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

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 interested in this topic will face.

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

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 significance of these associations has not been rigorously tested.

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

Invertebrates Mitochondrial Function and Energetic Challenges 203

crustacean apoptosis transcriptionally regulated, as in *Drosophila* (RHG ´killer´ proteins)? Or is it controlled by pro- and anti-apoptotic Bcl-2 family proteins, as in vertebrates? The issues that should be investigated in the short-term are whether the calcium-induced opening of the mitochondrion permeability transition pore (MPTP), commonly found on vertebrate species, also occurs in crustaceans. Furthermore, the study of the differences in the regulation of the intrinsic pathway of crustacean apoptosis will lead to an understanding of their adaptation to challenging environments; this is because marine organisms have to deal with seasonal as well as circadian changes in environmental variables. Some examples are UV radiation, temperature and dissolved oxygen, and even some biological stresses such as toxins that may vary over time. But this is not all: other variables that may inhibit apoptosis must be considered. "Characterisation of the players, pathways, and their significance in the core machinery of crustacean apoptosis is revealing new insights for the field of cell

Apoptosis is a key host response to viral infection. Viruses that can modulate a host's apoptotic responses are likely to gain important opportunities for transmission. Here, we review recent studies that demonstrate that the particles of Invertebrate Iridescent Virus6 (IIV-6) (Iridoviridae, genus Iridovirus), or an IIV-6 virion protein extract, are capable of inducing apoptosis in lepidopteran and coleopteran cells, at concentrations 1000-fold lower than that required to shut-off the host's macromolecular synthesis (Williams et al., 2009). Throughout the process of pathogen–host coevolution, viruses have developed a battery of distinct strategies to overcome the biochemical and immunological defences of the host. Thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. Since the elimination of infected cells via programmed cell death is one of the oldest defence mechanisms against infection, disabling host cell apoptosis might represent an almost obligatory step in the viral life cycle. Conversely, viruses may take advantage of stimulating apoptosis, either to kill uninfected cells from the immune system or else to induce the breakdown of infected cells, thereby

As stated by Van der Giezen in 2009 "over the last 5–10 years, it has become apparent that the organelle known as the mitochondrion is a much more fluid entity than generally believed," so "why should mitochondrion be the same in all eukaryotes while other cellular

It is our belief that since natural selection has given invertebrates the opportunity to evolve in quick steps, a large window is opening in the field of mitochondrial research among these species, giving an outstanding opportunity to researchers to contribute to an increase in knowledge, not only because there is scarce information, but also because many species

At this point, the information reviewed clearly shows that invertebrates display remarkable physiological capabilities, including highly specialised mechanisms for adjusting mitochondrial functions to solve their energetic demands under the stressful conditions they usually face. These species also include within their systems ancient and novel molecules and structures acting to reach an adaptive state, from the increasing number of

death"(Menze et al., 2010).

favouring viral dissemination (Galluzzi et al., 2008).

structures show such great evolutionary malleability?"

have shown special and unique characteristics that need to be explained.

mitochondria per cell to the highly complex function of the HIF system.

**7. Conclusion and future perspectives** 

other pathways through interaction of some initiator and effector proteins. Accordingly, apoptosis could have arisen simultaneously with – and as a by-product of – endosymbiosis (Kroemer, 1997). However, it has also been proposed that apoptosis may be the result of the acquisition of the aerobic metabolism by early eukaryotes (Frade & Michaelidis, 1997).

Apoptosis is a unique phenomenon of tissue kinetics as it can be said that life is critically controlled by the operational centre of cell, the nucleus. Instead, death is a process controlled by the power house of the cell, the mitochondria. Thus, even cells lacking nucleus commit apoptosis. In general, the two-step membrane depolarisation and free radical release taking place in the mitochondria may trigger apoptosis. This in fact is not so peculiar if we understand that mitochondria were once free-living bacteria which did not need an external gene control for achieving their functions. Once each came into symbiosis forming a eukaryotic cell, it retained some capacity to operate partially independently.

There are several major apoptotic pathways, but the most well-known and studied are the extrinsic and the intrinsic pathways, which respond to different environmental and cellular challenges in vertebrates. The intrinsic pathway is also called the mitochondrial pathway because of the involvement of mitochondria. There are mitochondrial proteins that induce this process (proapoptotic) and others that limit cell death (antiapoptotic). Both proteins interact so as to cooperate and govern the cell's fate. Also, the origin of the activation signals of apoptosis taking place on the mitochondria is a clue molecule, cytochrome C (Cyt C), which is released from the mitochondria to form the apoptosome complex. The intrinsic pathway – with some differences – is a mostly conserved pathway among metazoans (for a comprehensive review look at Wang & Youle, 2009). Cyt C is a key component of the apoptosome complex for activating the initiator caspase-9 after its release from mitochondria. Under non-apoptotic conditions, Cyt C is kept inside the respiratory chain. Against some cellular challenges, like the alteration of the DNA in the mitochondria or the nucleus, Cyt C is released from its membrane, crossing the external membrane and initiating the formation of the apoptosome complex. In essence, mitochondrial proteins – like Cyt C and caspases – are not hired guns and during non-apoptotic conditions they are responsible for various basic mitochondrial roles for normal cell functioning. The compartmentalisation of such mitochondrial proteins isolates them from interacting with partners or targets, a mechanism to prevent the unwanted activation of apoptosis in normal cells. Only after their appropriate release into the cytoplasm do such proteins play the role of triggers to initiate the cell's suicide.

The classical invertebrate model organisms for the study of apoptosis are *C. elegans* and *Drosophila*. In spite of the fact that the regulators of apoptosis have been found in such model organisms, the involvement of mitochondria in apoptosis is not conclusive. So far, no irrefutable evidence of the release of Cyt C from the intermembrane space has been found. Also, the involvement of Cyt C in the apoptosome formation in *Drosophila* is controversial, and some evidence suggests that Cyt C is not necessary (Rolland & Conradt, 2006).

The current evidence indicates that the whole process of apoptosis -including the involved proteins and the regulation mechanisms- in crustaceans is far more diverse than has been assumed from the studies with model organisms. Recent studies have shown that several proteins in the apoptotic network are quite conserved between mammals and arthropods; however, it is clear that the integration of such homologous proteins in the physiology and pathophysiology of crustaceans needs further experimental assessment. Some unresolved questions regarding this topic are: how does the regulation of the process occur? Is

other pathways through interaction of some initiator and effector proteins. Accordingly, apoptosis could have arisen simultaneously with – and as a by-product of – endosymbiosis (Kroemer, 1997). However, it has also been proposed that apoptosis may be the result of the acquisition of the aerobic metabolism by early eukaryotes (Frade & Michaelidis, 1997). Apoptosis is a unique phenomenon of tissue kinetics as it can be said that life is critically controlled by the operational centre of cell, the nucleus. Instead, death is a process controlled by the power house of the cell, the mitochondria. Thus, even cells lacking nucleus commit apoptosis. In general, the two-step membrane depolarisation and free radical release taking place in the mitochondria may trigger apoptosis. This in fact is not so peculiar if we understand that mitochondria were once free-living bacteria which did not need an external gene control for achieving their functions. Once each came into symbiosis forming a

There are several major apoptotic pathways, but the most well-known and studied are the extrinsic and the intrinsic pathways, which respond to different environmental and cellular challenges in vertebrates. The intrinsic pathway is also called the mitochondrial pathway because of the involvement of mitochondria. There are mitochondrial proteins that induce this process (proapoptotic) and others that limit cell death (antiapoptotic). Both proteins interact so as to cooperate and govern the cell's fate. Also, the origin of the activation signals of apoptosis taking place on the mitochondria is a clue molecule, cytochrome C (Cyt C), which is released from the mitochondria to form the apoptosome complex. The intrinsic pathway – with some differences – is a mostly conserved pathway among metazoans (for a comprehensive review look at Wang & Youle, 2009). Cyt C is a key component of the apoptosome complex for activating the initiator caspase-9 after its release from mitochondria. Under non-apoptotic conditions, Cyt C is kept inside the respiratory chain. Against some cellular challenges, like the alteration of the DNA in the mitochondria or the nucleus, Cyt C is released from its membrane, crossing the external membrane and initiating the formation of the apoptosome complex. In essence, mitochondrial proteins – like Cyt C and caspases – are not hired guns and during non-apoptotic conditions they are responsible for various basic mitochondrial roles for normal cell functioning. The compartmentalisation of such mitochondrial proteins isolates them from interacting with partners or targets, a mechanism to prevent the unwanted activation of apoptosis in normal cells. Only after their appropriate release into the cytoplasm do such proteins play the role of triggers to initiate

The classical invertebrate model organisms for the study of apoptosis are *C. elegans* and *Drosophila*. In spite of the fact that the regulators of apoptosis have been found in such model organisms, the involvement of mitochondria in apoptosis is not conclusive. So far, no irrefutable evidence of the release of Cyt C from the intermembrane space has been found. Also, the involvement of Cyt C in the apoptosome formation in *Drosophila* is controversial,

The current evidence indicates that the whole process of apoptosis -including the involved proteins and the regulation mechanisms- in crustaceans is far more diverse than has been assumed from the studies with model organisms. Recent studies have shown that several proteins in the apoptotic network are quite conserved between mammals and arthropods; however, it is clear that the integration of such homologous proteins in the physiology and pathophysiology of crustaceans needs further experimental assessment. Some unresolved questions regarding this topic are: how does the regulation of the process occur? Is

and some evidence suggests that Cyt C is not necessary (Rolland & Conradt, 2006).

eukaryotic cell, it retained some capacity to operate partially independently.

the cell's suicide.

crustacean apoptosis transcriptionally regulated, as in *Drosophila* (RHG ´killer´ proteins)? Or is it controlled by pro- and anti-apoptotic Bcl-2 family proteins, as in vertebrates? The issues that should be investigated in the short-term are whether the calcium-induced opening of the mitochondrion permeability transition pore (MPTP), commonly found on vertebrate species, also occurs in crustaceans. Furthermore, the study of the differences in the regulation of the intrinsic pathway of crustacean apoptosis will lead to an understanding of their adaptation to challenging environments; this is because marine organisms have to deal with seasonal as well as circadian changes in environmental variables. Some examples are UV radiation, temperature and dissolved oxygen, and even some biological stresses such as toxins that may vary over time. But this is not all: other variables that may inhibit apoptosis must be considered. "Characterisation of the players, pathways, and their significance in the core machinery of crustacean apoptosis is revealing new insights for the field of cell death"(Menze et al., 2010).

Apoptosis is a key host response to viral infection. Viruses that can modulate a host's apoptotic responses are likely to gain important opportunities for transmission. Here, we review recent studies that demonstrate that the particles of Invertebrate Iridescent Virus6 (IIV-6) (Iridoviridae, genus Iridovirus), or an IIV-6 virion protein extract, are capable of inducing apoptosis in lepidopteran and coleopteran cells, at concentrations 1000-fold lower than that required to shut-off the host's macromolecular synthesis (Williams et al., 2009). Throughout the process of pathogen–host coevolution, viruses have developed a battery of distinct strategies to overcome the biochemical and immunological defences of the host. Thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. Since the elimination of infected cells via programmed cell death is one of the oldest defence mechanisms against infection, disabling host cell apoptosis might represent an almost obligatory step in the viral life cycle. Conversely, viruses may take advantage of stimulating apoptosis, either to kill uninfected cells from the immune system or else to induce the breakdown of infected cells, thereby favouring viral dissemination (Galluzzi et al., 2008).
