative gt gene was identified from the assembled genomic database on BSFbase (https://insectomics.net/BSFbase/)⁶⁰. The open reading frame of gt was amplified using KOD plus polymerase (TOYOBO, Tokyo, Japan). The purified PCR products were confirmed by Sanger sequencing after cloning into the pJET1.2–T vector (Thermo Fisher Scientific, Ohio, USA). All primers used in this work are listed in Table S1.

Amino acid homology analysis of gt (XP_037916866.1) was performed using the NCBI protein–protein BLAST. Full–length proteins were aligned using the ClustalW program in MEGA X⁶¹, and the alignment presentation was generated with the ESPript 3 program⁶². The maximum likelihood trees were constructed and adjusted using MEGA X software and Evolview v3^61,63, and the statistical robustness was assessed through the bootstrap method with 1000 replicates.

SgRNA synthesis

To target gt, two 23–nucleotide single–guide RNAs (sgRNAs) were designed following the oligonucleotide criteria 5’–GG–(N)18–NGG–3’⁶⁴. sgRNAs were synthesized in vitro using the MEGAshortscript^TM T7 kit (Thermo Fisher Scientific, Ohio, USA) following the manufacturer’s instructions. Subsequently, sgRNAs were purified with chloroform and stored at −80 °C.

Generation of homozygote mutants

Fresh embryos were collected and injected with the mixture of Cas9 protein (300 ng/μL, TrueCut^TM Cas9 Protein v2, Thermo Fisher Scientific, Ohio, USA) and sgRNAs (150 ng/μL). Injected eggs were kept at 28 ± 1°C, 60 ± 10% RH until hatching. The larvae were reared on an artificial diet and adults were crossed with the WT insects. The genotype of the offspring was detected through PCR amplification and sequencing to select heterozygote mutants with large fragment deletion at the genomic locus. Homozygotes were screened after twice crossing of the heterozygotes with the same genotype. Two homozygote strains were obtained and self–crossed for at least three generations to keep the phenotypes stable.

Qualitative and quantitative real–time PCR analyses

Total RNA extracted from the brain of 10 mutants or WT larvae at the L5D1 stage with three replicates was used to assess ptth expression. And RNA from 10–15 whole body with three replicates of 0, 12, 24, 36 h neonate was used to detect ilps expression. First–strand cDNA was synthesized as described before. Quantitative Real–time PCR (qRT–PCR) was performed using SYBR Green Real–time PCR Master Mix (Yeason, Shanghai, China). rp49 gene was used as the internal control, and the transcription level was calculated using the 2^–ΔΔCT method. Three independent biological replicates were used. Reverse Transcription PCR (RT–PCR) was carried out using 2×Hieff^® PCR Master Mix (Yeasen, Shanghai, China).

Body weight, developmental timing, and mating behavior analyses

To evaluate the ability of BSFL growth and development, hatched neonates from two mutant colonies and WT were raised with the artificial diet for 4 days. Four replicates, each containing 20 BFSL from each colony, were randomly selected and placed in a uniform size box with 120 g feed. The diets were exchanged every 2 days. Larvae were weighed every 2 days, and the developmental stages were recorded until pupation. For mating behavior analysis, three replicates with fifteen pairs of mature adults from each colony were allowed to mate for 2 h in a mesh cage, with mating rate recorded every 20 min. The egg laying and fertilization rate of each group and the weight and length of the eggs were recorded. The experiments were repeated three times.

Conversion performance analyses

The conversion experiments were conducted under different treatments. Under the laboratory condition, four replicates of 20 BSFL of 4–day mutant or WT were fed on 250 g of WB (65% moisture) in a 500 mL plastic box. For production condition, 1 kg FW was provided for 600 BSFL of 4–5 d mutant and WT in a plastic crate (20 × 33 × 10 cm) with three replicates. To investigate the stability and universality of the strains, 600, 300 and 100 BSFL of the mutant and WT were fed in the plastic crates with 1 kg FW, 900 g DG and 300 g VW, respectively with three replicates, to test the ability to conversion different types of wastes. The experiment was conducted for two consecutive generations. The experiment continued until the first prepupae appeared in the rearing units (WB: 8 d; FW: 6 d; VW, DG: 10 d). The mutant line 1 was used for all experiments.

The conversion ability of the mutant was evaluated by BSF larval growth indices, including BSF larval yield, bioconversion efficiency, and substrate reduction, fat, and protein content. Briefly, the initial weight of the larvae was recorded before inoculating in the feed. After conversion, the BSFL and frass were separated using a vibrating grading sieve, weighed, and air–dried for dry weight. Waste reduction rate (WRR), survival rate (SR), and bioconversion efficiency corrected for residue (BER) were calculated using the following equations:

• Larval yield = Final larval weight – Initial larval weight WRR (%) = (Initial substrate weight – Residue weight)/Initial substrate weight × 100 SR (%) = Final amount of larvae/Initial amount of larvae × 100

• BER (%) = (Final larval weight – Initial larval weight)/(Initial substrate weight – Residue weight) × 100

Measurement of protein content

The total protein content of larvae after treatment with WB and FW was determined using a Dumas combustion assay, following the procedure outlined by Huang et al⁶⁵. Briefly, the samples with three replicates were dried to a constant weight at 70 °C and then powdered using a grinder (TL–48 R, Shanghai Jingxin Industrial Development, Shanghai, China; 90 Hz, 60 s). Approximately 50–60 mg of the powdered sample was encased in tin foil for testing. The total nitrogen content was determined using the Dumas rapid nitrogen analyzer (rapid N exceed, Elementar, Frankfurt, Germany). The program settings were configured as follows: O₂ dosing time, 60 s; O₂ dosing flow, 120 ml min⁻¹; O₂ cut–off threshold, 15%; Autozero delay, 30 s; and peak anticip., 90 s. At the same time, the packaged samples were placed into the sample tank according to the corresponding serial number. The protein content was calculated from total nitrogen using the nitrogen–to–protein conversion factor (Kp) of 4.76.

Measurement of fat content

The fat content and FA profile of BSFL was determined according to standard methods (GB5009.168–2016) with slight modifications. Briefly, about 0.5 g of the dried sample with three replicates was placed in a 4 mL vial and thoroughly ground, and the weight of the vial before and after grinding was recorded accurately. Then 3 mL of hexane was added to the vial, shaking at 25 °C, 1000 rpm to extract the total fat. After a 6 h extraction, the solution was centrifuged at 3000 × g for 5 min and transferred 2 mL of the supernatant to a new vial for nitrogen blowing at 60 °C for 30 min. The difference in the weight of the extract in the vial before and after blowing was precisely recorded. Using the quality data and hexane density, the crude fat content was calculated.

To determine the FA profile, 5–10 mg of the above crude extract was transferred to a new vial of known mass. Then 1 mL of 2% NaOH–MeOH was added, and the vial was shaken at 1000 rpm for 1 h. Subsequently, 1 mL of 10% BF₃–MeOH was added and shaken at 1000 rpm for 30 min. Next, 1 mL of hexane and 0.5 mL of saturated NaCl was added accurately, and the mixture was extracted by shaking. After centrifugation at 3000 × g for 3 min, 200 µL of supernatant was mixed with Copper (II) sulfate for water removal. After instantaneous centrifugation, 100 µl of supernatant was analyzed by GC2010 pro GC systems (Shimadzu, Suzhou, China) coupled to a flame ionization detector (FID), and a TG–5MS capillary column (30 × 0.25 × 0.25 µm, Thermo Scientific). The GC program was as follows: initial temperature of 120 °C; ramp to 165 °C at a rate of 30 °C min⁻¹; then increased by 2 °C min⁻¹ to 215 °C. The temperature of the injector port and detection were 250 °C and 260 °C, respectively. The sample volume per injection was 2 µL in a split ratio of 20. The flow rate of carrier gas (nitrogen) was kept at 1.24 mL min^–1.

Bioinformatic analysis

Total RNA was extracted from the whole body from day one in the third larval stage WT and gt mutants. The mRNA was enriched, and then fragmented and used for cDNA synthesis and library construction. The library was sequenced using Illumina Hiseq Sequencing 2000 System. Sequence data are available from the NCBI Short–Read Archive (SRA) database (accession numbers PRJNA1132557). The raw reads were filtered using Trimmomatic v0.39⁶⁶, and then were mapped to the reference genome of BSF using STAR 2.7.6a^67,68. FeatureCounts (https://subread.sourceforge.net/featureCounts.html) was applied to generate expression matrix, and downstream analysis of differentially expressed genes was performed using DESeq2⁶⁹. Genes that change over 2 folds with significant P–value are recognized as differentially expressed genes. Proteins of BSF are annotated with InterProScan⁷⁰. The GO enrichment analysis was performed using ClusterProfiler2⁷¹.

The BED file of gt binding sites of D. melanogaster was retrieved from ENCODE and converted to FASTA format using bedtools with dm6 assembly⁷². The candidate binding sites of D. melanogaster were used as the query sequences for performing BLAST in H. illucens genome.

Paraffin sectioning and hematoxylin–eosin staining

Epidermis of WT and the mutant were dissected from the day one in the third instar larvae and prefixed with Qurnah’s fixative (anhydrous ethanol: acetic acid: chloroform, 6:1:3 (v/v/v)) for 24 h. Samples were dehydrated using anhydrous ethanol for three times (1 h per time), followed by clearing for three times (10 min per time) using xylene. Tissues were embedded in paraffin overnight and were cut into cross–sections (5 μm) with a Leica RM2235 micro–tome. The sections were rehydrated and then stained using a mixture of hematoxylin and eosin to visualize morphology. The stained sections were photographed by using an Olympus BX53 microscope.

Photographs were aligned and measured using Adobe Illustrator, and the unit was converted to micrometers (μm), with a bar representing 100 μm for scale. On each sample’s cuticle intersection, three points were selected for measurement, and the mean value was used for plotting. The values of the two groups were compared statistically using a Mann–Whitney U–test.

Competitive reproductive fitness assay

To assess potential ecological risks associated with strain dissemination through escaped adult BSF, we conducted a competitive reproductive fitness evaluation of the gt strain using a dual experimental approach. The study comprised the larva and adult experimental groups with three replicates. For the former, Equal biomass (0.01 g) of egg of WT and homozygous gt mutants was co–reared to adulthood, and transferred to mating cages. The same cage of adults was maintained for continuous ad libitum mating at post–eclosion days 2 (D2, sexual maturity), 4, 6, and 8. The mating assay was conducted under artificial lighting. Trials continued until 20 successful mating pairs were established per replicate. Post–trial, adults were transferred to darkness to terminate mating activity. After a 2–day interval, they were re–exposed to light to resume mating until D8. For the latter, age–synchronized WT and gt adults (four groups: D2, D4, D6 and D8 unmated adults after eclosion; 20 males plus 20 females per strain) were co–housed and mated in mating cages. The virgin adults were maintained in darkness until reaching the target age, then directly transferred to light conditions for the mating assay. The termination criterion was the same as the larvae group. Fifteen F1 progeny per replicate with both groups were randomly selected for genotyping using target–site PCR (WT: 590 bp; mutant: 278 bp). Competitive fitness was quantified by chi–square tests comparing the observed versus expected frequency of the mutation in progeny from competitive mating assays (expected values: WT: 25%; gt heterozygote: 50%; gt homozygote: 25%).

Statistics and reproducibility

Statistical analysis was conducted using GraphPad Prism 8.0.0. Statistical significance was assessed using a two–tailed Student’s t–test for comparing two groups and a one–way analysis of variance for comparisons involving three or more groups, followed by Tukey’s multiple comparison test. The data are presented as means ± SD, and statistical significance was assumed for P < 0.05. The exact number of insects and repetitions for each experiment are indicated in the Supplementary Data file, Figures and the Figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

S.Z.W., Z.K., Y.J., and S.T.W. carried out experiments and analyzed the data. J.K.T. helped interpret the results. Y.H., S.T.W., and W.Z. designed the study. S.Z.W. and S.T.W. wrote the manuscript with input from all coauthors. All authors proofread and approved the manuscript.

Peer review

Peer review information

Communications Biology thanks Lianhai Ren and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: Ophelia Bu.

Data availability

Uncropped gel images are included as Supplementary Information. The source data behind the graphs in the paper are provided as Supplementary Data 1–2. Sequence data are updated to SRA database (PRJNA1132557).

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

No ethical approval was required.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-025-08571-1.