All organisms face the possibility of enduring limited access to food resources, posing a potential risk of mortality from starvation19. The recurrent utilization of metabolic rate depression as an adaptive survival strategy in different animals suggests that the regulation of metabolic arrest follows fundamental principles and mechanisms. These principles are not only evident in all cell types within an individual but are also conserved across phylogenetic lines20. Blood glucose level stands out as the most frequently measured physiological variable in starving animals. Initially, circulating glucose levels decrease in humans and most other mammals experiencing starvation 21,22.
A common response to acute hypoglycemia in mammals involves the secretion of glucagon from the pancreas, which facilitates the restoration of blood glucose to pre-starvation levels by utilizing stored hepatic glycogen21–23. Mammalian glucagon functions to stimulate gluconeogenesis while insulin leads to glycogenesis, using the newly produced glucose. Glycolytic enzymes such as hexokinase, pyruvate kinase and phosphofructokinase24–26 and gluconeogenic enzymes such as glucose-6-phosphorylase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glycerol-3-phosphate dehydrogenase, as well as various transaminases24,25,27 may be used to study the regulation of carbohydrate metabolism during starvation. Down-regulation of glycolytic enzymes and up-regulation of gluconeogenic enzymes have been observed in numerous starving animal species. However, the extent and timing of such changes vary significantly among different species.
Ticks, among other animals, must constantly deal with long periods of starvation28. Amblyomma spp. tick larvae can endure starvation for 6 months, while nymphs of the same species can survive up to 12 months without feeding29. Similarly, under harsh climatic circumstances, the larval stages of R. microplus can endure more than 6 months without feeding30. In this context, a dynamic regulation of energy metabolism is crucial for ticks to survive extended periods without nutrients. Importantly, tick egg development takes place entirely in the absence of external nutritional input7. Considering this, it is of fundamental importance to understand the metabolic remodeling processes undertaken by ticks to meet their energy demands for survival and development. To this end, we used tick embryonic cell line BME26 subjected to prolonged fasting as a model. Our study focused particularly on the use of energy reserves and the gluconeogenesis pathway, which is highly expressed in the embryonic phase of some arthropods7,10,31. We observed that BME26 cells exhibit high tolerance to starvation (Figure 1A and 1B), further endorsing its value as a model to study prolonged starvation in R. microplus. Apoptosis, a programmed cell death mechanism, intricately modifies cellular morphology through orchestrated events, including shrinkage, nuclear condensation, and apoptotic body formation. These processes eventually lead to controlled disassembly, breaking the cell into smaller fragments32. Flow cytometry analysis was conducted to assess cell death through apoptosis and necrosis in BME26 cells subjected to starvation. Interestingly, no apoptotic cell death was observed, despite the presence of late-stage apoptosis markers in these cells (Figure 2A and 2B). While substantial morphological transformations were not observed under the conditions of nutritional restriction, a notable trend to gradually reduce cell size over time was detected (Figure 3A and B). The subtle morphological changes observed in BME26 cells during starvation present an intriguing aspect of our findings. It suggests that these cells employ a conservative approach to save resources, potentially by reducing their size.
Potential energy sources that sustain the viability of BME26 cells during prolonged starvation were investigated. The protein content remained unchanged by starvation (Figure 4B), suggesting the activation of intricate adaptive mechanisms to preserve cellular integrity and metabolic function during nutrient scarcity. This phenomenon may involve mTORC1 signaling regulation, inhibiting protein synthesis to conserve energy while potentially reducing protein degradation, thus maintaining stable protein levels33. Additionally, autophagy activation, a cellular recycling mechanism, likely plays a crucial role in maintaining protein levels by targeting damaged or unnecessary proteins for degradation and subsequent recycling during starvation34.
TLC analysis detected no significant changes in the levels of different lipid classes (Figure 6). Surprisingly, the presence of triacylglycerol was not detected by TLC. These results show that starvation did not induce changes in BME26 lipid composition, highlighting a stable lipid profile under the tested conditions. Studies have shown that arthropods tend to conserve carbohydrate reserves initially during periods of fasting, leaving degradation for situations of more prolonged starvation6. Importantly in this context, ticks are capable of synthesizing glycogen again through partial gluconeogenesis during embryogenesis, as do BME26 embryonic cells in culture10,12. The glycogen content was evaluated (Figure 4B), and a marked reduction was observed at 24 and 48 hours of starvation, suggesting its mobilization as a source of energy during this challenge. The size of lipids droplets was evaluated through confocal microscopy, revealing no significant difference in droplet size over the observed starvation period (Figure 5 A and B). However, a reduction in cell nucleus size was noted during this starvation interval (Figure 5A and C). Glucose metabolism plays a pivotal role in the development, reproduction, and survival of ticks. Carbohydrate reserves serve as the primary stored energy source, particularly during extended periods without blood feeding10.
Studies performed in vivo have demonstrated that GSK3 participates in processes related to cell viability, oviposition, and hatching rate in R. microplus, demonstrating an important role in metabolism35. However, the functions performed by this enzyme in other biological and molecular targets, mainly its role during an extreme nutritional challenge in arthropod cells, remain unknown. Martins and colleagues (2015) observed that GSK3 may be indirectly involved in the gluconeogenic pathway, as PEPCK has its expression reduced after GSK3 silencing12. In this present work, the expression level of GSK3 gene was evaluated in BME26 cells during the course of fasting. It was observed that there was no increase in GSK3 expression during starvation (Figure 7A). The gene expression levels of GS and GDE are also unchanged (Figure 7B and C). These data suggest that, while fasting, there was no need for the activation of glycogen synthesis, since the availability of glucose in this situation is low. Also, further studies on the activity of these enzymes are needed to conclusively determine these biochemical interactions.
Cellular amino acid metabolism is essential for preserving redox equilibrium and safeguarding against oxidative harm caused by reactive oxygen species (ROS)36. ROS, natural by-products of cellular metabolism37, play essential roles in cellular processes like signaling and homeostasis38. At low to moderate levels, ROS act as secondary messengers in signaling pathways and support vital cellular functions 39,40. However, exceeding cellular antioxidant defenses leads to oxidative stress36,38,39. BME26 cells have a very impressive metabolic response to oxidative stress17,41. They exhibit high tolerance to hydrogen peroxide challenge, being able to maintain redox balance17. To determine if BME26 cells would be able to maintain redox balance even after the induced nutritional stress, we sought to evaluate the transcription of NADPH-dependent G6PDH and IDH. G6PDH is an enzyme classically described as responsible for maintaining cellular NADPH levels, thereby maintaining the reducing potential. Glucose-6-phosphate dehydrogenase (G6PDH) gene expression demonstrated a marked upregulation (Figure 8A), implying an augmented capability for preserving redox balance, which could be important in countering oxidative stress during fasting. Conversely, isocitrate dehydrogenase (IDH) expression was reduced (Figure 8B), suggestive of a potential redirection in metabolic pathways, likely oriented towards conserving energy stores and enabling adaptive responses in the face of nutrient scarcity. These observations are valuable insights into the metabolic adaptability under starvation conditions in BME26 cells, when these cells utilize amino acids to maintain the homeostasis, probably involving autophagic mechanisms.
Gluconeogenesis, insulin signaling pathway, and glycogen synthesis pathways stand out as highly conserved pathways among disease vectors7,10,12,31. Under normal physiological conditions, such as starvation, when glucose levels decrease, the gluconeogenic flux increases. Therefore, an increase in the expression of gluconeogenic enzymes is expected. In this study, a significant increase in the transcription of PEPCK was observed in BME26 cells after 24 and 48 hours of fasting (Figure 9D). Changes in PEPCK expression were found to correlate with its enzymatic activity in the liver and renal cortex of rats, tissues that are involved in glucose synthesis13. Studies on PEPCK have shown that transcriptional changes regulate the total activity of the enzyme13,14,42. Our results thus suggest the activation of gluconeogenesis during starvation, aiming to synthesize glucose to supply the necessary energy demand for cell viability. PEPCK is the enzyme responsible for catalyzing the conversion of oxaloacetate into phosphoenolpyruvate, playing an important role in the intersection of glycolysis, gluconeogenesis (Figure 9E), and the Krebs cycle13,15 Pyruvate kinase (PK) expression levels were significantly reduced after nutritional restriction, indicating that the conversion of phosphoenolpyruvate to pyruvate could be occurring at a smaller scale, prioritizing the gluconeogenic pathway over the glycolytic pathway during fasting (Figure 9B). Moreover, the observed decline in expression levels of glucose-6-phosphatase gene (Figure 9C), a key gluconeogenic enzyme, at both examined time points suggests a controlled reduction in glucose production. This adaptive response likely aims to optimize the utilization of available glucose resources during periods of nutrient scarcity. Furthermore, the diminished transcription of hexokinase (Figure 9A) and pyruvate kinase (Figure 9B) after 48 hours of starvation underscores a deceleration in glycolysis, highlighting a strategic cellular adjustment to prolonged nutrient deprivation (Figure 9D).
Autophagy is a vital catabolic process that helps maintain cellular homeostasis by degrading cytosolic material within lysosomes43. The ATG8 family of proteins, which are ubiquitin-like molecules, associate with autophagosomal membranes by binding with the lipid phosphatidylethanolamine. ATG8 is thought to facilitate the expansion and closure of the autophagosomal membranes (Figure 10D)44–46. Wang et al. conducted studies which demonstrated that embryonic cells from Ixodes scapularis and R. microplus can undergo autophagy in response to prolonged starvation47. Additionally, Moura et al. observed an increase in the expression of the ATG8 gene in BME26 cells following starvation48. The activation of this process was evaluated in BME26 cells during a period of imposed starvation. The survival mechanisms during long periods of starvation in ticks are still poorly understood. However, it is believed that autophagy may play a significant role as a strategy for coping with adverse environmental conditions47,49,50. In our current study, the expression of ATG8 was increased at 24 h and 48 h of starvation (Figure 10B). Other genes also related to the autophagic process, such as ATG6 and ATG4, were evaluated, showing a significant increase 48 h (Figure 10 A and C). These findings suggest a dynamic regulation of key autophagy-related genes (ATGs) in BME26 tick embryonic cells during starvation. The upregulation of ATG6, ATG8, and ATG4 at different time points implies a potential role for autophagy in cellular adaptation to nutrient scarcity43. Autophagy, as a catabolic process, may play a vital role in recycling cellular components and providing essential nutrients, contributing to the survival and resilience of BME26 cells during starvation conditions. This pathway may also provide a substrate for the gluconeogenic pathway. Further investigation is warranted to elucidate the precise mechanisms and functional implications of autophagy in this context.
In conclusion, our study on BME26 cells highlights their remarkable resilience to prolonged starvation, as evidenced by high cell viability and minimal morphological changes. The observed metabolic adaptations, including the mobilization of glycogen and upregulation of key genes related to autophagy and gluconeogenesis, shed light on the strategies employed by these tick embryonic cells to endure nutrient scarcity. These findings provide valuable insights into the metabolic flexibility and survival mechanisms of ticks, paving the way for further exploration of their unique adaptations to environmental challenges.