**4. Invertebrate challenges and how marine species spend energy**

In most animal species, high energy levels in their bodies reveal fast growth, adequate energy storage, effective reproduction strategies and viable descendants with characteristic short life spans; however, reduced energy levels in a biological system results in affected gene expression, low survival rates and reduced metabolic rates and, therefore, a need on the part of physiological mechanisms to slow the ageing rate until environmental conditions are enhanced and higher energy levels are again reached (Stuart & Brown, 2006). In their natural habitat, many invertebrate species must undergo endogenous physiological processes during their life cycle, such as molting, starvation, quiescence and metamorphosis, among others. Many of these processes imply high energetic expense, causing a low energy status that reduces their ability to reach the adult stage (Hochachka & Somero, 2002).

The role of metamorphosis – one of the most amazing physiological endogenous processes in nature – becomes strikingly important when considering the large number of animal species that undergo metamorphic changes. Frequently, the energetic balance of holometabolous insects during metamorphosis is negative, because there is no energy gain and species must face all these changes by using any energetic reserves previously stored (Nestel et al., 2003).

During their larval stages, insects – such as Lepidopterans – show fast growth rates, as observed in the tobacco worm larvae of *Manduca sexta* which increases its mass 10,000-fold in just 16 days at the final larval instar (Goodman et al., 1985). The midgut epithelium of this species is a highly aerobic tissue that digests and absorbs nutrients, and transports ions at high rates. During metamorphic changes, the midgut epithelium is programmed to die and the larval midgut should maintain structural and functional integrity until the pupal epithelium is formed. During this process, ATP synthesis and mitochondrial function must be obligatorily maintained. Thus, organisms resolve this by reducing mitochondrial

Invertebrates Mitochondrial Function and Energetic Challenges 195

been suggested that the amazing aerobic capabilities of insects are based on a highly efficient mode of oxygen delivery that includes their oxygen transport system in a well

In addition, several studies have demonstrated that the function and energy needs of certain tissues are highly correlated with the number of mitochondria per cell (Robin & Wong, 1988). This agrees with the large quantities of mitochondria with pronounced cristae and large surface areas that are found in the flight muscle cells of the honey bee *Apis mellifera* (Suarez et al., 2000). To date, it is well-known that oxygen uptake rates in mitochondria cristae are much higher in the flying muscle of *A. mellifera* than that observed in mammals' mitochondria – this can explain the higher electron transport rates observed in such enzymes as cytochrome c oxidase, whose maximum catalytic capacity was recorded in this

Besides the increase on the ATP hydrolysis rate during flight, other mitochondrial adaptations to the highly and continuous energy requirements of flying species have been reported, such as the remarkable dependence on the synthesis of energy-rich phosphate compounds like phosphoarginine. Phosphoarginine, as mentioned above, constitutes a usable pool of high energy phosphate (Hird, 1986) so as to maintain the high rate of ATP

In addition to the various metamorphic changes in their life, crustaceans undergo a frequent and cyclic process: molting. During the molt cycle, crustaceans are exposed to a temporary scarcity of food since they lack the ability to handle food until their new exoskeleton is synthesised. Several adaptive strategies have been recognised as being employed by these organisms so as to avoid the adverse effects of starvation, such as the storage of fuel compounds in their midgut gland (Sanchez-Paz et al., 2007), changes in locomotor activity (Hervant & Renault 2002), and a decrease in oxygen consumption (Morris et al., 2005). However, little attention has been paid to the bioenergetic consequences of starvation in shrimp; since the composition of food plays an important role in oxidative phosphorylation, the nutritional status of shrimp species, such as *Litopenaeus vannamei*, may affect its major

In our lab, we have hypothesised that, due of its central role in the cell energy metabolism, the expression of genes encoding the different polypeptide subunits that compose ATP synthase during unpredictable episodes of food shortage may ultimately be modulated. Thus, we experimentally evaluate the effect of starvation in the gene expression of subunits

deprivation (5 days). Our results (Figure 2) show that the mRNA amounts from subunits

increased; however, no significant changes were observed in the mRNA amounts of *atp9*,

Sanchez-Paz et al., (2007) reported a gradual decrease of glycogen in the midgut gland of the white shrimp as starvation progressed. After a 24 h starvation period, the glycogen content dropped by about 50%, which correlates with an increase of the *atp*9 subunit after 24 h of starvation, suggesting that glycogen may be used as fuel to generate ATP and pyruvic acid. As glycogen stores become depleted, the organism must increasingly rely on fatty acid catabolism as a source for ATP synthesis. In general, starved shrimp showed a sharp decrease in their midgut gland lipidic constituents for up to 120 h (more noticeable in

which forms the oligomeric ring from Fo in the shrimp ATP-synthase.

and *atp*in the shrimp midgut gland, during a period of short-term food

which directly participate during ATP synthesis decreased as starvation time

distributed system of tracheae and tracheoles (Wegener, 1996).

species during flight - (Suarez et al., 2000).

turnover in flying insects (Wegener, 1996).

bioenergetic functions.

acylglycerides).

*atp, atp*

*atp*and *atp*

substrate oxidation, a clear indication that the electron transport chain may be a site of modulation during metamorphosis (Chamberlin, 2004).

Quiescence and estivation are also two responses that some species may display during unfavourable environmental conditions in which insufficient energy is available to grow and breed. These dormant states allow species to survive by reversibly down-regulating their metabolism to low levels for up to several years. Among invertebrates, many species show quiescent states at stress conditions, including nematodes, crustaceans such as the brine shrimp *Artemia franciscana* (Hand, 1998), the estivating pulmonate snail *Helix aspersa* (Pedler et al., 1996), and various insect species entering in diapause, such as *Helicoverpa armigera.* Studies have proposed that a coordination mechanism is required when animals enter into the dormant state so as to maintain cellular homeostasis by both energyconsuming and energy-producing pathways. During quiescence, *A. franciscana* can reduce its metabolism essentially to zero, this metabolic-rate suppression affects the mitochondrial respiratory capacity and the rates of ATP-consuming processes (Barger et al., 2003). In the embryos of *Artemia franciscana*, anoxia provokes the organism to enter into a quiescent state. During experimental gradual oxygen removal, various biochemical responses are observed, such as a pH decrease, the reduction of heat production and the depression of ATP levels. Also, genetic responses, such as the down-regulation of RNA transcription, are observed during quiescence (Hand, 1998).

Often, metabolic rates have been inversely related to the life span of mammals. Moreover, when mitochondrial respiration has been inhibited by RNAi techniques, the life span extends in *C. elegans* (Lee et al., 2003), and long-lived mutants of this nematode concomitantly show decreased metabolic rates (Stuart & Brown, 2006).

The process by which mitochondrial respiration affects or extends life span has been studied in several organisms, including yeasts, worms, flies and mice (Lee et al., 2010). Electron transport in mitochondria is the main producer of superoxide anion (O- ), which in turn generates several types of reactive oxygen species (ROS), as has been mentioned (mitochondrial Complex III). In fact, according to various studies, ROS are not only undesirable toxic metabolites promoting organism oxidative stress, but they are also molecules that participate in the mitochondria-nucleus's signalling pathways (Storz, 2006). Emerging data on *C. elegans* suggests a new described pathway where superoxide serves as an intracellular messenger, whereby with increasing superoxide concentration a signal transduction pathway is triggered, resulting in changes in the pattern of gene expression of nuclear proteins and which finally results in an increased life span (Yang & Hekimi, 2010). However, different mechanisms have also been proposed as being implicated in the aging process, such as diet restriction, ubiquinone deficiency and the hypoxic response (Klimova & Chandel, 2008).

At this point, this chapter would not be complete if the energetic costs of flying for insect species were to be omitted. This activity is probably the most expensive process recorded in nature. It is by now a well-known and remarked-upon fact that the metabolic rate during insect flight increases over 50-100 fold above the resting rate (Ellington, 1985). Thus, it is clear that the flight muscle of insects is the model tissue that many researchers have adopted in order to understand mitochondrial function since it is capable of effectively producing and hydrolysing large amounts of ATP (Sherwood et al., 2005). Insect flight is a highly oxygen-dependent process, and the flight muscle metabolism is fully aerobic; thus, it has

substrate oxidation, a clear indication that the electron transport chain may be a site of

Quiescence and estivation are also two responses that some species may display during unfavourable environmental conditions in which insufficient energy is available to grow and breed. These dormant states allow species to survive by reversibly down-regulating their metabolism to low levels for up to several years. Among invertebrates, many species show quiescent states at stress conditions, including nematodes, crustaceans such as the brine shrimp *Artemia franciscana* (Hand, 1998), the estivating pulmonate snail *Helix aspersa* (Pedler et al., 1996), and various insect species entering in diapause, such as *Helicoverpa armigera.* Studies have proposed that a coordination mechanism is required when animals enter into the dormant state so as to maintain cellular homeostasis by both energyconsuming and energy-producing pathways. During quiescence, *A. franciscana* can reduce its metabolism essentially to zero, this metabolic-rate suppression affects the mitochondrial respiratory capacity and the rates of ATP-consuming processes (Barger et al., 2003). In the embryos of *Artemia franciscana*, anoxia provokes the organism to enter into a quiescent state. During experimental gradual oxygen removal, various biochemical responses are observed, such as a pH decrease, the reduction of heat production and the depression of ATP levels. Also, genetic responses, such as the down-regulation of RNA transcription, are observed

Often, metabolic rates have been inversely related to the life span of mammals. Moreover, when mitochondrial respiration has been inhibited by RNAi techniques, the life span extends in *C. elegans* (Lee et al., 2003), and long-lived mutants of this nematode

The process by which mitochondrial respiration affects or extends life span has been studied in several organisms, including yeasts, worms, flies and mice (Lee et al., 2010). Electron

generates several types of reactive oxygen species (ROS), as has been mentioned (mitochondrial Complex III). In fact, according to various studies, ROS are not only undesirable toxic metabolites promoting organism oxidative stress, but they are also molecules that participate in the mitochondria-nucleus's signalling pathways (Storz, 2006). Emerging data on *C. elegans* suggests a new described pathway where superoxide serves as an intracellular messenger, whereby with increasing superoxide concentration a signal transduction pathway is triggered, resulting in changes in the pattern of gene expression of nuclear proteins and which finally results in an increased life span (Yang & Hekimi, 2010). However, different mechanisms have also been proposed as being implicated in the aging process, such as diet restriction, ubiquinone deficiency and the hypoxic response (Klimova

At this point, this chapter would not be complete if the energetic costs of flying for insect species were to be omitted. This activity is probably the most expensive process recorded in nature. It is by now a well-known and remarked-upon fact that the metabolic rate during insect flight increases over 50-100 fold above the resting rate (Ellington, 1985). Thus, it is clear that the flight muscle of insects is the model tissue that many researchers have adopted in order to understand mitochondrial function since it is capable of effectively producing and hydrolysing large amounts of ATP (Sherwood et al., 2005). Insect flight is a highly oxygen-dependent process, and the flight muscle metabolism is fully aerobic; thus, it has

), which in turn

concomitantly show decreased metabolic rates (Stuart & Brown, 2006).

transport in mitochondria is the main producer of superoxide anion (O-

modulation during metamorphosis (Chamberlin, 2004).

during quiescence (Hand, 1998).

& Chandel, 2008).

been suggested that the amazing aerobic capabilities of insects are based on a highly efficient mode of oxygen delivery that includes their oxygen transport system in a well distributed system of tracheae and tracheoles (Wegener, 1996).

In addition, several studies have demonstrated that the function and energy needs of certain tissues are highly correlated with the number of mitochondria per cell (Robin & Wong, 1988). This agrees with the large quantities of mitochondria with pronounced cristae and large surface areas that are found in the flight muscle cells of the honey bee *Apis mellifera* (Suarez et al., 2000). To date, it is well-known that oxygen uptake rates in mitochondria cristae are much higher in the flying muscle of *A. mellifera* than that observed in mammals' mitochondria – this can explain the higher electron transport rates observed in such enzymes as cytochrome c oxidase, whose maximum catalytic capacity was recorded in this species during flight - (Suarez et al., 2000).

Besides the increase on the ATP hydrolysis rate during flight, other mitochondrial adaptations to the highly and continuous energy requirements of flying species have been reported, such as the remarkable dependence on the synthesis of energy-rich phosphate compounds like phosphoarginine. Phosphoarginine, as mentioned above, constitutes a usable pool of high energy phosphate (Hird, 1986) so as to maintain the high rate of ATP turnover in flying insects (Wegener, 1996).

In addition to the various metamorphic changes in their life, crustaceans undergo a frequent and cyclic process: molting. During the molt cycle, crustaceans are exposed to a temporary scarcity of food since they lack the ability to handle food until their new exoskeleton is synthesised. Several adaptive strategies have been recognised as being employed by these organisms so as to avoid the adverse effects of starvation, such as the storage of fuel compounds in their midgut gland (Sanchez-Paz et al., 2007), changes in locomotor activity (Hervant & Renault 2002), and a decrease in oxygen consumption (Morris et al., 2005). However, little attention has been paid to the bioenergetic consequences of starvation in shrimp; since the composition of food plays an important role in oxidative phosphorylation, the nutritional status of shrimp species, such as *Litopenaeus vannamei*, may affect its major bioenergetic functions.

In our lab, we have hypothesised that, due of its central role in the cell energy metabolism, the expression of genes encoding the different polypeptide subunits that compose ATP synthase during unpredictable episodes of food shortage may ultimately be modulated. Thus, we experimentally evaluate the effect of starvation in the gene expression of subunits *atp, atp* and *atp*in the shrimp midgut gland, during a period of short-term food deprivation (5 days). Our results (Figure 2) show that the mRNA amounts from subunits *atp* and *atp* which directly participate during ATP synthesis decreased as starvation time increased; however, no significant changes were observed in the mRNA amounts of *atp9*, which forms the oligomeric ring from Fo in the shrimp ATP-synthase.

Sanchez-Paz et al., (2007) reported a gradual decrease of glycogen in the midgut gland of the white shrimp as starvation progressed. After a 24 h starvation period, the glycogen content dropped by about 50%, which correlates with an increase of the *atp*9 subunit after 24 h of starvation, suggesting that glycogen may be used as fuel to generate ATP and pyruvic acid. As glycogen stores become depleted, the organism must increasingly rely on fatty acid catabolism as a source for ATP synthesis. In general, starved shrimp showed a sharp decrease in their midgut gland lipidic constituents for up to 120 h (more noticeable in acylglycerides).

Invertebrates Mitochondrial Function and Energetic Challenges 197

Various studies have shown that during starvation-induced lipolysis there is a decrease in the amount of ATP, which was accompanied by a fall in some subunits of the FoF1-ATP synthase (Vendemiale et al., 2001). It is well-known that starvation tampers with cellular detoxification systems and may expose cells to oxidative injury (Di Simplicio et al., 1997; Vendemiale et al., 2001), leading to an impaired production of ATP and a reduced uptake of substrates for mitochondrial metabolism. The results from our study, together with results from previous studies, prompt us to suggest that shrimp are capable to satisfy their energy demands through a complex combination of mechanisms that enables them to survive the

Due to its density, viscosity (800 times more dense and 50 times more viscous than air) and low oxygen solubility, water – as a respiratory medium – imposes difficulties for aquatic breathers in obtaining the necessary supply of oxygen from their surrounding environment so as to keep breathing and bringing oxygen into their systems. This process becomes more complicated when considering additional parameters (such as temperature, salinity and depth) affecting the dissolved oxygen concentration of seawater, causing additional constraints on marine species' development (Sherwood et al., 2005). All the species inhabiting marine environments should face these dynamic environmental conditions, which in over the last few decades have been seriously affected by a wide variety of

Several studies have found that marine invertebrates may respond to stress conditions by changes at the transcriptional level. In crustacean species such as the crab *Eriocheir sinensis*, different gene expression profiles from gills were characterised during acclimation to high cadmium concentrations in water. Analyses have revealed over-expressed genes, such as disulphide isomerase, thioredoxin peroxidase and glutathione S-transferase. Under the same conditions, ATP synthase beta, alpha tubulin, arginine kinase, glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase were down-regulated. The results demonstrated that acute and chronic exposure to waterborne cadmium induced a decreased abundance of the transcript-encoding enzymes involved in energy transfer; this suggests that chronic

anthropogenic activities, such as industrial and agricultural runoffs (Wu, 2002).

metal exposure induced an important metabolic reorganisation (Silvestre et al., 2006).

the mitochondrial system of ectotherm species.

Some other species which face high cadmium concentrations are marine intertidal molluscs, such as oysters, which live in estuaries were fluctuating temperatures and levels of trace metals are known to directly affect mitochondrial function. Isolated mitochondria from the oyster *Crassostrea virginica* which were exposed to low cadmium concentrations (1 µmol.

resulted in a progressive uncoupling that increased with the increasing dose of cadmium; this response agrees with that observed in mammals. However, unlike mammals, molluscs are ectotherms and the exposure to the combined effects of high temperatures and cadmium concentrations severely affected mitochondrial function since elevated temperatures increased the sensitivity of this organelle to cadmium and promoted an increase in the rate of ROS production (Sokolova, 2004). These results highlight the key role of temperature in

Most invertebrates are described as ectotherm species because their body temperatures vary with the environment. At very low temperatures, polar marine invertebrates were expected to show low metabolic rates, as previously observed in Antarctic fish; however, in 1999 Sommer & Portner found important intraespecific differences in the mitochondrial function of the polychaete *Arenicola marina* from the North Sea and the colder White Sea. Their results

L-1)

adverse effects of food scarcity.

Fig. 2. The relative expression of A) ATP, B) ATP and C) ATP9 mRNA in the midgut gland of the white shrimp *Litopenaeus vannamei* in response to a short-term starvation period. Expression values are given based on normalisation to L8. The data is represented as the mean and standard deviation of triplicate determinations. (\*) Statistical significance was considered at *P*  0.05.

(A)

(B)

(C)

Fig. 2. The relative expression of A) ATP, B) ATP and C) ATP9 mRNA in the midgut gland of the white shrimp *Litopenaeus vannamei* in response to a short-term starvation period. Expression values are given based on normalisation to L8. The data is represented as the mean and standard deviation of triplicate determinations. (\*) Statistical significance was

considered at *P*  0.05.

Various studies have shown that during starvation-induced lipolysis there is a decrease in the amount of ATP, which was accompanied by a fall in some subunits of the FoF1-ATP synthase (Vendemiale et al., 2001). It is well-known that starvation tampers with cellular detoxification systems and may expose cells to oxidative injury (Di Simplicio et al., 1997; Vendemiale et al., 2001), leading to an impaired production of ATP and a reduced uptake of substrates for mitochondrial metabolism. The results from our study, together with results from previous studies, prompt us to suggest that shrimp are capable to satisfy their energy demands through a complex combination of mechanisms that enables them to survive the adverse effects of food scarcity.

Due to its density, viscosity (800 times more dense and 50 times more viscous than air) and low oxygen solubility, water – as a respiratory medium – imposes difficulties for aquatic breathers in obtaining the necessary supply of oxygen from their surrounding environment so as to keep breathing and bringing oxygen into their systems. This process becomes more complicated when considering additional parameters (such as temperature, salinity and depth) affecting the dissolved oxygen concentration of seawater, causing additional constraints on marine species' development (Sherwood et al., 2005). All the species inhabiting marine environments should face these dynamic environmental conditions, which in over the last few decades have been seriously affected by a wide variety of anthropogenic activities, such as industrial and agricultural runoffs (Wu, 2002).

Several studies have found that marine invertebrates may respond to stress conditions by changes at the transcriptional level. In crustacean species such as the crab *Eriocheir sinensis*, different gene expression profiles from gills were characterised during acclimation to high cadmium concentrations in water. Analyses have revealed over-expressed genes, such as disulphide isomerase, thioredoxin peroxidase and glutathione S-transferase. Under the same conditions, ATP synthase beta, alpha tubulin, arginine kinase, glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase were down-regulated. The results demonstrated that acute and chronic exposure to waterborne cadmium induced a decreased abundance of the transcript-encoding enzymes involved in energy transfer; this suggests that chronic metal exposure induced an important metabolic reorganisation (Silvestre et al., 2006).

Some other species which face high cadmium concentrations are marine intertidal molluscs, such as oysters, which live in estuaries were fluctuating temperatures and levels of trace metals are known to directly affect mitochondrial function. Isolated mitochondria from the oyster *Crassostrea virginica* which were exposed to low cadmium concentrations (1 µmol. L-1) resulted in a progressive uncoupling that increased with the increasing dose of cadmium; this response agrees with that observed in mammals. However, unlike mammals, molluscs are ectotherms and the exposure to the combined effects of high temperatures and cadmium concentrations severely affected mitochondrial function since elevated temperatures increased the sensitivity of this organelle to cadmium and promoted an increase in the rate of ROS production (Sokolova, 2004). These results highlight the key role of temperature in the mitochondrial system of ectotherm species.

Most invertebrates are described as ectotherm species because their body temperatures vary with the environment. At very low temperatures, polar marine invertebrates were expected to show low metabolic rates, as previously observed in Antarctic fish; however, in 1999 Sommer & Portner found important intraespecific differences in the mitochondrial function of the polychaete *Arenicola marina* from the North Sea and the colder White Sea. Their results

Invertebrates Mitochondrial Function and Energetic Challenges 199

biochemical levels – of the mitochondrial proteins and enzymes that participate in the

The main physiological responses from invertebrates to hypoxia are somewhat similar to those from vertebrates since in the reduction or absence of oxygen, animal cells are not able to produce enough energy to survive. Such general responses are clearly a legacy of the evolutionary past from ancestral forms and they serve adaptive ends. In marine species, such as crustaceans and molluscs, reduced oxygen consumption and metabolic rates have been confirmed during hypoxia; in addition, glucose utilisation and lactate accumulation as indicators of a switch to anaerobic metabolism have been detected at low oxygen concentrations in water (Racotta et al., 2002; Martinez-Cruz, 2007; Soldatov et al., 2010). In the brine shrimp *A. Franciscana*, the intracellular pH falls at anoxia, heat production is reduced and

ATP concentrations are also depressed to low levels (Hand, 1998; Eads & Hand, 2003).

A large amount of information is now available about the changes at the transcriptional level promoted by hypoxia in invertebrates, most of it concerning aquatic species. In our lab, we have evaluated the effects of hypoxia in the gene expression of FOF1 ATP synthase

shrimp *L. vannamei*. Results show a general trend towards increase the amount of mRNA as oxygen concentrations decrease (Martinez-Cruz, 2007; Martinez-Cruz et al., 2011; Martinez-Cruz et al. in preparation). Also, significant changes in the amount of mRNA from the mitochondrial- and nuclear- encoded subunits of the ATP synthase were detected at different molt stages and tissues, according to the energy requirements of each stage and the specific requirements of the function of each tissue (Muhlia-Almazan et al., 2008). Chronic exposure to severe hypoxia (1.5 mg/mL during 7 days) also causes the increased transcription of mitochondrial-encoded genes, such as the 16S, CO1, and CO2 subunits from the cytochrome C oxidase in the grass shrimp *Palaemonetes pugio* (Brouwer et al., 2008). To date, microarray technologies have revealed a set of genes that are up- and down-regulated in *P. pugio* during chronic, acute and moderate hypoxia; the results revealed that various

In the absence of oxygen, animal cells activate transcription factors – such as the wellstudied vertebrates hypoxia-inducible factor (HIF) – which has been reported in invertebrates from worms to flies (Semenza, 2007). When activated, HIF leads the organism to exhibit metabolic adaptation to hypoxia by regulating the genetic expression of some proteins and enzymes involved in central biological processes such as glycolysis, erythropoiesis, breathing and angiogenesis so as to maintain cell homeostasis (Klimova & Chandel, 2008). In the shrimp *P. pugio*, a homolog protein to HIF- called gsHIF was found in this hypoxia-tolerant species. It includes all the conserved domains of vertebrates' HIF proteins, and an additional polypeptide sequence of 130 residues that has not been found in databases, and its participation in the functional properties of the protein has not yet been determined (Li & Brouwer, 2009). In the white shrimp *L. vannamei*, HIF-1 is a heterodimer formed by two subunits: HIF-1which is constitutively expressed in shrimp cells and HIF-1which is differentially expressed in hypoxic conditions. HIF-1 is suggested in crustaceans to be the master regulator that senses decreased oxygen availability and transmits signals promoting the physiological responses mentioned above (Soñanez-Organis et al., 2009). Additional functions have been attributed to HIF in coral species, such as *Acropora millepora*, where the diel cycle in the central metabolism appear to be governed by the circadian clock and regulated by the HIF system operating in parallel (Levy et al., 2011).

in different tissues of the white

respiration process are still poorly studied for most invertebrate species.

*, atp, atp, atp, and atp*

genes encoding mitochondrial proteins were affected (Li & Brouwer, 2009).

subunits, such as *atp9, atp6, atp*

concluded that invertebrate life is more costly at higher latitudes, where oxygen uptake, tissues mitochondrial densities and mitochondrial capacities were higher.

Remarkable abilities have been recorded in invertebrate species inhabiting extreme environments. The term "metabolic plasticity" perfectly describes such organisms as the intertidal periwinkle snail *Littorina littorea*, which has the ability to deal with very low temperatures and also to tolerate the changing environmental conditions imposed by the tidal cycle, implying continuous oxygen deprivation (Storey, 1993). Besides the biochemical and physiological mechanisms previously identified in this species, the over-expressed gene encoding a metallothionein (MT) was recently found during the exposure to low temperature and anoxic conditions of the tissues of *L. littorea*. Since thermogenesis is a process that requires high oxygen consumption and since it is also accompanied by a sharp rise in reactive oxygen species (ROS) generation, the authors describe the ability of MT to function as an antioxidant and as a reservoir of essential metals that contributes to survival under these conditions (English & Storey, 2003).

The deep sea hydrothermal vents are a different type of extreme environment where thermophilic species such as the Pompeii worm *Alvinella pompejana* inhabit. Shin et al. (2009) studied the structure and biochemical characteristics of the Cu,Zn-superoxide dismutase (SOD) of this species and found striking similarities between this enzyme and that of humans, but with an enhanced stability and catalysis – characteristics that may mean that this enzyme is potentially suitable for scientific and medical application. Other mitochondrial proteins have been proposed as a part of gene therapy for devastating human diseases by preventing the cell damage caused by oxidative stress. AOX – the mitochondrial alternative oxidase previously mentioned – is suggested to work in any cell, becoming chemically active only when it is required. AOX is provided to the cell by engineering a gene from a marine invertebrate snail *Ciona intestinalis*; this protein is under analysis as a therapeutic tool tested in mammalian disease models (Hakkaart et al., 2006).

## **5. How do invertebrates face hypoxia?**

Hypoxia is probably one of the most studied factors affecting the central metabolic pathways of living organisms, including invertebrates. Aquatic species usually face hypoxic events in freshwater or marine environments as a daily cyclic routine in the shallow waters of lagoons, estuaries and mangroves during the dark hours, when plants and algae do not produce oxygen and organic matter is continuously oxidised (Dall et al., 1990). However, nowadays the frequency, abundance and severity of hypoxic events in coastal waters have increased due to anthropogenic activities resulting in deteriorating environments affecting marine organisms (Diaz, 2001). It is well known that hypoxia depresses the growth rate of marine animals, as it disturbs metabolic pathways and promotes the reallocation of energy resources (Wei et al., 2008; Wang et al., 2009).

Several studies have examined the physiological responses of invertebrate species to hypoxia, such as growth, stress resistance and even behaviour patterns in aquatic species able to vertically and horizontally migrate through the water column to reach more oxygenated zones (Eads & Hand, 2003; Burgents et al., 2005; Abe et al., 2007; Seibel, 2011). In fact, among invertebrates there are hypoxia-tolerant species, such as bivalve molluscs and annelids, with highly adapted structures and mechanisms to deal with hypoxia, and some others, such as crustaceans, whose tolerance to hypoxia depends on their habitat, food, and energy needs. Unfortunately, the responses to hypoxic conditions – at the molecular and

concluded that invertebrate life is more costly at higher latitudes, where oxygen uptake,

Remarkable abilities have been recorded in invertebrate species inhabiting extreme environments. The term "metabolic plasticity" perfectly describes such organisms as the intertidal periwinkle snail *Littorina littorea*, which has the ability to deal with very low temperatures and also to tolerate the changing environmental conditions imposed by the tidal cycle, implying continuous oxygen deprivation (Storey, 1993). Besides the biochemical and physiological mechanisms previously identified in this species, the over-expressed gene encoding a metallothionein (MT) was recently found during the exposure to low temperature and anoxic conditions of the tissues of *L. littorea*. Since thermogenesis is a process that requires high oxygen consumption and since it is also accompanied by a sharp rise in reactive oxygen species (ROS) generation, the authors describe the ability of MT to function as an antioxidant and as a reservoir of essential metals that contributes to survival

The deep sea hydrothermal vents are a different type of extreme environment where thermophilic species such as the Pompeii worm *Alvinella pompejana* inhabit. Shin et al. (2009) studied the structure and biochemical characteristics of the Cu,Zn-superoxide dismutase (SOD) of this species and found striking similarities between this enzyme and that of humans, but with an enhanced stability and catalysis – characteristics that may mean that this enzyme is potentially suitable for scientific and medical application. Other mitochondrial proteins have been proposed as a part of gene therapy for devastating human diseases by preventing the cell damage caused by oxidative stress. AOX – the mitochondrial alternative oxidase previously mentioned – is suggested to work in any cell, becoming chemically active only when it is required. AOX is provided to the cell by engineering a gene from a marine invertebrate snail *Ciona intestinalis*; this protein is under analysis as a

Hypoxia is probably one of the most studied factors affecting the central metabolic pathways of living organisms, including invertebrates. Aquatic species usually face hypoxic events in freshwater or marine environments as a daily cyclic routine in the shallow waters of lagoons, estuaries and mangroves during the dark hours, when plants and algae do not produce oxygen and organic matter is continuously oxidised (Dall et al., 1990). However, nowadays the frequency, abundance and severity of hypoxic events in coastal waters have increased due to anthropogenic activities resulting in deteriorating environments affecting marine organisms (Diaz, 2001). It is well known that hypoxia depresses the growth rate of marine animals, as it disturbs metabolic pathways and promotes the reallocation of energy

Several studies have examined the physiological responses of invertebrate species to hypoxia, such as growth, stress resistance and even behaviour patterns in aquatic species able to vertically and horizontally migrate through the water column to reach more oxygenated zones (Eads & Hand, 2003; Burgents et al., 2005; Abe et al., 2007; Seibel, 2011). In fact, among invertebrates there are hypoxia-tolerant species, such as bivalve molluscs and annelids, with highly adapted structures and mechanisms to deal with hypoxia, and some others, such as crustaceans, whose tolerance to hypoxia depends on their habitat, food, and energy needs. Unfortunately, the responses to hypoxic conditions – at the molecular and

therapeutic tool tested in mammalian disease models (Hakkaart et al., 2006).

tissues mitochondrial densities and mitochondrial capacities were higher.

under these conditions (English & Storey, 2003).

**5. How do invertebrates face hypoxia?** 

resources (Wei et al., 2008; Wang et al., 2009).

biochemical levels – of the mitochondrial proteins and enzymes that participate in the respiration process are still poorly studied for most invertebrate species.

The main physiological responses from invertebrates to hypoxia are somewhat similar to those from vertebrates since in the reduction or absence of oxygen, animal cells are not able to produce enough energy to survive. Such general responses are clearly a legacy of the evolutionary past from ancestral forms and they serve adaptive ends. In marine species, such as crustaceans and molluscs, reduced oxygen consumption and metabolic rates have been confirmed during hypoxia; in addition, glucose utilisation and lactate accumulation as indicators of a switch to anaerobic metabolism have been detected at low oxygen concentrations in water (Racotta et al., 2002; Martinez-Cruz, 2007; Soldatov et al., 2010). In the brine shrimp *A. Franciscana*, the intracellular pH falls at anoxia, heat production is reduced and ATP concentrations are also depressed to low levels (Hand, 1998; Eads & Hand, 2003).

A large amount of information is now available about the changes at the transcriptional level promoted by hypoxia in invertebrates, most of it concerning aquatic species. In our lab, we have evaluated the effects of hypoxia in the gene expression of FOF1 ATP synthase subunits, such as *atp9, atp6, atp, atp, atp, atp, and atp* in different tissues of the white shrimp *L. vannamei*. Results show a general trend towards increase the amount of mRNA as oxygen concentrations decrease (Martinez-Cruz, 2007; Martinez-Cruz et al., 2011; Martinez-Cruz et al. in preparation). Also, significant changes in the amount of mRNA from the mitochondrial- and nuclear- encoded subunits of the ATP synthase were detected at different molt stages and tissues, according to the energy requirements of each stage and the specific requirements of the function of each tissue (Muhlia-Almazan et al., 2008). Chronic exposure to severe hypoxia (1.5 mg/mL during 7 days) also causes the increased transcription of mitochondrial-encoded genes, such as the 16S, CO1, and CO2 subunits from the cytochrome C oxidase in the grass shrimp *Palaemonetes pugio* (Brouwer et al., 2008). To date, microarray technologies have revealed a set of genes that are up- and down-regulated in *P. pugio* during chronic, acute and moderate hypoxia; the results revealed that various genes encoding mitochondrial proteins were affected (Li & Brouwer, 2009).

In the absence of oxygen, animal cells activate transcription factors – such as the wellstudied vertebrates hypoxia-inducible factor (HIF) – which has been reported in invertebrates from worms to flies (Semenza, 2007). When activated, HIF leads the organism to exhibit metabolic adaptation to hypoxia by regulating the genetic expression of some proteins and enzymes involved in central biological processes such as glycolysis, erythropoiesis, breathing and angiogenesis so as to maintain cell homeostasis (Klimova & Chandel, 2008). In the shrimp *P. pugio*, a homolog protein to HIF- called gsHIF was found in this hypoxia-tolerant species. It includes all the conserved domains of vertebrates' HIF proteins, and an additional polypeptide sequence of 130 residues that has not been found in databases, and its participation in the functional properties of the protein has not yet been determined (Li & Brouwer, 2009). In the white shrimp *L. vannamei*, HIF-1 is a heterodimer formed by two subunits: HIF-1which is constitutively expressed in shrimp cells and HIF-1which is differentially expressed in hypoxic conditions. HIF-1 is suggested in crustaceans to be the master regulator that senses decreased oxygen availability and transmits signals promoting the physiological responses mentioned above (Soñanez-Organis et al., 2009). Additional functions have been attributed to HIF in coral species, such as *Acropora millepora*, where the diel cycle in the central metabolism appear to be governed by the circadian clock and regulated by the HIF system operating in parallel (Levy et al., 2011).

Invertebrates Mitochondrial Function and Energetic Challenges 201

Besides the various functions just described, mitochondria also acts as the arsenal of the cell. Numerous and complex processes, still poorly understood, can trigger the release of mitochondrial components into the cytoplasm and subsequently induce cellular apoptosis of the organelle (Hengarter, 2000). It is not our intent here to provide exhaustive coverage of all the issues relating to apoptosis in great detail, but rather to give the reader a basic description of the process – to highlight its importance and to show the challenges that those

As has been mentioned, studies in invertebrate biology are paramount to an understanding of biodiversity and to the search for potential uses for their metabolic capabilities and products for biotechnologies. Besides, comparative sciences may facilitate the use of invertebrate models in understanding the biology and pathology of farmed animals and humans. This is due – in spite of differences in the biochemical, physiological, and cellular characteristics that make invertebrates and vertebrates so obviously different – to the fact that most parts of such grades of their biology have remained similar in both groups through their evolution. For example, invertebrate cells – whether wounded by harsh environments or by the expression of abnormal proteins – die as do vertebrate cells, indicating that the powerful advantages of invertebrate molecular genetics might be successfully used for testing specific hypotheses about human diseases, for the discovery of drugs and for non-biased screens for suppressors and enhancers of maladies (Driscoll & Gerstbrein, 2003). The same criteria apply for all cellular functioning, as for apoptosis. Apoptosis (from the Greek: "falling off") – or programmed and regulated cell death and elimination – is a pivotal process in embryogenesis, the orderly elimination of wounded or infected cells, and the maintenance of tissue homeostasis. The process is so important that it is estimated that on a daily basis the human body must get rid of approximately 1010 cells. Through apoptosis, cells die quietly in a controlled, regulated fashion; while in another forms of cell death – such as in necrosis – a series of uncontrolled events occur leading to serious and irreversible damage. Given the proper conditions, apoptosis destroys the cell swiftly and neatly. In contrast, necrosis causes the rupture of the cell, releasing its content into the surrounding tissue. Tampering with apoptosis may result in devastating health problems, such as cancers, immune diseases, neurodegenerative disorders and the proliferation of viruses. Apoptosis is executed by a variety of membrane, organelle, cytoplasmic and nucleus signalling, and initiator and effector molecules, including a

**6. The role of mitochondria in invertebrate programmed cell death** 

subfamily of cysteine proteases known as caspases (Jiang & Wang, 2004).

significance of these associations has not been rigorously tested.

In mammals, the active role of mitochondria in apoptosis induction has been wellestablished. In invertebrate models of apoptosis, such as the fly *Drosophila melanogaster* and the worm *C. elegans*, the role that mitochondria play during apoptosis and, in particular, during apoptosis initiation is less clear (Rolland & Conradt, 2006). While key regulators of apoptosis in *Drosophila* and *C. elegans* have been found in association with mitochondria, the

The regulated destruction of a cell is a basic process in Metazoa, as multicellular animals are obligated to remove damaged or harmful cells. During apoptosis, cells die in an orderly, regulated sequence of molecular, biochemical, and cellular processes. According to the endosymbiotic theory, the origin of apoptosis is currently regarded as the result of molecular interactions in which some components of a signal transduction pathway affects

**(Apoptosis)** 

interested in this topic will face.

As a part of the HIF-regulated metabolic responses to hypoxia in invertebrates, the activities of specific enzymes – most of them part of the central metabolism – are known to increase. In bivalves such as *Anadara inaequivalvis*, the increased activities of enzymes – such as malate and lactate dehydrogenases – were detected at hypoxia (Soldatov et al., 2010). Also, increases in the catalase and GST activities during anoxia in the estuarine crab *Chasmagnathus granulate* have been observed. It has been suggested that such responses may be a strategy to prepare the organisms for oxidative stress in an effort to protect tissues against oxidative damage during re-oxygenation. An important decrease in SOD activity (which occurred after aerobic recuperation) was also detected; and it could have been caused by the accumulation of hydrogen peroxide production during re-oxygenation (de Oliveira et al., 2005).

At normoxia, the small levels of ROS produced by the metabolism in normal animal mitochondria come from carrying electrons along the mitochondrial complexes I, II, and III (Turrens, 2003). However, when oxygen levels are reduced, the presence of the final electron acceptor in the mitochondrial respiratory chain fails, producing a reduction in the rate of electron transport and a decrease in oxygen consumption. Under these conditions, the membrane potential increases as does ROS production (Guerrero-Castillo et al., 2011).

It has been reported that in invertebrate species considered to be hypoxia-tolerant, the absolute rate of H2O2 production is at least an order of magnitude less per mg of mitochondrial protein than that measured on mammalian species (Abele & Puntarulo, 2004). However, some other species which are not tolerant to hypoxia tend to produce higher levels of ROS at low oxygen levels; thus, it is suggested that they display alternate pathways in order to maintain the mitochondrial respiratory rate and avoid an over-production of ROS (Guerrero-Castillo et al., 2011).

Nowadays, the alternative mechanism of proton sinks has been evidenced in invertebrates since uncoupling proteins (UCPs) have been identified in these species (Abele et al., 2007). Such proteins have been involved in various functions, including thermoregulation, body composition, antioxidant defence and apoptosis. UCPs are thought to dissipate the proton gradient across the inner mitochondrial membrane and may help in controlling ROS production (Yu et al., 2000).

In *Drosophila*, an UCP5 protein over-expressed in a heterologous system has shown to have similar functional abilities to an uncoupling protein (Fridell et al., 2004), while in the marine eastern oyster, *Crassostrea virginica*, UCP5 is represented by two transcript forms: UCP5S (small) and UCP5L (large). However, their function has not been determined since its gene expression is not affected by hypoxia, cadmium exposure or different temperatures (Kern et al., 2009). In addition, a novel protein (UCP6) in invertebrates is considered to be an ancestral form of the vertebrates UCP1, UCP2, and UCP3 (Sokolova & Sokolov, 2005).

In mammals, it is known that less-severe hypoxia induces protective mechanisms. This phenomenon – called hypoxic preconditioning (HP) – appears in two forms: immediate preconditioning (which occurs only a few minutes after a sub-lethal hypoxic episode and declines after 4 h) and delayed preconditioning (which requires gene expression changes and takes place 12 to 24 h later and can last for days) (Dirnagl et al., 2009). In the nematode *C. elegans*, the delayed form of HP has been found to induce unfolded protein response pathways – at this point, misfolded proteins serve as early hypoxic sensors that trigger signalling pathways to induce a hypoxia protective response (Mao & Crowder, 2010).

As a part of the HIF-regulated metabolic responses to hypoxia in invertebrates, the activities of specific enzymes – most of them part of the central metabolism – are known to increase. In bivalves such as *Anadara inaequivalvis*, the increased activities of enzymes – such as malate and lactate dehydrogenases – were detected at hypoxia (Soldatov et al., 2010). Also, increases in the catalase and GST activities during anoxia in the estuarine crab *Chasmagnathus granulate* have been observed. It has been suggested that such responses may be a strategy to prepare the organisms for oxidative stress in an effort to protect tissues against oxidative damage during re-oxygenation. An important decrease in SOD activity (which occurred after aerobic recuperation) was also detected; and it could have been caused by the accumulation of hydrogen peroxide production during re-oxygenation (de

At normoxia, the small levels of ROS produced by the metabolism in normal animal mitochondria come from carrying electrons along the mitochondrial complexes I, II, and III (Turrens, 2003). However, when oxygen levels are reduced, the presence of the final electron acceptor in the mitochondrial respiratory chain fails, producing a reduction in the rate of electron transport and a decrease in oxygen consumption. Under these conditions, the membrane potential increases as does ROS production (Guerrero-Castillo et al., 2011). It has been reported that in invertebrate species considered to be hypoxia-tolerant, the absolute rate of H2O2 production is at least an order of magnitude less per mg of mitochondrial protein than that measured on mammalian species (Abele & Puntarulo, 2004). However, some other species which are not tolerant to hypoxia tend to produce higher levels of ROS at low oxygen levels; thus, it is suggested that they display alternate pathways in order to maintain the mitochondrial respiratory rate and avoid an over-production of

Nowadays, the alternative mechanism of proton sinks has been evidenced in invertebrates since uncoupling proteins (UCPs) have been identified in these species (Abele et al., 2007). Such proteins have been involved in various functions, including thermoregulation, body composition, antioxidant defence and apoptosis. UCPs are thought to dissipate the proton gradient across the inner mitochondrial membrane and may help in controlling ROS

In *Drosophila*, an UCP5 protein over-expressed in a heterologous system has shown to have similar functional abilities to an uncoupling protein (Fridell et al., 2004), while in the marine eastern oyster, *Crassostrea virginica*, UCP5 is represented by two transcript forms: UCP5S (small) and UCP5L (large). However, their function has not been determined since its gene expression is not affected by hypoxia, cadmium exposure or different temperatures (Kern et al., 2009). In addition, a novel protein (UCP6) in invertebrates is considered to be an ancestral form of the vertebrates UCP1, UCP2, and UCP3 (Sokolova & Sokolov, 2005). In mammals, it is known that less-severe hypoxia induces protective mechanisms. This phenomenon – called hypoxic preconditioning (HP) – appears in two forms: immediate preconditioning (which occurs only a few minutes after a sub-lethal hypoxic episode and declines after 4 h) and delayed preconditioning (which requires gene expression changes and takes place 12 to 24 h later and can last for days) (Dirnagl et al., 2009). In the nematode *C. elegans*, the delayed form of HP has been found to induce unfolded protein response pathways – at this point, misfolded proteins serve as early hypoxic sensors that trigger

signalling pathways to induce a hypoxia protective response (Mao & Crowder, 2010).

Oliveira et al., 2005).

ROS (Guerrero-Castillo et al., 2011).

production (Yu et al., 2000).
