An Acyl Carrier Protein Gene Affects Fatty Acid Synthesis and Growth of Hermetia illucens

Simple Summary Fatty acids play essential roles in insect growth and development. The key fatty acid synthesis reaction is catalyzed by fatty acid synthase (FAS). As a cofactor of fatty acid synthesis, however, little is known about the functions of acyl carrier protein (ACP) in insects and how it modulates the composition of fatty acids. We isolated an ACP from the black soldier fly Hermetia illucens (Diptera: Stratiomyidae) and investigated the expression patterns as well as functions in fatty acid synthesis and growth of insects. HiACP has a classical conserved region of DSLD. Based on the RNAi approach, we found that HiACP could regulate the fatty acid synthesis, in particular, reducing the composition of saturated fatty acids. Meanwhile, HiACP could also affect the growth and mortality of H. illucens. Abstract Acyl carrier protein (ACP) is an acyl carrier in fatty acid synthesis and is an important cofactor of fatty acid synthetase. Little is known about ACP in insects and how this protein may modulate the composition and storage of fatty acids. We used an RNAi-assisted strategy to study the potential function of ACP in Hermetia illucens (Diptera: Stratiomyidae). We identified a HiACP gene with a cDNA length of 501 bp and a classical conserved region of DSLD. This gene was highly expressed in the egg and late larval instars and was most abundant in the midgut and fat bodies of larvae. Injection of dsACP significantly inhibited the expression level of HiACP and further regulated the fatty acid synthesis in treated H. illucens larvae. The composition of saturated fatty acids was reduced, and the percentage of unsaturated fatty acids (UFAs) was increased. After interfering with HiACP, the cumulative mortality of H. illucens increased to 68.00% (p < 0.05). H. illucens growth was greatly influenced. The development duration increased to 5.5 days, the average final body weights of larvae and pupae were decreased by 44.85 mg and 14.59 mg, respectively, and the average body lengths of larvae and pupae were significantly shortened by 3.09 mm and 3.82 mm, respectively. The adult eclosion rate and the oviposition of adult females were also severely influenced. These results demonstrated that HiACP regulates fatty acid content and influences multiple biological processes of H. illucens.


Introduction
Fatty acids play essential roles in animal health and development [1]. They can be esterified by glycerol and serve as energy sources (triglycerides) within adipose tissue. Fatty acids are also present in cell membrane phospholipids involved in cell structure and membrane fluidity. They stimulate signaling processes, including immune response or metabolic functions [2,3]. In insects, fatty acids are crucial for embryonic development, metamorphosis, and reproductive processes [4][5][6]. To obtain fatty acids, insects have evolved a system for their de novo synthesis.

Sequence Analysis and Quantification of the HiACP Gene
The nucleotide sequence of cDNA was transformed into an amino acid sequence by the Translate tool implicated in Expasy. HiACP gene sequences of other insect species were downloaded by the NCBI protein database [39]. The physicochemical properties of protein were predicted by Expasy [40]. The signal peptide was predicted using SignalP [41]. Protein transmembrane structure was predicted by TMHMM [42]. Subcellular localization was performed by Euk-multi-2 [43]. Prediction of phosphorylation sites was performed by NetPhos [44]. Protein secondary structure was predicted by Cgi-bin. Multi-sequence alignment was obtained by DNAMAN software [45].
Quantification of the HiACP gene was performed by Real-time PCR method. The cDNA was diluted to 100 ng/µL. PCR reactions were prepared by TB GREENTM premix ex taq (Takara). The reaction conditions were set as follows: pre-denaturation at 95 • C for 30 s; 95 • C, 10 s; 60 • C, 30 s, 39 cycles; 95 • C, 15 s; 65 • C, 5 s; 95 • C, 5 s. We used β-actin as a housekeeping gene [46]. Table 1 shows the primers used for qRT-PCR. Three biological repeats were prepared in total, and each was repeated three times (technical repeats). The relative transcription level was analyzed using the 2 −∆∆Ct method [47].

RNAi Treatment
The DNA of HiACP was amplified from the cDNA of H. illucens larvae using the primer set "HiACP" (Table 1). PCR products were purified by the gel extraction kit (Vazyme, Nanjing, China). The concentration of the final DNA product was greater than 60 ng/µL, and the A260/A280 value ranged from 1.8 to 2.0 [48]. The DNA fragments were further used for dsRNA synthesis. Synthesis of dsRNA was performed using T7 high-yield RNA transcription Kit (Vazyme Biotech Co., Ltd.). Primers used in this step were shown in Table 1 (dsACP and dsGFP). For dsRNA injection, the 3rd-instar larvae were starved for 24 h, then washed, dried, and settled on the ice. Before injection, 75% alcohol was used to disinfect the 10 µL microinjector. Subsequently, the tip of the injector was burned by a burner, and the injection was preceded after the needle was cooled down [49]. A total of 1.5 µL of dsRNA was injected into the membrane of the 7-8th segments of the larvae. The treated insects were reared in an incubator for further tests. Daily cumulative mortality (Daily cumulative mortality (%) = cumulative number of dead worms per day/total number of worms × 100%) [50] of RNAi-treated larvae was noted every day. Body weight was measured daily, and larval body length was measured by Leica M165 C stereo microscope. The eclosion rate was calculated as follows: Adult eclosion rate (%) = adult number/total pupae number × 100%. Oviposition rate (%) = the number of eggs/the number of female adults × 100% [51].

Fatty Acid Contents in H. illucens
Larvae were ground to powder with liquid nitrogen. Totally 0.2 g of larvae powder was collected into a glass tube, and 1 µg/mL of C17:0 was added as the internal standard. Total Fatty Acids were extracted by chloroform and dried by N 2 . Methyl esterification was carried out with the methanol-H 2 SO 4 method; n-hexane was used to extract fatty methyl esters (FAME) [52]. The FAME products were measured by an Agilent 6890N Gas Chromatography gas phase analyzer with a DB-23 column (60 m × 250 m × 0.25 m). The sampling parameter was set as follows: detector temperature, 260 • C; inlet temperature, 270 • C. The heating program was started initially at 100 • C for 13 min, then 10 • C/min to 180 • C for 5 min, 1 • C/min to 200 • C for 20 min, and finally heated at 4 • C/min to 240 • C for 10 min [53]. Helium was used as the carrier gas. The C4-C24 FAME mixture (Supelco 37 F. A. M. E. Mix, Sigma-Aldrich, St. Louis, MO, USA) was used as the standard solution.

Statistical Analysis
All statistical analysis was performed by Prism 9.0. Variations of test groups were calculated by Student's t-test (for two groups) and One-Way ANOVA followed by Tukey's post-hoc test (pair-wise analysis of multi-groups).

Expression at Growth Stages and Tissues
The relative expression levels of HiACP in different developmental stages and tissues

Expression at Growth Stages and Tissues
The relative expression levels of HiACP in different developmental stages and tissues of H. illucens were determined by qRT-PCR. The midgut, integument, hemolymph, fat body, head of 5th instar larvae, and the egg, 1st, 2nd, 3rd, 4th, 5th, and 6th instar larvae, pupae, female, and male adults were tested. The relative expression levels of HiACP in the different developmental stages of H. illucens were as follows: The relative transcription level in the egg stage was significantly greater than that in other developmental stages (p < 0.05). The relative transcript levels of HiACP increased as the larval instar increased, but it decreased from the pupal stage to the period after adult eclosion ( Figure 2A). The results showed the relative transcript level of HiACP in different tissues. The highest expression level was in the midgut (p < 0.05), followed by the expression in the fat body (p < 0.05) ( Figure 2B).
Insects 2023, 14, x FOR PEER REVIEW 6 of 14 level in the egg stage was significantly greater than that in other developmental stages (p < 0.05). The relative transcript levels of HiACP increased as the larval instar increased, but it decreased from the pupal stage to the period after adult eclosion ( Figure 2A). The results showed the relative transcript level of HiACP in different tissues. The highest expression level was in the midgut (p < 0.05), followed by the expression in the fat body (p < 0.05) ( Figure 2B).

Effects of RNAi of HiACP
We injected dsRNA into one-day-old 3rd instar larvae to determine the optimal concentration and repression time. When the concentration of dsACP was 2000 µg·mL −1 , the silencing efficiency was greatest at 61.5% (p < 0.05). There was no significant difference when the concentration of dsACP was 500 µg·mL −1 ( Figure 3A). The relative expression of the HiACP gene on different days following the dsACP injection was also analyzed. Silencing efficiency was best on the 3rd day (1.5 µL, 2000 µg*mL −1 )) and reached 81.9% (p < 0.05) ( Figure 3B). Lowercase letters (a-e) represent significant differences (p < 0.05) using Tukey's multiple range test. The relative transcript levels of HiACP in different developmental stages and different tissues were detected by qRT-PCR (Using β-actin as the reference housekeeping gene). The Y-axis values are mean ± SE of relative expression levels. Different lowercase letters on the bars indicate a significant difference between the treatments.

Effects of RNAi of HiACP
We injected dsRNA into one-day-old 3rd instar larvae to determine the optimal concentration and repression time. When the concentration of dsACP was 2000 µg·mL −1 , the silencing efficiency was greatest at 61.5% (p < 0.05). There was no significant difference when the concentration of dsACP was 500 µg·mL −1 ( Figure 3A). The relative expression of the HiACP gene on different days following the dsACP injection was also analyzed. Silencing efficiency was best on the 3rd day (1.5 µL, 2000 µg·mL −1 )) and reached 81.9% (p < 0.05) ( Figure 3B).
The changes in fatty acid content in dsACP-injected 3rd instar larvae on the 3rd day after interference were studied using the RNAi method (Table 2). After interference, the total fatty acid content decreased by 13.13% (p < 0.05), and lauric acid (C12:0) decreased by 20.22% (p < 0.05). After interference with dsACP, the percentage of SFA in total fatty acid decreased by 2.44% (p < 0.05), while the percentage of UFA in total fatty acid increased by 4.94% (p < 0.05). centration and repression time. When the concentration of dsACP was 2000 µg·mL −1 , the silencing efficiency was greatest at 61.5% (p < 0.05). There was no significant difference when the concentration of dsACP was 500 µg·mL −1 ( Figure 3A). The relative expression of the HiACP gene on different days following the dsACP injection was also analyzed. Silencing efficiency was best on the 3rd day (1.5 µL, 2000 µg*mL −1 )) and reached 81.9% (p < 0.05) ( Figure 3B).  We studied the influence of HiACP interference on the growth of 3rd instar larvae of H. illucens. After injecting dsACP, the 3rd instar larvae were continuously observed for 30 d. The 3rd instar larvae injected with dsACP grew to 5th instar larvae. Compared to the control 5th instar larvae, the treated larvae had an average body weight that was 44.85 mg less and an average body length that was 3.09 mm shorter ( Figure 4A). These values were significantly different from the control group (p < 0.05). When the 3rd instar larvae grew to pre-pupae, we observed that the weight of the average pre-pupa was 14.59 mg less, and the average pre-pupa length was 3.82 mm less than the control ( Figure 4B). These reductions were significantly less than the control (p < 0.05).  The photos of larva and pupa were collected at the 3rd day of the corresponding instar. Mean ± SE was calculated from three biological replicates, and each biological replicate contained 50 3rd instar larvae.
After the 3rd instar larvae interference treatment, individuals grew to the adult stage, the adult male weight was reduced by 34.06 mg, and the adult female weight was reduced by 33.05 mg compared to the control. These changes were significantly different from the control group (p < 0.05) ( Figure 5A). The experimental group's adult male body length was reduced by 5.9 mm, and the adult female body length was reduced by 5.8 mm. These reductions were significantly different from the control group (p < 0.01) ( Figure 5B). The adult eclosion rate was reduced by 8.86%, and adult female oviposition was reduced by 18.52% ( Figure 5C). These values were significantly less than those in the control (p < 0.05). After the 3rd instar larvae interference treatment, individuals grew to the adult stage, the adult male weight was reduced by 34.06 mg, and the adult female weight was reduced by 33.05 mg compared to the control. These changes were significantly different from the control group (p < 0.05) ( Figure 5A). The experimental group's adult male body length was reduced by 5.9 mm, and the adult female body length was reduced by 5.8 mm. These reductions were significantly different from the control group (p < 0.01) ( Figure 5B). The adult eclosion rate was reduced by 8.86%, and adult female oviposition was reduced by 18.52% ( Figure 5C). These values were significantly less than those in the control (p < 0.05). ). Mean ± SE was calculated from three biological replicates, and each biological replicate contained 50 one-day-old 3rd first instar larvae that grew to the adult stage. Bars marked with different letters indicate significant differences between treatment and control (Student's t-test, p < 0.05).
On day 40, after injecting dsACP, the treated 3rd instar larvae had developed into adults. The cumulative mortality of the experimental group was 68.00%, which was significantly greater than the control group (p < 0.01) ( Figure 6A). We also found that after injecting dsACP into the 3rd larvae, there was a significant increase in instar duration. The duration of 4th instar larvae, pupae, and adults was significantly longer than the control group. The duration of 4th instar larvae increased by 1.3 d, pupal duration increased by 1.5 d, and the adult stage increased by 2.7 d. These increases were significantly different from the control group (p < 0.01) ( Figure 6B). ). Mean ± SE was calculated from three biological replicates, and each biological replicate contained 50 one-day-old 3rd first instar larvae that grew to the adult stage. Bars marked with different letters indicate significant differences between treatment and control (Student's t-test, p < 0.05).
On day 40, after injecting dsACP, the treated 3rd instar larvae had developed into adults. The cumulative mortality of the experimental group was 68.00%, which was significantly greater than the control group (p < 0.01) ( Figure 6A). We also found that after injecting dsACP into the 3rd larvae, there was a significant increase in instar duration. The duration of 4th instar larvae, pupae, and adults was significantly longer than the control group. The duration of 4th instar larvae increased by 1.3 d, pupal duration increased by 1.5 d, and the adult stage increased by 2.7 d. These increases were significantly different from the control group (p < 0.01) ( Figure 6B). The duration of 3rd, 4th, 5th, 6th instar larvae, pupae, and adults. Y-axis values are the means of duration at different instars. Mean ± SE was calculated from three biological replicates, and each biological replicate contained 50 one-day-old 3rd instar larvae grown to different instars. Bars marked with different letters indicate significant differences between treatment and control (Student's t-test, p < 0.05).

Discussion
Fatty acid synthase plays a crucial role in the fatty acid metabolism of insects, and ACP is an important functional cofactor that directs the production of various fatty acids in insect tissues. We studied ACP deficient H. illucens to determine the potential function of this key FAS cofactor. Inhibition of ACP expression during the larva stage directly down-regulated the content of common fatty acids stored in the tissues. This effectively reduced the growth of H. illucens. The effects included a reduced survival rate, reduced body size and mass, increased development duration, and reduced female reproduction. These results indicated that ACP could have a great impact on fat storage and the growth of H. illucens. Advance editing of this gene might help improve fatty acid production and conversion efficiency of organic waste by H. illucens.
The development of H. illucens larvae can be affected by various extrinsic factors such as temperature, gut microbes, food nutrition, and xenobiotics [23,54,55]. Intrinsic factors, such as the expression level of metabolic genes, also perform vital roles during the developmental process. Our result exhibited that HiACP was expressed at the highest level during the egg stage of H. illucens (Figure 2A). During embryogenesis, a large amount of energy is needed to complete the transformation from egg to 1st instar larva. Lipids, consisting of glycerol and fatty acids, are the major fuel source during this development stage [56]. Similar to H. illucens, a large number of fatty acids occur in the oocytes of mosquitos. These have been termed maternal fatty acids [57]. Fatty acid deficiency can reduce the egg lifespan of insects and hence impair the fitness of eggs [57,58]. These results confirm that, in H. illucens, HiACP regulates the biosynthesis of intra-egg fatty acids and is involved in the development of the embryo to larval stages. After the egg hatched, the expression level of the HiACP gene increased during the H. illucens growth. This indicated a gradually increased storage of fatty acids in the gut and fat body of larvae ( Figure 2B). This energy source could be used for pupation and subsequent adult eclosion. In adult female Bombyx mori, the ACP gene was highly expressed in the unfertilized egg, flight muscle, and fat body tissues. The adult B. mori midgut had a relatively lower expression level than those found in other tissues [59], which is inconsistent with our results. This could be caused by the degenerated gut tract of the silkworm, which does not participate in food digestion and fatty acid metabolism. The duration of 3rd, 4th, 5th, 6th instar larvae, pupae, and adults. Y-axis values are the means of duration at different instars. Mean ± SE was calculated from three biological replicates, and each biological replicate contained 50 one-day-old 3rd instar larvae grown to different instars. Bars marked with different letters indicate significant differences between treatment and control (Student's t-test, p < 0.05).

Discussion
Fatty acid synthase plays a crucial role in the fatty acid metabolism of insects, and ACP is an important functional cofactor that directs the production of various fatty acids in insect tissues. We studied ACP deficient H. illucens to determine the potential function of this key FAS cofactor. Inhibition of ACP expression during the larva stage directly down-regulated the content of common fatty acids stored in the tissues. This effectively reduced the growth of H. illucens. The effects included a reduced survival rate, reduced body size and mass, increased development duration, and reduced female reproduction. These results indicated that ACP could have a great impact on fat storage and the growth of H. illucens. Advance editing of this gene might help improve fatty acid production and conversion efficiency of organic waste by H. illucens.
The development of H. illucens larvae can be affected by various extrinsic factors such as temperature, gut microbes, food nutrition, and xenobiotics [23,54,55]. Intrinsic factors, such as the expression level of metabolic genes, also perform vital roles during the developmental process. Our result exhibited that HiACP was expressed at the highest level during the egg stage of H. illucens (Figure 2A). During embryogenesis, a large amount of energy is needed to complete the transformation from egg to 1st instar larva. Lipids, consisting of glycerol and fatty acids, are the major fuel source during this development stage [56]. Similar to H. illucens, a large number of fatty acids occur in the oocytes of mosquitos. These have been termed maternal fatty acids [57]. Fatty acid deficiency can reduce the egg lifespan of insects and hence impair the fitness of eggs [57,58]. These results confirm that, in H. illucens, HiACP regulates the biosynthesis of intra-egg fatty acids and is involved in the development of the embryo to larval stages. After the egg hatched, the expression level of the HiACP gene increased during the H. illucens growth. This indicated a gradually increased storage of fatty acids in the gut and fat body of larvae ( Figure 2B). This energy source could be used for pupation and subsequent adult eclosion. In adult female Bombyx mori, the ACP gene was highly expressed in the unfertilized egg, flight muscle, and fat body tissues. The adult B. mori midgut had a relatively lower expression level than those found in other tissues [59], which is inconsistent with our results. This could be caused by the degenerated gut tract of the silkworm, which does not participate in food digestion and fatty acid metabolism.
In mammals, fatty acid synthase mainly produces C16 fatty acids, whereas insect fatty acid products are evenly distributed in carbon chain lengths, reaching C18 [60]. Large amounts of C18 fatty acids, such as stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18: 3), have been identified in many insects, including crickets, grasshoppers, mealworms, and houseflies [61][62][63]. H. illucens larvae contain a large amount of lauric acid (C12:0), palmitic acid (C16:0), and oleic acid. Our results demonstrated that SFAs are dominant in the H. illucens larva, which is consistent with previous studies of this species [33,64]. Lauric acid (C12:0) was the most abundant fatty acid in H. illucens larvae, a result that differs from previous data. H. illucens contains high levels of lauric acid and serves as a useful SFA supplier for the food, cosmetics, and poultry industries [65]. Using the H. illucens RNAi system, we found that lauric acid content decreased by 20.22% in the 3rd instar larva after silencing of HiACP. However, other C16 and C18 SFA and MUFA compounds were also simultaneously down-regulated. No fatty acid was observed to decrease significantly compared with its other analogs. This result implies that HiACP mainly participates in the elongation step of fatty acid biosynthesis in H. illucens and might not be involved in the decoration and termination reaction of the hydrocarbon chain. Functional genes that contribute to the composition of specific fatty acid types (e.g., lauric acid) may be uncovered by future RNAi studies.
RNAi treatment had significant effects on the growth and development of 3rd instar H. illucens larvae. Inhibition of HiACP decreased fatty acid storage in tissues and impaired triglyceride synthesis in RNAi-treated individuals. When the energy storage ability of insects was compromised, their body size, duration of development, and fertility were also affected. This result indicated that HiACP is essential for the nutrient digestibility and energy storage of H. illucens. ACPs have exhibited similar functions in other animals. For example, the downregulation of genes related to fatty acid synthesis can induce dysfunctions in adipose tissue and lead to fat loss and weight loss in rats [66]. Similarly, RNAi treatment of the FAS gene disturbed the rate of midgut digestion and caused reduced lipid synthesis in Aedes aegypti [67]. The fatty acid synthesis pathway is a basic biological process, the products of which are widely involved in the physiology of the immune and other cell signaling systems [68]. For example, lauric acid has bactericidal activity [69], so a decrease in lauric acid content triggered by ACP interference, as observed here, might suppress the immune function of H. illucens against bacterial pathogens. The chain extension reaction of the fatty acid synthesis process is also used by the insect hormone biosynthesis process [70][71][72], which indicates that the ACP downregulated induced disturbance of the fatty acid elongation reaction might diminish the hormone homeostasis of H. illucens. We assume that this hormone dysfunction might have a wider influence on insect physiology and ecology.
RNAi treatment of H. illucens demonstrated that HiACP is a core gene involved in the fatty acid synthesis process. This gene cannot precisely govern the production of specific fatty acid content and the composition of fatty acids stored in H. illucens tissues. It appears to act as a "general controller" of the fatty acids that regulate fatty acid composition and storage in H. illucens. This knowledge could be used for expanding the fatty acids produced by the H. illucens rearing industry.

Conclusions
We demonstrated that the HiACP gene regulates the biosynthesis of fatty acids. This gene affects the growth of H. illucen larvae, adult eclosion rate, and adult oviposition. These effects helped reveal the molecular mechanism and function of the HiACP gene and the process of fatty acid regulation. Future research should focus on the optimization of the fatty acid regulatory mechanisms and the expression of high-quality fatty acids in vitro.