The energy stored in fatty acids is important for several critical activities of insects, such as embryogenesis, oviposition, and flight. However, to access this energy, these fatty acids need to be broken down in a pathway that makes maximum use of this energy by channeling it toward ATP production in the mitochondria. This pathway is mitochondrial β-oxidation. This pathway is well described in mammals, and the enzyme carnitine palmitoyltransferase I (CPT1), which is part of the carnitine shuttle and responsible for the formation of acyl-carnitine moieties from acyl-CoA species, plays a central role in β-oxidation. CPT1 is located on the exterior face of the mitochondria, and performs the limiting step of the pathway (Houten and Wanders, 2010). There are three different isoforms in mammals, CPT1A, CPT1B, and CPT1C, each with its own particular characteristics regarding expression in tissues and physiologic function (Price et al., 2002; Luiken et al., 2009). Insect models were of great importance in the past for understanding the β-oxidation pathway, with the demonstration that Tenebrio molitor requires an essential growth factor, which was initially called vitamin B_t but was later revealed to be carnitine, a substrate of CPT1 (Fraenkel, 1948; Carter et al., 1952). Despite this important first contribution, information about this pathway in insects is scarce.

Unlike mammals, insects and other arthropods have only one isoform of CPT1 (Lavárías et al., 2009; Price et al., 2010; Majerowicz et al., 2017). Insect development and adaptability to nutritional and environmental stresses both depend on the proper operation of the β-oxidation pathway. This is exemplified by the fact that D. melanogaster withered (whd) mutants, in which CPT1 is knocked out, are very sensitive to starvation (Strub et al., 2008). Additionally, the knockout of other components of the pathway downstream of CPT1 results in a reduction in life expectancy, deficiencies in locomotor activity, reduced oviposition and a reduced capacity of fatty acid oxidation in D. melanogaster (Kishita et al., 2012). However, some data indicate that CPT1 may not perform the rate-limiting step of the pathway, at least in some insects, such as the moths Prodenia eridania and Amphion floridensis (Stevenson, 1968; O’Brien and Suarez, 2001).

R. prolixus is a hematophagous insect with great economic and medical importance since it is one of the main vectors of Chagas disease in Central and South America (Antinori et al., 2017). Like other insects, it possesses most of the enzymes involved in β-oxidation, as shown by bioinformatic analyses (Majerowicz et al., 2017), and there are only a few studies concerning lipid oxidation in this model. The last three reactions of this pathway are catalyzed by the mitochondrial trifunctional protein (MTP), and the inhibition of the gene expression of the alpha subunit (HADHA, hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha) in R. prolixus impairs lipid mobilization during starvation and results in higher contents of triacylglycerol (TG) and larger lipid droplets (LDs) in the fat body, where lipids are stored after a blood meal (Arêdes et al., 2022). In addition, oviposition is decreased in these insects, as is the flight capacity. The knockdown of RhoprACSL2, a long-chain acyl-CoA synthetase that is one out of two isoforms responsible for the conversion of fatty acids to acyl-CoA moieties in this insect, also decreases oviposition and leads to a 90% reduction in β-oxidation rates, indicating that this enzyme activates fatty acids channeled for β-oxidation in R. prolixus (Alves-Bezerra et al., 2016). These results show that the proper activity of mitochondrial fatty acid oxidation is critical for reproductive success. On the other hand, the knockdown of RhoprGPAT1, a glycerol-3-phosphate acyltransferase that catalyzes the first step of de novo TG synthesis, leads to a 2-fold increase in β-oxidation rates and a 65% decrease in TG content in the fat body (Alves-Bezerra, et al., 2017). Thus, when fatty acids are not efficiently directed to TG synthesis, they are oxidized, and reserves are not properly formed, showing that there is a fine balance between lipid synthesis and degradation.

Although the β-oxidation pathway is fundamental for the processes of reproduction and locomotor activity and to guarantee metabolic homeostasis, the role of the CPT1 ortholog of R. prolixus (RhoprCPT1) in adaptation to starvation is unclear. R. prolixus is an intermittent feeder, and weeks can go by between blood meals. Therefore, the proper mobilization of lipid reserves in the fat body between feeding bouts is critical for survival, and RhoprCPT1 likely plays a central role in this process. Failure to mobilize these reserves may have implications for survival, oviposition, and flight. Thus, as we previously identified its gene in the R. prolixus genome (Majerowicz et al., 2017), we investigated the role of RhoprCPT1 in insect physiology, especially under starvation conditions, by assessing its gene expression and β-oxidation biochemical activity. We found that after inhibiting RhoprCpt1 gene expression using RNAi, survival during starvation and lipid mobilization were affected.

Stored lipids are very important for maintaining energetic homeostasis and contribute to organismal viability. When necessary, TG is mobilized from LDs, and the released fatty acids can be directed to the mitochondrial β-oxidation pathway. To enter mitochondria, activated fatty acids (acyl-CoA esters) are converted to acyl-carnitine esters, by the enzyme CPT1, and this reaction is commonly considered a critical step for the control of β-oxidation activity.

The phylogenetic analysis of the proteins with the choline/carnitine o-acyltransferase domain showed that R. prolixus has only one CPT1 ortholog, similar to all the other insects analyzed. This result confirms the previous findings of our group (Majerowicz et al., 2017). Moreover, this phylogenetic analysis revealed several gene duplication and loss patterns across different lineages. The presence of two independent duplications of the CPT2 gene in R. prolixus and B. tabaci suggests positive selection or functional divergence. Furthermore, extensive gene loss in the CROT clade suggests that the function of this gene may not be essential in many lineages, possibly due to redundancy with other enzymes or metabolic pathways. The diversification of the CRAT gene through multiple paralogs suggests functional divergence or neofunctionalization. These results provide a basis for generating hypotheses about the evolution and function of these enzymes and their associated domains. Further studies are needed to test these hypotheses and reveal the mechanisms driving the diversification and specialization of these enzymes across different lineages.

RhoprCpt1 expression in the flight muscle was much higher than that in any other analyzed organ, indicating that the flight muscle probably has a high oxidative capacity, which was confirmed when β-oxidation activity was measured in the flight muscle and the fat body. Indeed, it seems that, in R. prolixus, lipids importantly contribute to energy generation during flight (Ward et al., 1982; Oliveira et al., 2006). These rates of β-oxidation activity during starvation were expressed as normalized per protein, and under this condition, the total protein content in the flight muscle was approximately 15 times higher than that in the fat body (∼750 µg versus ∼50 µg) (Santos-Araujo et al., 2020). Thus, we could estimate that, considering the whole organ, the capacity of the flight muscle to oxidize fatty acids can be approximately 35 times higher than that of the fat body. Although gene expression was very high in the flight muscle, it was not modulated by a blood meal, in contrast to what occurred in the fat body, where RhoprCpt1 expression was higher in starved insects, and dramatically decreased after a blood meal. In D. melanogaster, the expression of diverse genes involved in lipid oxidation, including CPT1, was also higher in starved insects (Palanker et al., 2009). Surprisingly, despite the observed variation in RhoprCpt1 gene expression, β-oxidation activity was constant before and at various days after a blood meal, indicating that the fat body of R. prolixus adults has a similar capacity to oxidize lipids throughout the digestive cycle.

Fatty acid oxidation was not affected by the addition of etomoxir, an inhibitor of the mammalian isoform CPT1A (Kiorpes et al., 1984), similar to what was observed in cardiac myocytes of rats chronically treated with etomoxir, where CPT1B enzymatic activity is reduced, but β-oxidation rates are not (Luiken et al., 2009). Another point to be considered is that etomoxir needs to be converted to its acyl-CoA derivative, etomoxiryl-CoA to exert its inhibitory activity, but the specific enzyme responsible for this conversion in mammals has not been identified thus far, partly due to the lack of a crystal of CPT1 with bound etomoxir (Selby and Sherratt, 1989; Rufer et al., 2009). Thus, it is not known whether insects possess the enzymatic tools required for this conversion. Additionally, etomoxir has other intracellular targets, such as interferon γ (INF-γ), interleukin-17 α (IL-17α), tumor necrosis factor-α (TNF-α), and pyruvate dehydrogenase (Lopaschuk et al., 1990; Mørkholt et al., 2017), showing that it is not a specific inhibitor of CPT1. The addition of malonyl-CoA also had no effect on β-oxidation rates in the fat body samples. Malonyl-CoA is produced from acetyl-CoA, by ACC. Vertebrates have two isoforms of this enzyme, ACC1 and ACC2. In mammals, ACC1 is more highly expressed in lipogenic tissues, where generated malonyl-CoA is used for lipid synthesis, and ACC2 is expressed mostly in the heart and skeletal muscle, where malonyl-CoA acts as a CPT1 inhibitor and controls fatty acid oxidation (McGarry et al., 1983; Tong, 2005). Insects and other arthropods have only one ACC isoform (Saraiva et al., 2021), and to our knowledge, no physiological effect of malonyl-CoA on the β-oxidation rate has been demonstrated. CPT1 from D. melanogaster was expressed in the yeast Pichia pastoris, and the activity of the recombinant protein was inhibited by malonyl-CoA (Jackson et al., 1999), but whether this possible CPT1 inhibition may affect the insect β-oxidation rate was not evaluated. Thus, it is still not possible to say if fatty acid oxidation is regulated by this metabolite in these animals. In R. prolixus, de novo lipid synthesis in the fat body is triggered by a blood meal (Saraiva et al., 2021), and it is therefore possible that the malonyl-CoA concentration increases after feeding, because ACC catalyzes the first step in this process, but this has not been evaluated. On the other hand, β-oxidation activity was constant before and after the blood meal, suggesting that it was not regulated by malonyl-CoA in the concentration range found in the fat body cell. Even in the mammalian heart, the effect of malonyl-CoA on the β-oxidation rate is still being discussed. This effect can also be modulated by the presence of fatty acids, since increasing concentrations of palmitoyl-CoA reduce the malonyl-CoA inhibitory capacity, as shown in mitochondria and permeabilized muscle fibers (Smith et al., 2012). The effect of malonyl-CoA on CPT1 can also be modulated by other more complex mechanisms. Hepatic CPT1A interacts with the voltage-dependent anionic channel (VDAC) and ACSL to form a complex that channels acyl-CoA toward oxidation in the mitochondria (Lee et al., 2011). As VDAC also interacts with microtubules and tubulin, alterations in the cytoskeleton of hepatocytes and other cells may affect the activity of CPT1 and its sensitivity to malonyl-CoA (Velasco et al., 1998; Miotto et al., 2017). Notably, the availability of malonyl-CoA differs between mammals, as human muscles have lower levels of this metabolite than rat muscles, despite showing the same IC ₅₀ values for their respective CPT1 proteins (Eaton, 2002).

It should also be considered that peroxisomes could have contributed to the measured fatty acid oxidation rates, as we did not use isolated mitochondria, and the peroxisomal enzyme acyl-CoA oxidase (ACOX) can use fatty acids of 12–18 carbons, as demonstrated in rat and human models (Osumi et al., 1980; Chu et al., 1995). However, peroxisomes do not always fully oxidize fatty acids, instead generating 8- to 12-carbon fatty acids that are then exported to the mitochondria for further oxidation (Houten et al., 2020). Whereas mitochondrial β-oxidation requires carnitine for the import of fatty acids via CPT1, the transport of fatty acids into peroxisomes in D. melanogaster, and likely in R. prolixus as well, is mediated by the ATP-binding cassette transporters (ABC) of subfamily D (ABCD) and is not dependent on carnitine (Tawbeh et al., 2021). The fact that the addition of carnitine significantly improved the rate of fatty acid oxidation in the fat body of starved females confirmed that a large part of the oxidation we determined in our assays likely resulted from mitochondrial β-oxidation.

The higher TG content detected in the fat bodies of RhoprCpt1 knockdown females was also observed in D. melanogaster adults, when the gene expression of splicing factors that act on the CPT1 gene product was altered (Gingras et al., 2014; Bennick et al., 2019). The retention of TG in the silenced females was confirmed by the increase in the average diameter of the LDs and their size distribution, where a higher proportion of larger LDs was found in these insects, than in the controls. In insects, as in any other organism, lipids stored in LDs need to be mobilized to be oxidized, and both lipolysis and lipophagy may take part in this process (Zechner et al., 2012; Cingolani and Czaja, 2016; Lu et al., 2018; Santos-Araujo et al., 2020). In D. melanogaster, during starvation, the nuclear receptor hepatocyte nuclear factor 4 (HNF4) is activated by fatty acids freed from TG and induces the expression of enzymes involved in both lipid mobilization and β-oxidation for ATP production (Palanker et al., 2009). In line with these results, in Aedes aegypti, the knockdown of the HNF4 gene resulted in TG accumulation. In these mosquitoes, the expression of this nuclear receptor is activated by ecdysone, and the silencing of the ecdysone receptor causes a lower CPT1 gene expression level as well as larger LDs and a higher TG content (Wang et al., 2017). This link between lipid mobilization and oxidation was also noted when HADHA, an enzyme that takes part in the β-oxidation pathway as a component of the MTP, was silenced, and TG accumulated in the fat body of adults of R. prolixus (Arêdes et al., 2022). Additionally, similar to what is observed after RhoprCpt1 knockdown, the silencing of HADHA had no impact on flight muscle TG content but led to a reduced forced flight time when compared with control insects (Arêdes et al., 2022). It is noteworthy that despite the observed retention of TG in the fat body of the knockdown females and the decrease in survival during starvation, we were not able to detect any difference in β-oxidation activity. In mammals, fatty acid oxidation decreases under high levels of exercise despite a lack of increase in malonyl-CoA levels in both human and rat muscle, showing that CPT1 may not be the sole point of control of fatty acid oxidation in this tissue (Eaton, 2002). Therefore, perhaps the difference was too small in magnitude for detection, or some regulatory event acts to restore the oxidation flux. One possibility is that the fatty acid oxidation rates remained unchanged due to some unknown compensatory mechanism, such as the activity of CRAT, identified in the R. prolixus genome, which would generate acylcarnitine moieties in the absence of CPT1. In CRAT-knockout models, there is an increase in long-chain acylcarnitines, an indicator of incomplete β-oxidation (Muoio et al., 2012). Of course, these possibilities are highly speculative, and more studies are necessary to clarify this matter. On the other hand, we have previously shown that fatty acid oxidation is altered when other genes are silenced in R. prolixus. In adult females, ACSL2 knockdown results in a decrease in the β-oxidation rate, indicating that this enzyme activates fatty acids directed to this pathway (Alves-Bezerra et al., 2016). In contrast, when TG synthesis is inhibited due to GPAT1 silencing, β-oxidation is stimulated, probably because fatty acids are not used by the phosphatidic acid pathway at the same rate as in the control insects (Alves-Bezerra et al., 2017).

The reduced survival of the knockdown females under starvation conditions was similar to what is observed in the D. melanogaster HNF4 mutants, which show lower expression of the CPT1 gene and compromised TG mobilization (Palanker et al., 2009). In contrast to these results, when HADHA was silenced in adult females of R. prolixus, there was no difference in lifespan, possibly because the oxidation of other substrates, such as amino acids, counterbalanced this deficiency (Arêdes et al., 2022). Moreover, we cannot discard the possibility that other factors, in addition to energetic homeostasis, may also affect the response to starvation in the knockdown insects. In D. melanogaster, for instance, whitered (whd) CPT1 knockout mutants are very sensitive not only to starvation, but also to oxidative stress (Strub et al., 2008). Thus, despite the very different feeding habits of these two species, it is possible that the silenced R. prolixus adults have a shorter lifespan not only due to a deficiency in lipid utilization but also because of higher sensitivity to the oxidative stress regularly generated by insect metabolism. As RhoprCPT1 knockdown had a relatively small impact on the survival of the fasted insects, it did not significantly affect the vectorial capacity of the kissing bug. Thus, there are better targets than RhoprCPT1 for vector population control. We cannot rule out the possibility that RhoprCPT1 knockdown affects the parasite-vector interaction, but this possibility still needs to be investigated.

R. prolixus is an obligatory hematophagous insect during its whole life cycle. Blood is mainly composed of proteins, and carbohydrates and lipids are present in much lower amounts (Lynch et al., 2017). These insects feed infrequently on large meals, and amino acids released from the digestion of blood proteins are thus used for the de novo synthesis of lipids, which are stored in the fat body to be used until the next meal (Pontes et al., 2008; Saraiva et al., 2021; Moraes et al., 2022). Thus, lipids play a central role in the metabolism of this insect. This study confirmed that the dynamics of lipid mobilization from previously synthesized stores and their efficient oxidation are essential for R. prolixus survival.