**2. The highly conserved mitochondrial machinery of invertebrates: Same functions, different challenges**

Following the endosymbiotic origin from primitive bacteria – at least 2 billion years ago – when atmospheric oxygen levels rose and subsequently remained relatively steady, mitochondria have experienced large changes among species, from -proteobacteria to mammals. During the adaptation process of organisms to their new dynamic environment, some mitochondrial characteristics have remained highly conserved even among distantly related species, such as their rod shape - the overall structure including two phospholipid membranes – and, with some exceptions, their conserved characteristic genome content of 22 tRNAs, 2 rRNAs, and 13 genes encoding protein subunits of the enzymes from the oxidative phosphorylation system (OXPHOS) (Boore, 1999; Gray et al., 1999).

Besides mitochondrial encoded proteins, a significant fraction of the original mitochondrial genes have moved to the nucleus. Thus, in the mammalian mitochondria, approximately 76 subunits – which are part of the respiratory chain – are encoded by nuclear genes, and all of them must be imported into the mitochondria. The complete protein machinery involved in mtDNA replication, transcription and translation (including all of the ribosomal protein subunits) is encoded by nuclear genes (St. John et al., 2005; Falkenberg et al., 2005). Furthermore, several of these imported proteins are highly conserved among species, some of them accomplishing key roles as subunits alpha and beta of the ATP-synthase, which are part of the catalytic sites of the enzyme (Martinez-Cruz et al., 2011).

In addition to those key proteins that maintain a conserved function, hundreds of new proteins have been described among invertebrate species as being imported to mitochondria, each presumed to participate in at least one of the large number of metabolic pathways occurring in this organelle. However, its major conserved function allows mitochondrion to produce – from food assimilated compounds via oxidation – the proton motive force that drives ATP synthesis (Rich & Marechal, 2010). This complex process produces 95% of the cellular ATP that cells need for biosynthesis, transport and motility (Wilson et al., 1988; Dudkina et al., 2008; Diaz, 2010), and any significant change in the system could result in deleterious consequences for the whole cell metabolism and – consequently – reduce its efficiency or provoke its death (Mayevsky & Rogatsky, 2007).

Throughout the years (and mostly based in the study of human pathologies) researchers have found that mitochondria are involved in various critical functions – such as thermoregulation – in the synthesis of essential molecules – such as phospholipids and heme – in the programmed cell death or apoptosis of mediating multiple cellular signalling

Invertebrates Mitochondrial Function and Energetic Challenges 185

of them from the mitochondrial genome-, their proteins, and an interesting study of sitedirected mutagenesis aiming to understand the subunits' function in model insect species

In addition, the existence of an alternative oxidase (AOX) in the animal mitochondria has been confirmed. Previously, this enzyme – which catalyses the O2-dependent oxidation of ubiquinol, producing ubiquinone and H2O – was thought to be limited to plants, some fungi and protists. The major difference between complex I and AOX is that the electron flow from ubiquinol to AOX is not coupled to the generation of a proton motive force, decreasing energy conservation in oxidative phosphorylation. The complementary DNA sequence that encodes AOX in invertebrate species from the phyla Porifera, Cnidaria, Nematoda, Anellida, Mollusca, and Echinodermata, has been characterised and it has been suggested that it may contribute on the acclimation of animals to stress conditions, mainly when the cytochrome

Also called Succinate Dehydrogenase (SDH), is a functional member of the Krebs cycle and the aerobic respiratory chain, and it couples the oxidation of succinate to fumarate with the reduction of quinone to quinol (QH2). Most probably, this enzyme presents the most striking differences among the mitochondrial complexes in the electron transport chain and OXPHOS (Rich & Marechal, 2010). It must be noticed that the oxidation of succinate to fumarate is the only Krebs reaction that takes place in the mitochondrial inner membrane itself; this reaction does not participate in proton translocation from one side to the other of the inner mitochondrial membrane. The energy carrier flavin adenine dinucleotide (FAD) forms a part of complex II, and succinate oxidation begins after the binding of succinate to the enzyme. This covalent binding of FAD to the enzyme increases the redox potential to a

Contrary to the four human and yeast mitochondrial complexes, which include subunits that are encoded by the mitochondrial genome, the four subunits of SDH are encoded in the

Early studies of complex II (SDH) from invertebrates reported the isolation of mitochondrial fractions from the body muscles of the worm *Nereis virens* and from the tail muscle of the lobster *Homarus gammarus*, and reported high activity in both enzymes (Mattisson, 1965). Unfortunately, there is scarce new information available concerning complex II in invertebrates. However, the study of mitochondria from parasite species – used as animal models – can be considered a framework that has guided our knowledge in the understanding of such critical endogenous processes as aging, mitochondrial dysfunction and the role of the organelle in apoptosis (Grad et al., 2008; Wang & Youle, 2009). Thus, it has been suggested that mitochondria may influence the longevity of the nematode *Caenorhabditis elegans* through the rate of ROS production and by the stress-evoked signals that are known to act in a cell-non-autonomous manner during mitochondrial protein regulation (Durieux et al., 2011). Furthermore, *C. elegans* has been used as a model to investigate the mitochondrial mechanisms of human aging and tumourigenesis by studying the catalytic effects of mutation in the genes encoding the SDH iron-sulphur subunit. Promising results suggest that the SDH ubiquinone-binding site can become a source of superoxide and that the pathological consequences of SDH mutations can be diminished

with antioxidants, such as ascorbate and N-acetyl-l-cysteine (Huang & Lemire, 2009).

such as *Drosophila spp*. (Tovoinen et al., 2001; Sanz et al., 2010).

**2.2 Complex II, Succinate: Ubiquinone- Oxidoreductase (EC 1.3.99.1)** 

level that allows succinate oxidation (Rich & Marechal, 2010).

nuclear genome (SDH1 to SDH4; Figueroa et al., 2002).

pathway is inhibited (McDonald et al., 2009).

pathways (Ryan & Hoogenraad, 2007). Mitochondria are also essential in the cholesterol metabolism and the detoxification of ammonia in the urea cycle. In addition, there is a close relationship between mitochondria and different cell types. It is well known that the number of mitochondria in individual cell types varies according to their function and energy requirements (St. John et al., 2005; Chen & Chan, 2009). Thus, highly energetic tissues as the flight muscle of flying insects and the midgut gland of crustaceans are known to contain a large number of mitochondria, just as occurs in the skeletal muscles of vertebrates during endurance training (Harrison & Roberts, 2000).

Mitochondria are known as dynamic organelles that cannot be made *de novo*, and instead they divide through a highly regulated process called mitochondrial fission, mediated by a defined set of new proteins recruited from the cytoplasm, which are added to pre-existing sub-compartments and protein complexes to a point whereby the organelle grows and divides (Ryan & Hoogenraad, 2007). Furthermore, mitochondria are now seen as a set of organelles that are able to migrate throughout the cell, to fuse and divide regulating mitochondrial function (Chen & Chan, 2009).

Recent findings have also confirmed the existence of dynamic mitochondrial supercomplexes – defined as the association of protein complexes distributed along the inner mitochondrial membrane – on mammals, plants, yeasts (*Yarrowia lipolytica)*, and bacteria (Nübel et al., 2009; Wittig & Schägger, 2009; Dudkina et al., 2010). Complexes I, III and IV are able to associate in order to promote electron transport as single OXPHOS complexes or else as a supercomplex called respirasome (I + III2 + IV1-2) both of which can autonomously carry out respiration (Wittig et al., 2006). Furthermore, complex V – the mitochondrial F1FOATP-synthase – is associated to form dimeric, trimeric and tetrameric organisations (Dudkina et al., 2008). Unfortunately, to our knowledge, there are no reports confirming the existence of these mitochondrial protein associations from invertebrate species.

A general description of the most recent advances covering mitochondrial enzymes participating in the electron transport chain and the OXPHOS, including some particular findings on the enzymes of some invertebrate species, is presented below:

#### **2.1 Complex I, NADH: Ubiquinone-oxidoreductase (EC. 1.6.5.3)**

Is an enzyme which provides the input to the respiratory chain by catalysing the transfer of two electrons from NADH from - glycolysis - to ubiquinone, and which utilises the free energy released in this redox reaction for the translocation of four protons across the membrane, from the matrix to the intermembrane space. The proton translocation from the mitochondrial matrix generates the proton-motive force required for ATP synthesis at the end of the respiratory chain during oxidative phosphorylation (Friedrich & Weiss, 1997; Dudkina et al., 2008). However, this proton-pumping enzyme is the largest, most complicated and least-well understood of the respiratory chain (Zickermann et al., 2008). Another unconventional function of complex I is the generation of reactive oxygen species (ROS) – such as the superoxide ion (O2- ) – and, even if it is not a strong oxidant, it is a precursor of most other ROS and, consequently, contributes significantly to cellular oxidative stress. In mammalian mitochondria, the superoxide production is predominantly produced by complex I (Turrens, 2003).

The scarce information available concerning mitochondrial complex I from invertebrates includes basic descriptive reports of the nucleotide sequences of the NADH subunits - most

pathways (Ryan & Hoogenraad, 2007). Mitochondria are also essential in the cholesterol metabolism and the detoxification of ammonia in the urea cycle. In addition, there is a close relationship between mitochondria and different cell types. It is well known that the number of mitochondria in individual cell types varies according to their function and energy requirements (St. John et al., 2005; Chen & Chan, 2009). Thus, highly energetic tissues as the flight muscle of flying insects and the midgut gland of crustaceans are known to contain a large number of mitochondria, just as occurs in the skeletal muscles of vertebrates during

Mitochondria are known as dynamic organelles that cannot be made *de novo*, and instead they divide through a highly regulated process called mitochondrial fission, mediated by a defined set of new proteins recruited from the cytoplasm, which are added to pre-existing sub-compartments and protein complexes to a point whereby the organelle grows and divides (Ryan & Hoogenraad, 2007). Furthermore, mitochondria are now seen as a set of organelles that are able to migrate throughout the cell, to fuse and divide regulating

Recent findings have also confirmed the existence of dynamic mitochondrial supercomplexes – defined as the association of protein complexes distributed along the inner mitochondrial membrane – on mammals, plants, yeasts (*Yarrowia lipolytica)*, and bacteria (Nübel et al., 2009; Wittig & Schägger, 2009; Dudkina et al., 2010). Complexes I, III and IV are able to associate in order to promote electron transport as single OXPHOS complexes or else as a supercomplex called respirasome (I + III2 + IV1-2) both of which can autonomously carry out respiration (Wittig et al., 2006). Furthermore, complex V – the mitochondrial F1FOATP-synthase – is associated to form dimeric, trimeric and tetrameric organisations (Dudkina et al., 2008). Unfortunately, to our knowledge, there are no reports confirming the existence of these mitochondrial protein associations from invertebrate

A general description of the most recent advances covering mitochondrial enzymes participating in the electron transport chain and the OXPHOS, including some particular

Is an enzyme which provides the input to the respiratory chain by catalysing the transfer of two electrons from NADH from - glycolysis - to ubiquinone, and which utilises the free energy released in this redox reaction for the translocation of four protons across the membrane, from the matrix to the intermembrane space. The proton translocation from the mitochondrial matrix generates the proton-motive force required for ATP synthesis at the end of the respiratory chain during oxidative phosphorylation (Friedrich & Weiss, 1997; Dudkina et al., 2008). However, this proton-pumping enzyme is the largest, most complicated and least-well understood of the respiratory chain (Zickermann et al., 2008). Another unconventional function of complex I is the generation of reactive oxygen species (ROS) – such as the superoxide ion (O2-) – and, even if it is not a strong oxidant, it is a precursor of most other ROS and, consequently, contributes significantly to cellular oxidative stress. In mammalian mitochondria, the superoxide production is predominantly

The scarce information available concerning mitochondrial complex I from invertebrates includes basic descriptive reports of the nucleotide sequences of the NADH subunits - most

findings on the enzymes of some invertebrate species, is presented below:

**2.1 Complex I, NADH: Ubiquinone-oxidoreductase (EC. 1.6.5.3)** 

endurance training (Harrison & Roberts, 2000).

mitochondrial function (Chen & Chan, 2009).

produced by complex I (Turrens, 2003).

species.

of them from the mitochondrial genome-, their proteins, and an interesting study of sitedirected mutagenesis aiming to understand the subunits' function in model insect species such as *Drosophila spp*. (Tovoinen et al., 2001; Sanz et al., 2010).

In addition, the existence of an alternative oxidase (AOX) in the animal mitochondria has been confirmed. Previously, this enzyme – which catalyses the O2-dependent oxidation of ubiquinol, producing ubiquinone and H2O – was thought to be limited to plants, some fungi and protists. The major difference between complex I and AOX is that the electron flow from ubiquinol to AOX is not coupled to the generation of a proton motive force, decreasing energy conservation in oxidative phosphorylation. The complementary DNA sequence that encodes AOX in invertebrate species from the phyla Porifera, Cnidaria, Nematoda, Anellida, Mollusca, and Echinodermata, has been characterised and it has been suggested that it may contribute on the acclimation of animals to stress conditions, mainly when the cytochrome pathway is inhibited (McDonald et al., 2009).

## **2.2 Complex II, Succinate: Ubiquinone- Oxidoreductase (EC 1.3.99.1)**

Also called Succinate Dehydrogenase (SDH), is a functional member of the Krebs cycle and the aerobic respiratory chain, and it couples the oxidation of succinate to fumarate with the reduction of quinone to quinol (QH2). Most probably, this enzyme presents the most striking differences among the mitochondrial complexes in the electron transport chain and OXPHOS (Rich & Marechal, 2010). It must be noticed that the oxidation of succinate to fumarate is the only Krebs reaction that takes place in the mitochondrial inner membrane itself; this reaction does not participate in proton translocation from one side to the other of the inner mitochondrial membrane. The energy carrier flavin adenine dinucleotide (FAD) forms a part of complex II, and succinate oxidation begins after the binding of succinate to the enzyme. This covalent binding of FAD to the enzyme increases the redox potential to a level that allows succinate oxidation (Rich & Marechal, 2010).

Contrary to the four human and yeast mitochondrial complexes, which include subunits that are encoded by the mitochondrial genome, the four subunits of SDH are encoded in the nuclear genome (SDH1 to SDH4; Figueroa et al., 2002).

Early studies of complex II (SDH) from invertebrates reported the isolation of mitochondrial fractions from the body muscles of the worm *Nereis virens* and from the tail muscle of the lobster *Homarus gammarus*, and reported high activity in both enzymes (Mattisson, 1965). Unfortunately, there is scarce new information available concerning complex II in invertebrates. However, the study of mitochondria from parasite species – used as animal models – can be considered a framework that has guided our knowledge in the understanding of such critical endogenous processes as aging, mitochondrial dysfunction and the role of the organelle in apoptosis (Grad et al., 2008; Wang & Youle, 2009). Thus, it has been suggested that mitochondria may influence the longevity of the nematode *Caenorhabditis elegans* through the rate of ROS production and by the stress-evoked signals that are known to act in a cell-non-autonomous manner during mitochondrial protein regulation (Durieux et al., 2011). Furthermore, *C. elegans* has been used as a model to investigate the mitochondrial mechanisms of human aging and tumourigenesis by studying the catalytic effects of mutation in the genes encoding the SDH iron-sulphur subunit. Promising results suggest that the SDH ubiquinone-binding site can become a source of superoxide and that the pathological consequences of SDH mutations can be diminished with antioxidants, such as ascorbate and N-acetyl-l-cysteine (Huang & Lemire, 2009).

Invertebrates Mitochondrial Function and Energetic Challenges 187

assembly, stability and regulation of the enzyme (Rich & Marechal, 2010). Moreover, CO is also regulated by the existence of various isoforms from each nuclear-encoded subunit which is known to be tissue- and specie-specific (e. g. CO5a and CO5b, CO6a, CO6b and

The CO genes' expression and the activity of the enzyme are known to be affected by external factors. In crustacean species, such as the grass shrimp *Palaemonetes pugio*, the gene expression of subunits CO1 and CO2 is positively or negatively regulated by low dissolved oxygen concentrations in water (Brouwer et al., 2008). References and further reading may be available

In insects, as with the sweet potato hornworm *Agrius convolvuli*, diapause – the delay in development in response to regularly and recurring periods of adverse environmental conditions – is induced by low temperatures. During this physiological state, the neurological activity, oxygen consumption rate and metabolic levels are low compared to undiapause animals; and it has been found that the genetic expression of the CO1 subunit is down-regulated. When the organism terminates diapause, CO1 is up-regulated and the enzyme activity also increases (Uno et al., 2004). Other insect species, such as the cotton boll worm *Helicoverpa armigera*, show diverse responses during diapause: the levels of CO1 mRNA and enzyme activity are low, suggesting that the diapause state is different in each

In some species, CO participates in organism detoxification, as observed in the polychaetes *Hediste diversicolor* and *Marenzelleria viridis* which inhabit eutrophicated regions with low oxygen levels and high sulphide concentrations - where CO functions as an alternative pathway of oxidation - (Hahlbeck et al., 2000). In addition, when sulphide becomes hydrogen sulphide (HS) – a weak acid that occurs in marine and aquatic environments such as hydrothermal vents, mudflats and marshes – HS is known to reversibly inhibit CO activity, affecting the aerobic metabolism of certain species, such as the worm *Urechis caupo*

Is a multimeric enzyme that transforms the kinetic energy of the protons' electrochemical gradient to synthesise the high energy phosphate molecule ATP. Nowadays, it is wellknown that the enzyme can also hydrolyse ATP, functioning as an ATPase (Boyer, 1997; Tuena de Gomez-Poyou et al., 1999). This mitochondrial enzyme comprises a catalytic sector F1 (composed by 33 subunits), and a transmembrane hydrophobic sector FO (composed of at least three subunits: a, b2 and c10-12), both linked by a central and a peripheral stalk (Mueller et al., 2004). As in other mitochondrial complexes, this enzyme includes subunits encoded in both the nuclear and mitochondrial genomes, in a tightly coordinated process to

During the oxidative phosphorylation process in mitochondria, the electron transport chain generates a proton gradient that is proposed to drive the rotation of Fo, a central rotor located in the inner mitochondrial membrane. This rotation movement is believed to reverse the rotation of the F1 nanomotor, inducing – via a conformational change – the sequential release of ATP from three identical catalytic sites followed by the sequential synthesis of newly formed ATP from Pi +ADP at these sites (Cardol et al., 2005). Biochemical and structural studies of the F1 sector from bovine enzymes have demonstrated that catalytic sites are integrated mainly by three subunits that alternate with three subunits. The

assemble this multimeric complex (Itoi et al., 2003; Muhlia-Almazan et al., 2008).

CO6c, and CO7a, CO7b, CO7c, etc.; Diaz, 2010).

for this article.

species (Yang et al., 2010).

(Julian et al., 1998).

**2.5 Complex V, ATP synthase (EC 3.6.3.14)** 

#### **2.3 Complex III, Ubiquinol: Cytochrome C Oxidoreductase or Cytochrome BCI (EC 1.10.2.2)**

Is a multimeric enzyme complex involved in the transfer of electrons from ubiquinol to cytochrome C, and it is also coupled to electrons' transfer across the inner mitochondrial membrane. This bovine enzyme is formed by 10 nuclear encoded subunits, with only one encoded in the mtDNA (Xia et al., 1997). The catalytic mechanism of the enzyme includes the complex mechanism of the protonmotive Q-cycle that provides the additional efficiency of the energy conservation of the electrons transferred (Mitchell, 1976; Rich & Marechal, 2010).

In such species as mammals and yeasts it has been observed that as the rate of electron transfer is reduced, the enzyme may leak electrons to molecular oxygen, promoting the formation of the superoxide ion. This mitochondrial dysfunction has been widely studied, and its role in the O2 sensing pathway has been investigated because the increasing production of reactive oxygen species (ROS) is the result of organisms in hypoxic/anoxic conditions (Guzy et al., 2007). New evidence suggests that ROS generated by the mitochondrial complex III are required for the hypoxic activation of transcription factors such as HIF (Hypoxia Inducible Factor); however, this topic will be more extensively discussed below.

The mitochondrial complex III from invertebrates has been poorly studied, but recent reports about these species confirm the importance of studying its basis and applications. An interesting example is the study about the control of Chagas disease, which severely affects the health of the human population in Latin America and which is caused by the protozoan parasite *Trypanosoma cruzi*. Genes et al. (2011) reported such bacteria species as *Serratia marcescens* biotype A1a, which is regularly found in the gut of the vector insect *Rhodnius prolixus*, and which demonstrates the trypanolytic activity conferred by prodigiosin. Prodigiosin is a potent bacterial tripyrrolic compound with various biological activities. This study suggests the abnormal mitochondrial function of *T. cruzi* since prodigiosin inhibits the mitochondrial complex III, affecting subsequent oxidative phosphorylation.

#### **2.4 Complex IV, Cytochrome C oxidase (EC 1.9.3.1)**

Is the terminal enzyme of the electron transport chain and it catalyses the reduction of molecular oxygen to water. The reduction of oxygen by this enzyme – which is responsible for biological energy conversion in mitochondria (Belevich et al., 2010) – is also linked to the translocation (pumping) of four protons across the membrane. This movement of electrons is subsequently coupled to ATP synthesis by the ATP-synthase (Khalimonchuk & Rödel, 2005). The cytochrome C oxidase (CO) has been described as one of the electron transport chain elements which is highly affected by changes in oxygen levels – since cytochrome C reduction is oxygen-dependent – and becomes more reduced when oxygen levels increase (Wilson et al., 1988).

The CO from eukaryotes consists of 11-13 subunits, depending on the species. It belongs to the family of heme-cooper enzymes, some of them suggested as hypoxia sensors. The enzyme is highly regulated by transcription factors, hormones, lipid membranes and the second messengers that control its activity (Ripamonti et al., 2006; Semenza, 2007; Fontanesi et al*.*, 2008). As observed in other mitochondrial complexes, CO also includes mitochondrial encoded genes as subunits CO1, CO2, and CO3 which form the functional core of the enzyme; the rest are nuclear-encoded subunits and their functions – even in the most studied animal models – remain unclear, although they are assumed to participate in the

Is a multimeric enzyme complex involved in the transfer of electrons from ubiquinol to cytochrome C, and it is also coupled to electrons' transfer across the inner mitochondrial membrane. This bovine enzyme is formed by 10 nuclear encoded subunits, with only one encoded in the mtDNA (Xia et al., 1997). The catalytic mechanism of the enzyme includes the complex mechanism of the protonmotive Q-cycle that provides the additional efficiency of the energy conservation of the electrons transferred (Mitchell, 1976; Rich & Marechal, 2010). In such species as mammals and yeasts it has been observed that as the rate of electron transfer is reduced, the enzyme may leak electrons to molecular oxygen, promoting the formation of the superoxide ion. This mitochondrial dysfunction has been widely studied, and its role in the O2 sensing pathway has been investigated because the increasing production of reactive oxygen species (ROS) is the result of organisms in hypoxic/anoxic conditions (Guzy et al., 2007). New evidence suggests that ROS generated by the mitochondrial complex III are required for the hypoxic activation of transcription factors such as HIF (Hypoxia Inducible Factor); however, this topic will be more extensively

The mitochondrial complex III from invertebrates has been poorly studied, but recent reports about these species confirm the importance of studying its basis and applications. An interesting example is the study about the control of Chagas disease, which severely affects the health of the human population in Latin America and which is caused by the protozoan parasite *Trypanosoma cruzi*. Genes et al. (2011) reported such bacteria species as *Serratia marcescens* biotype A1a, which is regularly found in the gut of the vector insect *Rhodnius prolixus*, and which demonstrates the trypanolytic activity conferred by prodigiosin. Prodigiosin is a potent bacterial tripyrrolic compound with various biological activities. This study suggests the abnormal mitochondrial function of *T. cruzi* since prodigiosin inhibits the mitochondrial complex III, affecting subsequent oxidative

Is the terminal enzyme of the electron transport chain and it catalyses the reduction of molecular oxygen to water. The reduction of oxygen by this enzyme – which is responsible for biological energy conversion in mitochondria (Belevich et al., 2010) – is also linked to the translocation (pumping) of four protons across the membrane. This movement of electrons is subsequently coupled to ATP synthesis by the ATP-synthase (Khalimonchuk & Rödel, 2005). The cytochrome C oxidase (CO) has been described as one of the electron transport chain elements which is highly affected by changes in oxygen levels – since cytochrome C reduction is oxygen-dependent – and becomes more reduced when oxygen levels increase

The CO from eukaryotes consists of 11-13 subunits, depending on the species. It belongs to the family of heme-cooper enzymes, some of them suggested as hypoxia sensors. The enzyme is highly regulated by transcription factors, hormones, lipid membranes and the second messengers that control its activity (Ripamonti et al., 2006; Semenza, 2007; Fontanesi et al*.*, 2008). As observed in other mitochondrial complexes, CO also includes mitochondrial encoded genes as subunits CO1, CO2, and CO3 which form the functional core of the enzyme; the rest are nuclear-encoded subunits and their functions – even in the most studied animal models – remain unclear, although they are assumed to participate in the

**2.3 Complex III, Ubiquinol: Cytochrome C Oxidoreductase or Cytochrome BCI (EC** 

**1.10.2.2)** 

discussed below.

phosphorylation.

(Wilson et al., 1988).

**2.4 Complex IV, Cytochrome C oxidase (EC 1.9.3.1)** 

assembly, stability and regulation of the enzyme (Rich & Marechal, 2010). Moreover, CO is also regulated by the existence of various isoforms from each nuclear-encoded subunit which is known to be tissue- and specie-specific (e. g. CO5a and CO5b, CO6a, CO6b and CO6c, and CO7a, CO7b, CO7c, etc.; Diaz, 2010).

The CO genes' expression and the activity of the enzyme are known to be affected by external factors. In crustacean species, such as the grass shrimp *Palaemonetes pugio*, the gene expression of subunits CO1 and CO2 is positively or negatively regulated by low dissolved oxygen concentrations in water (Brouwer et al., 2008). References and further reading may be available for this article.

In insects, as with the sweet potato hornworm *Agrius convolvuli*, diapause – the delay in development in response to regularly and recurring periods of adverse environmental conditions – is induced by low temperatures. During this physiological state, the neurological activity, oxygen consumption rate and metabolic levels are low compared to undiapause animals; and it has been found that the genetic expression of the CO1 subunit is down-regulated. When the organism terminates diapause, CO1 is up-regulated and the enzyme activity also increases (Uno et al., 2004). Other insect species, such as the cotton boll worm *Helicoverpa armigera*, show diverse responses during diapause: the levels of CO1 mRNA and enzyme activity are low, suggesting that the diapause state is different in each species (Yang et al., 2010).

In some species, CO participates in organism detoxification, as observed in the polychaetes *Hediste diversicolor* and *Marenzelleria viridis* which inhabit eutrophicated regions with low oxygen levels and high sulphide concentrations - where CO functions as an alternative pathway of oxidation - (Hahlbeck et al., 2000). In addition, when sulphide becomes hydrogen sulphide (HS) – a weak acid that occurs in marine and aquatic environments such as hydrothermal vents, mudflats and marshes – HS is known to reversibly inhibit CO activity, affecting the aerobic metabolism of certain species, such as the worm *Urechis caupo* (Julian et al., 1998).

#### **2.5 Complex V, ATP synthase (EC 3.6.3.14)**

Is a multimeric enzyme that transforms the kinetic energy of the protons' electrochemical gradient to synthesise the high energy phosphate molecule ATP. Nowadays, it is wellknown that the enzyme can also hydrolyse ATP, functioning as an ATPase (Boyer, 1997; Tuena de Gomez-Poyou et al., 1999). This mitochondrial enzyme comprises a catalytic sector F1 (composed by 33 subunits), and a transmembrane hydrophobic sector FO (composed of at least three subunits: a, b2 and c10-12), both linked by a central and a peripheral stalk (Mueller et al., 2004). As in other mitochondrial complexes, this enzyme includes subunits encoded in both the nuclear and mitochondrial genomes, in a tightly coordinated process to assemble this multimeric complex (Itoi et al., 2003; Muhlia-Almazan et al., 2008).

During the oxidative phosphorylation process in mitochondria, the electron transport chain generates a proton gradient that is proposed to drive the rotation of Fo, a central rotor located in the inner mitochondrial membrane. This rotation movement is believed to reverse the rotation of the F1 nanomotor, inducing – via a conformational change – the sequential release of ATP from three identical catalytic sites followed by the sequential synthesis of newly formed ATP from Pi +ADP at these sites (Cardol et al., 2005). Biochemical and structural studies of the F1 sector from bovine enzymes have demonstrated that catalytic sites are integrated mainly by three subunits that alternate with three subunits. The

Invertebrates Mitochondrial Function and Energetic Challenges 189

protein (IntlP) – known as an antiviral factor – showed increased expression in virusresistant shrimp (He et al., 2005). Later, Rosa & Barraco (2008) suggested that the shrimp interferon-like protein (IntlP) is rather a region of the insect mitochondrial b subunit of the ATP-synthase, due to the high identity between both proteins (60–73%). Recently, Liang et al. (2010) have suggested the ATP-synthase subunit (*atp*) - earlier called BP53 – as a protein involved in the WSSV binding to shrimp cells that may play an important role in the

A) Ribbon lateral view, and B) Ribbon front view of the subunit ATP6 complex with three ATP9 subunits. The predicted functional residues are marked in both subunits, R160 from ATP6, and E99

Fig. 1. Molecular Model of the ATP9- ATP6 Subcomplex from the Shrimp *L. vannamei*.

for controlling ticks and tick-borne pathogens (Oleaga et al., 2007).

Transcriptomes and proteomes have provided a lot of information, not only about the characteristics of specific sequences of nucleotides or amino acids, but also about the proteins' structure and function in invertebrate organisms under diverse environmental conditions (Clavero-Salas et al., 2007). Moreover, novel proteins have been reported as accessories to the mitochondrial protein complexes in invertebrates species, such as the ticks *Ornithodoros moubata* and *O. erraticus*, where six novel proteins similar to the ATP synthase subunit 6 (*atp6*) were identified in the salivary glands. These proteins are attractive targets

Actually, and based in the mitochondrial highly conserved function, generic models of the electron transport chain in mitochondria have been constructed using bioinformatic tools to predict how the rate of oxygen consumption through the system – and the redox states of some intermediates such as NAD/NADH, ubiquinone, and cytochromes – respond to physiological stimuli such varying oxygen levels and other rapid energy demands (Banaji,

antiviral defence system of shrimp against WSSV.

from ATP9. (Taken from Muhlia-Almazan et al., 2008).

2006).

three catalytic sites formed by these three pairs of / subunits are grouped in segments forming a sphere, which is connected to the subunit which connects F1 to Fo (Lai-Zhang & Mueller, 2000).

Due to its complex structure and the dual role that the ATP synthase plays in cells, the current state of research concerning this mitochondrial enzyme is both abundant and relevant; however, for the majority of invertebrate taxa, the information regarding this enzyme appears to be almost non-existent, restricted to some insect species for the more studied models. Analyses of the mitochondrial transcriptome and proteome from these species – which have been exposed to different environmental conditions – have shown that the ATP-synthase subunits can be affected in their expression, and that specific subunits of this multimeric complex can also play additional roles in the mitochondrial function. These findings suggest that invertebrates are able to respond by changing their metabolism to maintain cell homeostasis.

In the fruit fly *Drosophila melanogaster* and the California purple sea urchin *Strongylocentrotus purpuratus* the gene expression of the ATP-synthase subunit alpha (*atp*) was measured at early developmental stages, and it was found that the amount of mRNA varies throughout development in both species. Contrary results showed that during the larval stage the nuclear and mitochondrially encoded ATP synthase genes appear to be temporally coregulated in *Drosophila*, although in the sea urchin this development pattern was not observed (Talamillo et al., 1998). In 2005, Kidd et al. analysed null mutants of the *A*TPsynthase subunit in *Drosophila* spp., and a dramatic delay in the growth rate of the first instar larvae that finally died was reported. In addition, in fly embryos the ATP-synthase activity had a six-fold reduction.

Most likely, the first two studies concerning the ATP synthase of crustacean species were published in 2001. The authors characterised the enzymatic properties of F1 and evaluated its sensitivity to specific inhibitors and modulators in the gills of the freshwater crayfish *Orconectes virilis*; they included, as an important contribution, the standardised methods for isolating mitochondria from crustacean tissues and some results about their enzyme stability at different temperatures and pH conditions (Li & Neufeld, 2001a, 2001b)*.* 

Recent reports on the most-studied shrimp species – *Litopenaeus vannamei* – have characterised and studied several mitochondrial and nuclear encoded subunits from tissues such as muscles, gills, pleopods and the midgut gland (Muhlia-Almazan et al., 2008; Martinez-Cruz et al., 2011). The complementary DNA sequences of the *atp6* subunit encoded in the mtDNA and the *atp9* (a nuclear encoded subunit) were characterised and their deduced proteins, as major components of the FO sector, were included in a molecular model which predicted that in the shrimp FOF1 ATP synthase the *atp9* oligomeric ring may contain 9-10 proteins (Figure 1; Muhlia-Almazan et al., 2008).

Over the last decade, the effects of a viral agent which provokes shrimp death have been deeply studied. The white spot syndrome virus (WSSV) is perhaps the most devastating shrimp disease, causing massive mortalities in global aquaculture systems (Sanchez-Paz, 2010). In 2006, Wang et al. analysed the gene expression profile of the fleshy prawn *Fenneropenaeus chinensis* in response to WSSV infection through cDNA microarrays. Genes including the ATP-synthase A chain and arginine kinase were found to be down-regulated during WSSV infection. Additional studies in other shrimp species, reported thirty additional genes which are involved in the antiviral process as part of the shrimp's defence system. One of the most interesting findings of these studies was that the interferon-like

three catalytic sites formed by these three pairs of / subunits are grouped in segments forming a sphere, which is connected to the subunit which connects F1 to Fo (Lai-Zhang &

Due to its complex structure and the dual role that the ATP synthase plays in cells, the current state of research concerning this mitochondrial enzyme is both abundant and relevant; however, for the majority of invertebrate taxa, the information regarding this enzyme appears to be almost non-existent, restricted to some insect species for the more studied models. Analyses of the mitochondrial transcriptome and proteome from these species – which have been exposed to different environmental conditions – have shown that the ATP-synthase subunits can be affected in their expression, and that specific subunits of this multimeric complex can also play additional roles in the mitochondrial function. These findings suggest that invertebrates are able to respond by changing their metabolism to

In the fruit fly *Drosophila melanogaster* and the California purple sea urchin *Strongylocentrotus* 

early developmental stages, and it was found that the amount of mRNA varies throughout development in both species. Contrary results showed that during the larval stage the nuclear and mitochondrially encoded ATP synthase genes appear to be temporally coregulated in *Drosophila*, although in the sea urchin this development pattern was not observed (Talamillo et al., 1998). In 2005, Kidd et al. analysed null mutants of the *A*TPsynthase subunit in *Drosophila* spp., and a dramatic delay in the growth rate of the first instar larvae that finally died was reported. In addition, in fly embryos the ATP-synthase

Most likely, the first two studies concerning the ATP synthase of crustacean species were published in 2001. The authors characterised the enzymatic properties of F1 and evaluated its sensitivity to specific inhibitors and modulators in the gills of the freshwater crayfish *Orconectes virilis*; they included, as an important contribution, the standardised methods for isolating mitochondria from crustacean tissues and some results about their enzyme

Recent reports on the most-studied shrimp species – *Litopenaeus vannamei* – have characterised and studied several mitochondrial and nuclear encoded subunits from tissues such as muscles, gills, pleopods and the midgut gland (Muhlia-Almazan et al., 2008; Martinez-Cruz et al., 2011). The complementary DNA sequences of the *atp6* subunit encoded in the mtDNA and the *atp9* (a nuclear encoded subunit) were characterised and their deduced proteins, as major components of the FO sector, were included in a molecular model which predicted that in the shrimp FOF1 ATP synthase the *atp9* oligomeric ring may

Over the last decade, the effects of a viral agent which provokes shrimp death have been deeply studied. The white spot syndrome virus (WSSV) is perhaps the most devastating shrimp disease, causing massive mortalities in global aquaculture systems (Sanchez-Paz, 2010). In 2006, Wang et al. analysed the gene expression profile of the fleshy prawn *Fenneropenaeus chinensis* in response to WSSV infection through cDNA microarrays. Genes including the ATP-synthase A chain and arginine kinase were found to be down-regulated during WSSV infection. Additional studies in other shrimp species, reported thirty additional genes which are involved in the antiviral process as part of the shrimp's defence system. One of the most interesting findings of these studies was that the interferon-like

stability at different temperatures and pH conditions (Li & Neufeld, 2001a, 2001b)*.* 

contain 9-10 proteins (Figure 1; Muhlia-Almazan et al., 2008).

) was measured at

*purpuratus* the gene expression of the ATP-synthase subunit alpha (*atp*

Mueller, 2000).

maintain cell homeostasis.

activity had a six-fold reduction.

protein (IntlP) – known as an antiviral factor – showed increased expression in virusresistant shrimp (He et al., 2005). Later, Rosa & Barraco (2008) suggested that the shrimp interferon-like protein (IntlP) is rather a region of the insect mitochondrial b subunit of the ATP-synthase, due to the high identity between both proteins (60–73%). Recently, Liang et al. (2010) have suggested the ATP-synthase subunit (*atp*) - earlier called BP53 – as a protein involved in the WSSV binding to shrimp cells that may play an important role in the antiviral defence system of shrimp against WSSV.

A) Ribbon lateral view, and B) Ribbon front view of the subunit ATP6 complex with three ATP9 subunits. The predicted functional residues are marked in both subunits, R160 from ATP6, and E99 from ATP9. (Taken from Muhlia-Almazan et al., 2008).

Fig. 1. Molecular Model of the ATP9- ATP6 Subcomplex from the Shrimp *L. vannamei*.

Transcriptomes and proteomes have provided a lot of information, not only about the characteristics of specific sequences of nucleotides or amino acids, but also about the proteins' structure and function in invertebrate organisms under diverse environmental conditions (Clavero-Salas et al., 2007). Moreover, novel proteins have been reported as accessories to the mitochondrial protein complexes in invertebrates species, such as the ticks *Ornithodoros moubata* and *O. erraticus*, where six novel proteins similar to the ATP synthase subunit 6 (*atp6*) were identified in the salivary glands. These proteins are attractive targets for controlling ticks and tick-borne pathogens (Oleaga et al., 2007).

Actually, and based in the mitochondrial highly conserved function, generic models of the electron transport chain in mitochondria have been constructed using bioinformatic tools to predict how the rate of oxygen consumption through the system – and the redox states of some intermediates such as NAD/NADH, ubiquinone, and cytochromes – respond to physiological stimuli such varying oxygen levels and other rapid energy demands (Banaji, 2006).

Invertebrates Mitochondrial Function and Energetic Challenges 191

**size (bp)** 

**Onychophora** *Oroperipatus sp.* 14,493 NC\_015890 Segovia et al.,

**Brachiopoda** *Laqueus rubellus* 14,017 AB035869 Noguchi et al.,

**Echinodermata** *Acanthaster planci* 16,234 NC\_007788 Yasuda et al.,

**Mollusca** *Crassostrea gigas* 18,225 EU672831 Ren et al., 2010

Table 1. Invertebrates' mitochondrial genome size of the species of different phyla.

1988) and the horseshoe crab *Limulus polyphemus* (Staton et al., 1997).

Because of the wide variability of environmental conditions in which a large number of invertebrate species are distributed, several specific mtDNA-rearrangements have been found when compared with those observed in the mtDNA of mammals. Such novel arrangements include the mitogenome from the blue mussel *Mytilus edulis* (Hoffmann et al., 1992), and that of the fruit fly *Drosophila melanogaster* (Clary & Wolstenholme, 1985; Garesse,

Also, some species – or groups of species – may lack some genes, such as nematodes whose mtDNA lacks a gene for ATP8 (Keddie et al., 1998), or cnidarians like the coral *Sarcophyton glaucum* which includes an unusual gene encoding an extra tRNA (Beaton et al., 1998). Moreover, major changes have been found in invertebrates' mtDNA, such as the mitochondrial genes of *Lumbricus terrestris*, which are all known to be encoded in the same strand and, unlike others, the genes coding A8 and A6 are separated by a long 2700

**GenBank Acc. No.** 

13,794 NC\_001328 Wolstenholme et

13,605 AJ417719 Hu et al., 2002

15,650 NC\_001453 Qureshi & Jacobs,

15,619 AF178678 Boore & Brown,

14,998 NC\_001673 Boore & Brown,

14,519 NC\_006896 Davila et al., 2005

15,989 DQ534543 Shen et al., 2007

14,922 NC\_005870 Negrisolo et al.,

*Cucumaria miniata* 17,538 NC\_005929 Arndt & Smith,

*Cepaea nemoralis* 14,100 NC\_001816 Terrett et al., 1996 *Octopus minor* 15,974 HQ638215 Cheng et al., 2011

*Apis mellifera* 16,343 NC\_001566 Crozier &

**References** 

al., 1994

2011

2000

2006

1993

1998

2000

1995

2004

Crozier, 1993

**Phylum Species mtDNA** 

*elegans* 

*Necator americanus* 

*Strongylocentrotus purpuratus* 

*dumerilii* 

*Lumbricus terrestris* 

*Centruroides limpidus* 

*Litopenaeus vannamei* 

*Scutigera coleoptrata* 

nucleotides fragment (Boore & Brown, 1995).

**Nematoda** *Caenorhabditis* 

**Annelida** *Platynereis* 

**Arthropoda Subphylum Chelicerata** 

**Subphylum Crustacea** 

**Subphylum Myriapoda** 

**Subphylum Hexapoda** 

Ultimately, it is remarkable that the mitochondrial function has remained in all animal species through its long and peculiar evolutionary history and under the influence of variable selective pressures. Moreover, structural and biochemical adaptations promoting highly effective mitochondrial functions have allowed organisms to inhabit unusual environments.
