Lipid quantity and composition vary between three European house fly strains

Lipids are an important class of nutrients for insects, as they are the main energy source during metamorphosis and crucial for survival and reproduction. Lipid reserves in insects are mostly accumulated during larval development and can be influenced by environmental factors such as diet and temperature. Genetic factors can also affect larval lipid content and composition. Larvae of the house fly, Musca domestica L. (Diptera: Muscidae), are a valuable emerging resource for the insect feed sector, fueling a growing interest in the factors regulating larval lipid content. We investigated whether strains of three geographical origins vary in ontogeny, quantity, and composition of stored lipids in response to temperature. Italian, Spanish, and Dutch house fly larvae were reared on a similar diet at 25 and 35 °C. Larval dry weight and total lipid content were determined in early‐, mid‐, and late‐third instars by extraction of soluble lipids. The fatty acid composition was analyzed in late‐third instars using an improved extraction and derivatization protocol. We found that strain, larval stage, and temperature affected larval dry weight and absolute and relative lipid content, with significant interaction effects between these three factors. All flies were reared in a common environment, indicating a genetic component to lipid storage. Analysis of lipid composition identified 11 fatty acids, including some rarely reported in the literature. Five fatty acids accounted for 80 and 81% of the total fatty acid methyl esters detected at 25 and 35 °C, respectively. An effect of temperature but not strain was evident on the composition of the fatty acids. The observed differences in lipid content among strains and temperature conditions could be of interest for commercial rearing of the house fly.


| 249
FAT STORAGE IN THE HOUSE FLY parasitoid Asobara tabida (Nees) (Ellers & van Alphen, 1997). Geographical differences in the amount of lipids could be an adaptation to differences in diet composition (McKenna et al., 2019;Rudman et al., 2019) or caused by divergent life cycles (Lehmann et al., 2016;Verheyen et al., 2018). Moreover, variation in the amount of stored lipids has strong heritability, as shown for the yellow dung fly, Scathophaga stercoraria (L.) (Blanckenhorn & Hosken, 2003). Similarly, variation in the composition of stored lipids among populations is genetically based and highly heritable in Drosophila melanogaster Meigen (Scheitz et al., 2013). Therefore, divergent selection could result in differences in lipid accumulation and composition between populations.
The main environmental determinants of lipid content are diet and temperature (Lee & Roh, 2010). Although insect species can biosynthesize most lipid classes, the majority of the lipid reserves is accumulated during the larval stage. Larval lipid content is determined by diet composition and reflects the lipid quality of the substrate (Liland et al., 2017;Oonincx et al., 2020). Developmental temperature also strongly influences the quantity and quality of lipids that are accumulated during the larval stage, mainly through its effect on metabolic rate, development time, and other life-history traits (Sohal et al., 1981;Lee & Roh, 2010;Asiri, 2017;Wang et al., 2018;Klepsatel et al., 2019;Francuski et al., 2020). Lower temperature increases the time the larvae require to reach maturity (Wang et al., 2018) and allows for longer lipid accumulation (Francuski et al., 2020). Depending on how strongly low temperature slows down the lipid accumulation process, the total amount of lipid accumulated may increase or decrease. Temperature also strongly influences the qualitative profile of larval fatty acids in flies (Ewald et al., 2020).
There has been a surge of interest in genetic and environmental effects on insect lipid metabolism because of the growing commercial application of insects as alternative protein and lipids sources. Insect larvae can convert low-quality resources, such as manure or industrial waste, into high-quality products, such as insect meal and insect oil rich in proteins and lipids. However, the applicability of insect larvae can be limited by their high lipid content, for example, when insect meal is included in the formulation of animal diets (Motte et al., 2019), as fish response to a defatted meal is better than that obtained with the full-fat ingredient (Fasakin et al., 2003). Therefore, it would aid the use of insects for feed if the rearing of insect larvae could be tailored for optimal nutritional content, or if strains with suitable nutritional composition would be available.
The larvae of the house fly, Musca domestica L. (Diptera: Muscidae), are a valuable emerging resource for the insect feed sector. House fly larvae are rich in protein and have a balanced amino acid profile resembling that of fish meal (Barroso et al., 2014). Furthermore, the house fly is a cosmopolitan species with a rich geographic variation as natural populations have evolved in different climatic regions. Considerable genetic differences in morphology have been reported among populations (Marquez & Krafsur, 2002;Pastor et al., 2014), but variation in lipid content among house flies from different geographical origins has not been investigated so far. The house fly has a remarkable phenotypic plasticity (Bryant et al., 1986;Alves & Bélo, 2002) and temperature influences the quality of the fatty acid profile (Robb et al., 1972). It is known that temperature can affect the amount of stored lipids in D. melanogaster adults (Klepsatel et al., 2019), but this information is still lacking for species with potential commercially relevant application such as the house fly. In this context, identifying the factors that determine the quantitative and qualitative lipid profile of their larvae might improve our understanding of the genetic component of lipid metabolism.
Here, we ask whether differences exist in the lipid quantity and composition of three house fly strains from various geographic origins and their response to two temperatures. We measured the accumulated lipid reserves in the larvae at three time intervals of the third instar stage (early-, mid-, and late-third instar). We expected to find differences in lipid composition between the strains due to geographical origin and applied temperature.

House fly strain origin and culture
Larvae of three European strains of house fly were used for the experiments: one from Italy (IT; collected in 2013 in Altavilla Silentina and Castellaneta Marina), one from Spain (SP; collected in 2015 in Girona and Barcelona), and one from The Netherlands (NL; collected in 2018 in Gerkesklooster) (Francuski et al., 2020). These strains were maintained with about 20 generations per year and 100-200 individuals per generation at the University of Groningen (The Netherlands) at 25 °C and 50 ± 10% r.h. on standard medium (Francuski et al., 2020). From each strain, a sample of larvae and pupae was brought to the experimental facilities at Vrije Universiteit Amsterdam (The Netherlands) in the summer of 2019, where they were subsequently cultured on similar medium in a climate room (25 °C, 60% r.h., and L16:D8 photoperiod). Adults were kept in a rearing cage (32.5 × 32.5 × 32.5 cm, model BD4M3030D; BugDorm, Taichung, Taiwan) and provided milk powder, sugar, brewer's yeast, and water (Francuski et al., 2020).

Diet preparation and nutritional composition
The larval medium was produced in bulk formulation (Table 1) from which aliquots were taken for each replicate (32 g). The inactivated yeast, milk powder, and flour were first mixed with a magnetic stirrer in a plastic beaker (1.5 L) with warm tap water to produce a solution composed of all the pre-weighted dry ingredients.
Once all the ingredients were completely dissolved, nipagin (Alfa Aesar, Ward Hill, MA, USA) was added to the solution. The final solution was then slowly (2 min) put into the bowl of a food processor (Bowl-Lift stand mixer K5SS; KitchenAid, Benton Harbour, MI, USA) containing the preweighted dry wheat bran (Table 1), which was then stirred at low velocity. Once completely mixed, the wet fresh formulated medium was stirred for two additional minutes before being used to prepare the replicates. The nutritional composition of each ingredient and that of the final wet formulated larval medium is shown in Table 2.

Temperature experiment
Larvae were grown at 25 and 35 °C. Freshly laid eggs were collected from each of the three strains by placing an oviposition cup in the cage for 4 h. The eggs were washed into a fine mesh (220 μm pore size) and manually separated under an optic stereomicroscope (Olympus, Tokyo, Japan) into batches of 200 eggs, which were placed on a wettened cellulose filter (1 cm diameter, retrieved after the eggs hatched) and put in a 300-ml glass jar (12 × 7 cm) on top of 32 g of medium (Table 1). The amount of substrate was based on Pieterse & Gloy (2013). Five replicate jars were prepared per temperature treatment for the NL and SP strains. Only three replicates per temperature treatment could be prepared for the IT strain due to the lower number of eggs available. The replicates were randomly assigned to each of two temperature treatments for each strain. Those reared at 25 °C were kept in the same climate room where the adults were held before the test (25 °C and 70% r.h.). Those assigned to the 35 °C treatment were placed in a CO 2 incubator (B 5060 EK; Heraeus, Hanau, Germany) equipped with a halogen lamp set on the same photoperiod. Humidity in the incubator was controlled using 300 mL of a saturated solution of NaCl in demi water solution, which in a preliminary test provided a constant 60-75% r.h. at 35 °C (equal to the r.h. in the climate room) (Greenspan, 1976).
Larvae were sampled from each replicate jar at the early-, mid-, and late-third instar stage for the quantitative lipid analysis, whereas the sampling for qualitative lipid analysis took place only at the late-third instar stage. Development is faster at higher temperatures, therefore the exact time of sampling for each stage depended on the temperature treatment and was estimated based on Wang et al. (2018) and calculated from the moment of egg laying (Table 3). Sampling took place by collecting one larva from each of the four opposite lateral sides of the jar and one from its center, resulting in five larvae per replicate per sample per larval stage. The larvae for the quantitative lipid analysis were placed individually in PCR vials (Axygen 0.2 mL with dome cap; Corning, Somerville, MA, USA), whereas those T A B L E 1 Recipe for the bulk formulation of the house fly larval substrate.
T A B L E 2 Nutritional composition (g per 100 g of product) of the ingredients used for the formulation of the medium and final nutritional composition of the formulated house fly larval medium used in the experiment. for the qualitative analysis were grouped (five individuals) in a single PCR vial per replicate. All the samples were immediately frozen in liquid nitrogen and preserved at −20 °C until analysis.

Quantitative lipid analysis
The lipid quantity of the early-, mid-, and late-third instars was determined as the total diethyl-ether soluble lipid content by calculating the difference of the larval dry weight before and after extraction. The larvae were firstly dried in a ventilated oven (50 °C) for 72 h directly in the PCR tube in which they were stored. Next, each larva was individually weighed on a precision scale (± 0.1 μg) (UMT2; Mettler Toledo, Greifensee, Switzerland) to determine its dry weight before being transferred to a glass tube (16 × 100 mm with NS 14/23 socket; Lenz, Wertheim, Germany) filled with diethyl ether (5 mL; VWR, Leuven, Belgium) to extract the lipids. After 48 h, the larvae were removed from the solvent, each washed with 3 mL of fresh diethyl ether, and placed again in the oven to dry for 24 h, after which their weight was measured again. Pilot testing showed that more than 99% of the ether-soluble lipids was extracted after 48 h of static immersion in diethyl ether. The total ether-soluble lipid quantity was calculated as the difference between the initial dry weight and that measured after the extraction. The relative lipid content was successively calculated as the percentage of lipids of the initial dry weight.

Qualitative lipid analysis
The fatty acid composition was determined following a modified procedure from Matyash et al. (2008). The samples (late-third instars) were extracted from the freezer and maintained on ice. The five larvae preserved in each tube were homogenized with a glass potter in 5 mL ammonium acetate (Sigma Aldrich, Steinheim, Germany), and 200 μL of the homogenate was sampled and poured into a culture tube (15 mL) with Teflon-lined cap previously filled with 1.5 mL of high-grade methanol. Methyl-tert-butyl ether (MTBE; Sigma Aldrich) was added (5 mL) and the tube was shaken at room temperature for 1 h (250 oscillations per min). To induce phase separation, 1.25 mL of mass spectrometry (MS)grade water (Milli-Q) was added to each tube and kept at room temperature for 10 min. The sample was then centrifuged at 1000 g for 15 min at 20 °C to allow the separation of possible residues. Next, the upper organic phase was collected with a glass pipette and placed in a clean culture tube. The solvent was evaporated in a water bath (40 °C) under an N flux until the tubes were dry. Unlike Matyash et al. (2008), we did not perform a double extraction. The derivatization of the fatty acid methyl esters (FAMEs) followed the protocol of van Dooremalen et al. (2011). The FAMEs were analyzed in a gas chromatography-mass spectrometer (GC-MS) (6890 GC with a 5975c MS; Agilent Technologies, Santa Clara, CA, USA) using a BPX70 column (60 m × 0.25 mm internal diameter, 0.25 μm film thickness; Trajan, Melbourne, Australia). The analysis of the MS spectra was conducted using the GC-MS associated software (Chemstation G1701DA; Agilent Technologies). The peaks were automatically calculated imposing the threshold at 18 and the spectrum area registered and used to determine the relative content of each fatty acid with respect to the total amount detected. The fatty acids were identified based on their retention time and compared to a standard (37-component FAME Mix; Supelco, Bellefonte, PA, USA) run along with the samples.

Statistical analysis
A linear mixed-effects model (LME) imposing strain, temperature, and larval stage as factors with jar as random effect (full model) was used to analyze the results of the quantitative lipid content of the larvae. To test for significance, the full model was compared using log-likelihood ratio tests against three null models where each of the factors was excluded, and against a model without the interactions of the factors. A post-hoc Tukey pairwise comparison was used to detect significant differences among strains, stages, and temperatures in the full models of each variable.
To determine the effect of strain and temperature on the qualitative results, analysis of similarities (ANOSIM) was used and the results were graphically represented with non-metric multidimensional scaling (NMDS). Furthermore, an ANOVA was conducted on each single fatty acid relative content to determine whether strain, temperature, or their interaction explained any of the observed differences in the relative content of each fatty acid. Given the multiple testing for all fatty acids (n = 11) we applied a Bonferroni correction resulting in a critical P value of 0.0045.
The larvae at 25 °C pupated before the estimated time and it was impossible to obtain late-third instars for all the strains. Therefore, a second experiment with an identical setup was conducted to obtain the missing samples for the 25 °C treatment. The results obtained from the second experiment were excluded from the quantitative statistics model (however, they are shown in the descriptive T A B L E 3 House fly larvae sampling time (h from egg laying) at the two rearing temperatures based on Wang et al. (2018). statistics) and are not included in the statistical analysis. All analyses were conducted in R v.4.2.0 (R Core Team, 2022).

Larval dry weight
A three-way interaction effect between the factors strain, larval stage, and temperature was found for larval dry weight (χ 2 = 161.56, d.f. = 9, P < 0.0001). Larval dry weight was affected by strain (χ 2 = 130.92, d.f. = 10), larval stage (χ 2 = 470.48, d.f. = 9), and temperature (χ 2 = 89.81, d.f. = 6, all P < 0.0001). The dry weight significantly increased between the early-and mid-third instar in larvae exposed to all treatments except for the NL strain at 25 °C ( Figure 1A). From mid-third instar onwards, only the IT strain showed a further increase in dry weight at the late-third instar at 35 °C (t = −3.734, d.f. = 336, P = 0.025) ( Figure 1A). Note that the data for the late-third instar at 25 °C were not included in the model, as these were collected in a separate experiment (see Materials and methods). Although temperature overall was a significant main factor in the full model, its effect on larval dry weight differed between the strains ( Figure 1A). Comparing between temperatures revealed that the dry weight of the NL larvae at the early-third instar was significantly higher at 25 than 35 °C (t = 5.428, d.f. = 336, P < 0.0001). In contrast, there was no difference for the early-third instars of IT and SP strains. However, a significantly higher dry weight was observed at 35 °C for both the SP (t = −6.600, P < 0.0001) and NL (t = −4.227, P = 0.0039, both d.f. = 336) mid-third instars, whereas the dry weight of IT larvae at this stage was similar between temperatures ( Figure 1A). Of the three strains, IT larvae reached the highest dry weight, both at 25 (mid-third instar) and 35 °C (late-third instar) ( Figure 1A).

Absolute and relative lipid content
A three-way interaction effect between strain, larval stage, and temperature was found for the absolute lipid content of the larvae (χ 2 = 46.86, d.f. = 9, P < 0.0001). This variable was affected by strain (χ 2 = 55.57, d.f. = 10), larval stage (χ 2 = 289.06, d.f. = 9, both P < 0.0001), and temperature (χ 2 = 18.17, d.f. = 6, P = 0.0058). The larvae of all strains showed an increase in lipid content across all stages of development ( Figure 1B). Similar to the results on larval dry weight, the IT strain obtained the highest lipid content at the late-third instar at 35 °C compared to the NL (t = 3.817, P = 0.018) and the SP (t = 5.109, P = 0.0001, both d.f. = 339) larvae of the same stage. The only other stage at which marked, yet not significant, differences among strains were observed was the mid-third instar at 25 °C, with IT larvae having a higher absolute lipid content than SP and NL larvae ( Figure 1B).
The relative lipid content varied least ( Figure 1C), but a three-way interaction effect was also found between strain, larval stage, and temperature (χ 2 = 45.87, d.f. = 9, P < 0.001). Strain (χ 2 = 24.59, d.f. = 10, P = 0.006), stage (χ 2 = 89.66, d.f. = 9), and temperature (χ 2 = 42.26, d.f. = 6, both P < 0.0001) all affected the relative lipid content of the larvae. At 25 °C, a difference between the early-and mid-third instars was found only for both the NL strain (t = −3.815, P = 0.019) and the SP (t = −3.983, P = 0.010, both d.f. = 336), which had a higher relative lipid content at the latter stage. Although not significant, a higher temperature resulted in early-third instars with a higher relative lipid content than those reared at 25 °C for all strains. No directional change was observed over stages at 35 °C. Still, the relative lipid content was the highest at the late-third instar, with the IT (t = −5.475, P < 0.0001) and SP (t = −4.559, P < 0.001, both d.f. = 336) larvae different from the mid-third instar ( Figure 1C).
Note that the results obtained for the late-third instars at 25 °C were obtained in a separate run of the experiment and were therefore not included in the statistical analysis. However, those results closely resemble the pattern observed for the late-third instars at high temperature for all three variables.
The ANOSIM analysis showed a strong effect of temperature (R = 0.94, P = 0.0001) on all the strains' fatty F I G U R E 1 (A) Dry weight (mg), (B) absolute lipid content (mg), and (C) relative lipid content (%) of early-, mid-and late-third instars of three strains of the house fly -from Italy (IT), The Netherlands (NL), and Spain (SP) -reared at 25 and 35 °C. Boxplots indicate the mean and median values (i.e., the line and dot within the boxes, respectively) and the first and third quartile (upper and lower box). The whiskers show 1.5× the interquartile range. The dots beyond the whiskers are outliers. Means capped with different letters differ significantly (LME: P < 0.05). Note that the boxplots of the data for the late-third instars at 25 °C have not been included in the statistical analysis, as these experiments were performed separately. acid profile. However, no differences among strains were detected (Figure 2). We also conducted an ANOVA on each singular fatty acid using a Bonferroni correction for multiple testing, imposing strain and temperature as factors. This analysis identified a significant effect of temperature but not of strain in 10 of the fatty acids detected. The only FAME that was significantly affected by strain (F 2,22 = 11.563, P = 0.00037) was C16.1n7, for which the post-hoc pairwise comparison showed that SP larvae had a higher relative content at 25 °C and differed from those of IT (P = 0.048) and NL (P = 0.0052). This was also the only fatty acid that was not significantly affected by temperature.

DISCUSSION
We investigated strain differences in lipid reserves of larval stages of the house fly reared at two temperatures. Finding differences among strains when reared in a common environment would indicate a genetic component to lipid quantity and quality. We found significant differences among strains in growth rate at both 25 and 35° C and in lipid accumulation at 35° C. In addition, the three strains differed in their response to temperature, with the IT strain having the highest growth rates and lipid accumulation at both temperatures. Given that these differences were observed in a common environment for all strains, it can be assumed that they reflect genetic differences between strains that evolved in different thermal environments.
As the strains were sampled from wild populations at various time points and were maintained under laboratory conditions for variable time periods (40-140 generations), they may have undergone changes. The differences observed in this study, however, suggest that the constant laboratory conditions have not induced a convergent selection towards specific strategies for lipid storage. Additionally, the strains have been maintained long enough under laboratory conditions to assume that acclimatization to the artificial conditions had occurred, also for the strain that was brought to the laboratory most recently. Therefore, the differences observed here can be attributed to genetic differences present in the strain before the domestication to laboratory conditions. Nevertheless, the observed differences may not completely reflect the natural situation, where additional genetic and environmental effects may T A B L E 4 Mean (± SD) relative content (%) of the fatty acid methyl esters (FAMEs) detected in late third-instars of house fly strains from Italy (IT), The Netherlands (NL), and Spain (SP) reared at two temperatures.   Table 4. play a role. Our results also reveal that selection for altered lipid components may be successful in the house fly. We found that the IT strain grew heavier and faster than the NL and SP strains under both 25 and 35 °C, suggesting that it evolved in an environment that favors tolerance to a broader range of temperatures. Developmental temperature can strongly influence the growth of insects (Lee & Roh, 2010;Wang et al., 2018), as well as affect the quantity and composition of the lipids stored by an individual (Dwivedy, 1977;Stefanov et al., 2002;Skorupa et al., 2008;Brückner et al., 2018;Klepsatel et al., 2019;Liu et al., 2021). Specifically, at 25 °C, the NL and SP strains had lower dry weight and lipid storage than the IT strain. Temperatures outside the optimal range have been reported to negatively affect the storage of reserve lipids in adult D. melanogaster flies (Klepsatel et al., 2019). Our results suggest that this is similar in house fly larvae.

25°
The relative lipid content of larvae was remarkably similar across strains and temperatures. At 25 °C, the lipid percentage increased between the early-and the mid-third instars, but only significantly for the NL and SP strain. The additional data collected for the late-third instars at this temperature suggest that relative lipid content would increase to 15-20% at the end of larval development. In the higher temperature treatment, the percentages of lipid content found in late-third instars were very similar to those at 25 °C. This strongly suggests that house fly larvae converge on an optimal lipid percentage of 15-20% before pupation. Convergence of lipid content at the end of the developmental period has been shown for other fly species as well (Kaspi et al., 2002;Nestel et al., 2004). For example, the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), regulates lipid content during metamorphosis to converge toward similar lipid content at emergence regardless of the larval diet history (Nestel et al., 2004). Our results suggest that house fly larvae tend to regulate this process before initiating pupation, which could be advantageous to avoid detrimental effects of diet-induced obesity in the adult stage. Amylose starch-enriched diets induce obesity and reduce the life span of D. melanogaster adults (Abrat et al., 2018), with negative repercussions on individual fitness. Further research into this regulatory process could reveal how house flies achieve convergence to a specific relative lipid content and what the limits are to this capacity.
Our qualitative lipid analysis did not show significant differences in fatty acid composition between the strains, except for palmitoleic acid (C16.1n7), which was also the only fatty acid not influenced by temperature. The abundance of the five dominant fatty acids was similar to previous reports (Makkar et al., 2014). Importantly, we used an improved derivatization protocol (van Dooremalen et al., 2011), which helped to identify two rarely reported long-chain poly-unsaturated fatty acids (C18.1n7 and C18.3n6).
The lack of differences in lipid composition between strains may not be entirely surprising, as the fatty acid profile of the diet is known to play an important role in determining the qualitative lipid profile of insect larvae (Robb et al., 1972;Liland et al., 2017;Oonincx et al., 2020). In our study, all strains were reared on the same diet, which may have resulted in highly similar fatty acid profiles. For fatty acid composition, however, previous studies have shown a genetic component in D. melanogaster (Scheitz et al., 2013), and house flies have the ability to synthesize many fatty acids de novo, with the exception of sterol, which is required in the diet to sustain growth (Silverman & Levinson, 1954;Dwivedy, 1975;Thompson & Simpson, 2009). Some fatty acids accumulated during the larval stage are carried over to the adult stage (Bridges, 1971), and the fat body represents an important center for pupa development and adult formation (Liu et al., 2009). However, the effect of the dietary lipid composition on house fly life-history traits has been poorly investigated to date. Therefore, further research on the effect of dietary lipids on house fly lifehistory traits might provide insightful results.
The relevance of investigating genetic variation in the biological and morphological parameters of the house fly has already been pointed out by Pastor et al. (2014). Genetic differentiation based on mitochondrial haplotypes has been reported for geographically distant house fly populations, particularly at the regional level (Marquez & Krafsur, 2002). Strains differ in morphotype (Bryant et al., 1986;Alves & Bélo, 2002), many life-history traits Martínez-Sánchez et al., 2007;Pastor et al., 2014;Francuski et al., 2020), and biochemical responses (Sohal et al., 1987). However, none of the previous studies addressed genetic differences among house fly strains originated from various geographical areas for lipid content and its ontogeny. With the results of the current study, we can now add to this list the genetic differences in larval growth rate, lipid storage, and thermal responses among geographic strains. Additionally, we highlight how the strains retain these differences, even after long-term maintenance under similar laboratory conditions.
In conclusion, our results suggest that geographic differences among house fly strains are most pronounced for lipid quantity and ontogeny at various temperatures, whereas the qualitative lipid content is only affected by temperature. An adaptive explanation for the stronger divergence in the absolute amount of stored lipids might be its immediate relevance for securing the energy required for various biological processes. Although lipid accumulation during the larval stage is essential to reaching adulthood, an excessive energy intake in insect larvae results in obesity and the pathologies associated with this condition will negatively influence their fitness (Warbrick-Smith et al., 2006;Skorupa et al., 2008;Rovenko et al., 2015). Our results support the idea that insects can evolve mechanisms to regulate the uptake and storage of lipids by either compensating for the lack of lipids through the consumption of synthesized lipids (Robb et al., 1972) or avoiding obesity by downregulating fat storage (Warbrick-Smith et al., 2006). Our results also shed light on the phenotypic variation and the genetic component regulating lipid storage, which might persist in a culture even after years of captivity breeding and affect commercial production. This opens the possibility for laboratory-assisted evolution of intake and storage of lipids at different temperatures. The use of insects for industrial purposes and particularly as a supplement in animal feed could be enhanced if we can harness this genetic variation to improve the production of house fly larvae for this purpose.