Abnormal energy metabolism can alter foraging behavior in termites in different social contexts

Foraging behavior, as an energy-consuming behavior, is very important for collective survival in termites. How energy metabolism related to glucose decomposition and ATP production influences foraging behavior in termites is still unclear. Here, we analyzed the change in energy metabolism in the whole organism and brain after silencing the key metabolic gene isocitrate dehydrogenase (IDH) and then investigated its impact on foraging behavior in the subterranean termite Odontotermes formosanus in different social contexts. The IDH gene exhibited higher expression in the abdomen and head of O. formosanus. The knockdown of IDH resulted in metabolic disorders in the whole organism, including the impairment of the NAD+-IDH reaction and decreased ATP levels and glucose accumulation. The dsIDH-injected workers showed significantly reduced walking activity but increased foraging success. Interestingly, IDH downregulation altered brain energy metabolism, resulting in a decline in ATP levels and an increase in IDH activity. Additionally, the social context obviously affected brain energy metabolism and, thus, altered foraging behavior in O. formosanus. We found that the presence of predator ants increased the negative influence on the foraging behavior of dsIDH-injected workers, including a decrease in foraging success. However, an increase in the number of nestmate soldiers could provide social buffering to relieve the adverse effect of predator ants on worker foraging behavior. Our orthogonal experiments further verified that the role of the IDH gene as an inherent factor was dominant in manipulating termite foraging behavior compared with external social contexts, suggesting that energy metabolism, especially brain energy metabolism, plays a crucial role in regulating termite foraging behavior. Author summary Foraging behavior plays a key role in collective survival in social insects, as found in termites. Worker termites are responsible for foraging duty and exhibit large foraging areas and long foraging distances, so they need to consume much energy during foraging. It is well established that energy can influence insect behaviors. However, how energy metabolism affects foraging behavior in termites remains unknown. Here, we found that the downregulation of the conserved metabolic gene IDH impaired whole-organism and the brain energy metabolism and further altered foraging behavior, resulting in decreased walking activity but increased foraging success in the termite O. formosanus, which is an important insect pest damaging embankments and trees in China. Additionally, the social context affected brain energy metabolism and obviously changed foraging behavior in O. formosanus, causing a decline in foraging success in the absence of nestmate soldiers and the presence of predator ants. However, the increasing number of nestmate soldiers strengthened social buffering to relieve the negative effect of predator ants on worker foraging behavior. Our findings provide new insights into the underlying molecular mechanism involved in modulating the sophisticated foraging strategy of termites in different social contexts from the perspective of energy metabolism.


Introduction
213 and the frequency in food zones in Treatment 8 was extremely significantly lower than 214 that in Treatment 1 and was markedly less than that in Treatment 2 (Tukey's HSD test, 215 F = 4.027, df = 8, 99, p < 0.001). Additionally, the cumulative duration of visits in food 216 zones in Treatment 8 was significantly shorter than that in Treatment 1, and the 217 cumulative duration of visits in food zones in Treatment 9 was also extremely 218 significantly shorter than that in Treatment 1 (Tukey's HSD test, F = 3.095, df = 8, 99, 219 p = 0.004).

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The impact degrees of three factors on the velocity and distance moved were as 232 workers, which supports the notion that the movement requires much energy [53], 233 particularly for foraging behavior, which requires integration across different sensory 234 modalities in worker termites. In our study, silencing IDH disrupted mitochondrial 235 metabolism, including the disruption of the NAD + -IDH reaction in the TCA cycle and 236 the reduction of ATP levels, NADH levels and IDH activity (Fig 2A, B and C), which 237 caused decreased walking activity, including shorter collective distances moved and 238 slower velocities. Additionally, the trajectories of worker termites showed lower 239 mobility (Fig 3B). These results suggest that mitochondrial metabolism plays a vital 240 role in the regulation of walking activity. Normally, glucose can be decomposed and 241 then produce ATP through the glycolysis pathway and TCA cycle [51]. However, a 242 significant increase in glucose content was found in this study after silencing IDH, 243 which was also recently described in association with the downregulation of human 244 IDH3α [54] and knockdown of termite IDH [52]. Our results indicated that silencing 245 IDH disrupted the process of glucose metabolism and resulted in the accumulation of 246 glucose [52]. Surprisingly, knocking down IDH increased the frequency and 247 cumulative duration in food zones within a certain amount of foraging time, suggesting 248 that termite colonies could ensure the food supply by increasing foraging success [35] 249 and prolonging the time of food acquisition to reduce the negative effects of energy 250 deficiency. The changes in the energy metabolism of the whole organism caused by 251 IDH knockdown in this study provided direct evidence of the importance of the rate-252 limiting enzyme IDH gene in regulating metabolic homeostasis and energy supply, 253 which is closely related to foraging behavior in termites.
IDH expression levels might change the energy metabolism pathway of the termite 255 brain, which is energetically demanding as well as a key regulator of energy metabolism 256 [55]. In this study, IDH knockdown significantly reduced ATP levels in the brains of 257 worker termites but dramatically improved IDH activity. These results suggested that 258 the production efficiency of ATP decreased. Usually, glucose is metabolized via 259 glycolysis and the TCA cycle to generate sufficient ATP through the process of 260 oxidative phosphorylation [56]. However, IDH downregulation disrupts the TCA cycle 261 and thereby reduces the generation of ATP. On the other hand, it has been demonstrated 262 that a decrease in the expression of IDH3α converts the metabolic mode from oxidative 263 phosphorylation to aerobic glycolysis in cancer-associated fibroblasts [54]. However, 264 glycolysis is far less efficient than the TCA cycle coupled to oxidative phosphorylation 265 in producing ATP [56]. Therefore, the decreasing ATP levels in the brain of dsIDH-266 injected termites suggests that the brain might choose an inefficient way of producing 267 ATP, such as aerobic glycolysis, resulting in a low efficiency of ATP generation.
268 Actually, ATP is necessary to maintain homeostasis and cell survival, and the loss of 269 intracellular ATP may result in cell necrosis or apoptosis [56,57]. In addition, energetic 270 constraints play a major role in neural plasticity and brain health [58]. Thus, an increase 271 in IDH activity might reduce the damage caused by ATP deficiency in dsIDH-injected 272 termites.

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Energy metabolism in the termite brain is not only impacted by IDH expression 274 levels but is also influenced by the social context. Some researchers have suggested that 275 social information can change gene expression in the brain to influence behaviors in 276 social insects [59]. We also found that social information could affect foraging behavior 277 by changing brain energy metabolism in termites. When worker termites are exposed 278 to predation risks during foraging, they may exhibit an aggressive state and consume 279 much energy [29]. At this moment, ATP levels decreased remarkedly, but the IDH 280 activity increased significantly in the brains of dsIDH-and dsGFP-injected foragers in 281 the social context with predators, suggesting that the degree of aerobic glycolysis might 282 be enhanced, as reported for the metabolic pathway in the brain of aggressive honey 283 bees [57]. In other words, the brains of worker termites may prefer an inefficient but 284 faster pathway for ATP production [56] to meet the high energy demands of foraging 285 behavior when they are threatened by predators. On the other hand, the addition of 286 nestmate soldiers during foraging may enhance the group cognition involved in the 287 emergent aggregation behavior of workers and social networks [60,61], which is 288 energetically costly in brains [12]. Therefore, the decrease in ATP became more 289 significant, and the increase in IDH activity was greater when soldiers and ants were 290 present concurrently. In addition, the social context may regulate the expression of 291 genes associated with encoding neuropeptides in the brain, such as gonadotropin-292 releasing hormone (GnRH), vasotocin (VT) and vasopressin (VP) [62,63]. The sensory 293 and higher-order integrative processing mechanisms that are used when insects behave 294 in complex social contexts are also associated with nervous systems of the brain [64], 295 and the energetic basis of these behaviors is a bridge between behavioral ecology and 296 neuroscience [16]. Moreover, intermediates of energy metabolism can impact the 297 concentration of neurotransmitters by regulating the synthesis of precursors such as 298 alpha-ketoglutarate, which is the precursor of the excitatory neurotransmitter glutamate 299 produced via the TCA cycle [57,65]. Thus, the changes in energy metabolism in the 300 brain caused by IDH downregulation and social context might alter the energetic and 301 nervous system bases of foraging behavior in termites.

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The abnormal energy metabolism in the whole organism and the brain caused by 303 IDH downregulation and the social context could modulate foraging behavior in 304 termites. Silencing IDH reduced the walking activity of worker termites and simplified 305 their foraging trajectory (Treatments 7, 8 and 9 in Table 1). This reduction in walking 306 activity decreases significantly when the number of nestmate soldiers was increased but 307 no ants were present (Treatment 7 in Table 1) compared to the water-or dsGFP-injected 308 workers, with normal energy metabolism (Treatments 1, 2, 3 and 6 in Table 1), 309 indicating that worker termites may choose a more profitable foraging strategy, such as 310 reducing their foraging area, to relieve the energy shortage [66,67] because more 311 energy may be needed to cover a larger movement space [68,69]. The foraging success 312 of dsIDH-injected workers significantly increased (Fig 3E and F Table 1), illustrating that soldiers can provide social buffering to cover 321 the damage caused by the increase in ants during foraging [24,32,70].

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However, the foraging trajectory of worker termites with normal energy metabolism 323 became complex when ants were present (Treatments 3 and 6 in Table 1), indicating 324 that the increase in ants led to more purposeless walking, which is energetically 325 demanding [19,22]. In addition, the walking activity and foraging success of the worker 326 termites showed no significant changes as the number of soldiers was increased 327 together with the number of ants (Treatments 1, 2 and 3 in Table 1). Therefore, the 328 presence of nestmate soldiers is crucial to reduce the purposeless walking of foraging 329 workers and relieve the predation pressure from ants to help foragers to accurately find 330 food sites [24]. Even though the increase in nestmate soldiers could decrease the 331 purposeless walking caused by a small number of ants, foraging success still decreased 332 when the number of soldiers increased (Treatment 5 in Table 1). In addition, orthogonal 333 experiments showed that IDH expression extremely significantly impacted both 334 walking activity and foraging success in foraging workers (Table 1), but predator ants 335 only significantly influenced foraging success in foraging workers (Table 1), strongly 336 suggesting that the inherent gene plays the dominant role in modulating foraging 337 behavior in termites compared to the external social context [35].
In summary, we confirmed that the metabolic gene IDH was an important regulator 339 of foraging behavior in termites, which can be influenced by different social contexts.
340 IDH downregulation reduced ATP levels and IDH activity, and disrupted the NAD + -341 IDH reaction in the TCA in the whole organism but increased IDH activity and 342 decreased ATP levels in the brains of termites (Fig 6).   377 formosanus workers [48]. The nested polymerase chain reaction (PCR) amplification 498 analyzed after 5 min of recording, which was also the procedure for the foraging assay 499 without the social context described above.

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Four important phenotypic parameters of foraging behavior were measured, 501 including the velocity and distance moved, and the frequency and cumulative duration 502 in food zones. In this study, the velocity and distance moved were used to describe the 503 walking activity of the worker termites, and the frequency and cumulative duration in 504 food zones were used to describe the foraging success of the worker termites [35,75]. 519 The significance level in this study was set at p < 0.05.

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Acknowledgments 521 We would like to thank Dr. Zhao Xincheng from Henan Agriculture University for 522 technical help with the brain tissue anatomy and immunocytochemistry of termites.    formosanus. IDH silencing impaired the NAD + -IDH reaction in the TCA, leading to a decrease in ATP levels, IDH activity and the NADH levels but an increase in glucose levels in the whole organism, resulting in increased IDH activity but decreased the ATP level in the brain. When dsIDH-injected workers foraged together, their velocity and distance moved decreased, but their frequency and cumulative duration in food zones increased, suggesting that IDH downregulation reduced walking activity but enhanced foraging success. The social context could also alter the brain energy metabolism of foraging workers, including decreasing ATP levels but the increasing IDH activity in the social context with ants and soldiers, which further changed the foraging behavior of the workers. When predator ants were present, the dsIDH-injected workers decreased their frequency and cumulative duration in food zones, showing a significant decline in foraging success. However, the increase in the number of nestmate soldiers strengthened social buffering to relieve the negative effect of predator ants on worker foraging behavior and, thus, improved the foraging success of dsIDH-injected workers. Our orthogonal experiments verified that the role of the IDH gene as an inherent factor was dominant in modulating termite foraging behavior compared to the external social context (predator ants and nestmate soldiers).
Thus, abnormal energy metabolism mediated by IDH altered termite foraging behavior in different social contexts. R indicates the impact degree of the three factors on the features of foraging behavior obtained from the L 9 (3 3 ) orthogonal experiment. A higher R value for a factor suggests a stronger effect on foraging behavior. The data in the table are the mean ± SEM, and different letters in a column indicate significant differences by Tukey's HSD test (p < 0.05, n = 12). Asterisks indicate significant differences by GLM (*** < 0.001, 0.01 < * < 0.05).