**1. Introduction**

180 Bioenergetics

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*anaerobic distance capacity, maximal instantaneous velocity and aerobic inertia in sprint and endurance young swimmers.* Eur J Appl Physiol (2010) 110:121–131 Doi The term "invertebrate" recalls all animal species lacking a backbone or a bony skeleton. Although "invertebrate" is not a scientific term that encloses a taxonomic rank, this group includes animal species represented by over 30 phyla and it includes the first animals that successfully inhabited the earth, an event that – according to the fossil evidence – dates back to around 600 million years ago. This group is composed of several different phyla, such as annelids, molluscs, sponges, cnidarians, echinoderms, and all species from the phylum Arthropoda – which is the largest among invertebrates and is comprised by insects, arachnids and crustaceans (nearly reaching 1,033,160 species).

Since they appeared for the first time during the Cambrian period, invertebrates have played an important ecological role since they are frequently the key constituents of many trophic chains and they occupy virtually every available ecosystem on Earth, being characterised by notable variations in temperature, oxygen concentrations, food availability and food quality. Also, many species occupy highly specific and important roles in nature as pollinators, parasites or vectors for parasitic diseases affecting human and animal health.

It is clear that the ability of invertebrates to inhabit almost every ecosystem – as well as the diverse array of morphological and behavioural strategies used to obtain nutrients from the environment – is an accurate reflection of the enormous ability of these organisms to solve their most basic energetic requirements. From blood-suckers such as mosquitoes, intestinal nematodes and leeches (hirudin), to small plankton marine feeders such as cnidarians and marine benthic bivalves, all species face changes in food availability throughout their life cycle which affect their energy stores and growth rates (Peck, 2002; Popova-Butler & Dean, 2009). A beautiful example of highly specific energy stores – crucial during invertebrates' life cycle and important to human health – is that of the female mosquito (*Anopheles gambiae*), which usually feeds on sugar to gain energy to fly and to cope with metabolic

Invertebrates Mitochondrial Function and Energetic Challenges 183

Somero, 2002; Abele et al., 2007). Nevertheless, other invertebrate species are highly adapted to live in extreme conditions such as those living in hypoxic or even anoxic environments,

As has previously been stated, this chapter reviews the current state of knowledge of the mitochondrial function of invertebrate species. It asks two central questions: 1) How are invertebrates able to adapt to such diverse environmental conditions by using a common set of structures and mechanisms – their mitochondrial machinery – to fulfil their energy requirements along their entire life cycle? 2) Is it really important to understand the role of mitochondria in the life history of invertebrates? This chapter also includes original data on crustacean responses to the external factors affecting such mitochondrial functions as

**2. The highly conserved mitochondrial machinery of invertebrates: Same** 

oxidative phosphorylation system (OXPHOS) (Boore, 1999; Gray et al., 1999).

part of the catalytic sites of the enzyme (Martinez-Cruz et al., 2011).

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

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

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

like the brine shrimp *Artemia franciscana* (Eads & Hand, 1999; 2003).

hypoxia, starvation and the energetically expensive molt cycle.

**functions, different challenges** 

requirements; however, anautogenous mosquitoes require the energy resulting from blood digestion in order to produce eggs, and it is during blood sucking that *Plasmodium vivax* (the parasite from infected females) enters into the vertebrate host to produce Malaria, a major health problem around the world (Das et al., 2010).

Large energetic demands during external work are observed throughout the life of several invertebrate species, and a clear example may be found in insect flight, which is considered to be one of the most energetically demanding processes of animal locomotion (Harrison & Roberts, 2000). Besides this, being an aerobic process that requires a permanent oxygen supply and depends upon ATP cellular production, the high energetic cost of flying is related to the frequency of the flight muscles' contraction (Vishnudas & Vigoreaux, 2006). In vertebrate species, the existence of high-energetic molecules in the muscle (phosphocreatine) during its exercise has been well documented (Jubrias et al., 2001); however, in invertebrate species, the presence of phosphagen-kinases that catalyse the synthesis of these highenergetic phosphorylated molecules has not been widely distributed (Ellington & Hines, 1991). The insect flight muscle seems to lack such molecules, but some flying species are able to surpass such energy needs by the proximity of mitochondria to muscle myofibrils, thus facilitating the export of energy rich nucleotides – such as ATP – to myofibrils (Vishnudas & Vigoreaux, 2006).

Some other invertebrate phyla – such as crustaceans – are able to synthesise phosphagens differently from that of vertebrates, like phosphocreatine. Phosphoarginine – a phosphagen of L-arginine found in the tail muscle of shrimp and crabs as well as in the flight muscle of flying insects – is the chemical energy storage system of these tissues, and thus animals are able to rapidly produce ATP when it is required (Wegener, 1996; Kotlyar et al., 2000). The enzyme responsible for the synthesis of phosphoarginine from ATP and L-arginine in invertebrates is named 'arginine kinase' and it is also considered to be a major allergen protein for shrimp-allergic individuals (Garcia-Orozco et al., 2007).

Since energetics are considered to be a key factor in limiting organisms' adaptation to extreme temperatures, several invertebrate species inhabiting marine polar environments are known to show a remarkable plasticity as regards their cellular system. Such adaptations may include an increasing number of mitochondria per cell as the temperature decreases as well as differences in the mitochondrial characteristics relating to the species' lifestyle, from motile species to sedentary ones (Peck, 2002). Studies in the mitochondrial function of the eurythermal polychaete *Arenicola marina* have concluded that invertebrates inhabiting higher latitudes – and consequently exposed to cold temperatures – showed higher oxygen consumption, mitochondrial densities and mitochondrial capacities when compared with those organisms living at lower latitudes with higher temperatures (Sommer & Portner, 1999; Peck, 2002). This adaptation of cold-acclimatised organisms is thought to occur in order to equate the level of metabolic activity present at warmer temperatures.

Among other important environmental factors affecting the bioenergetic state of organisms, marine invertebrates face large daily fluctuations in the dissolved oxygen concentrations of water, as well as wide salinity changes between open ocean and coastal waters - where many species live at least during one specific stage of their life cycle - (Dall et al., 1990). Such variations can adversely affect some species whose physiological mechanisms usually do not allow them to cope with low oxygen levels (as oxyregulators) or to handle salinity changes (as osmoregulators). However, several species are able to swim or move from one place to other, searching for a suitable site to grow, reproduce and survive (Hochachka &

requirements; however, anautogenous mosquitoes require the energy resulting from blood digestion in order to produce eggs, and it is during blood sucking that *Plasmodium vivax* (the parasite from infected females) enters into the vertebrate host to produce Malaria, a major

Large energetic demands during external work are observed throughout the life of several invertebrate species, and a clear example may be found in insect flight, which is considered to be one of the most energetically demanding processes of animal locomotion (Harrison & Roberts, 2000). Besides this, being an aerobic process that requires a permanent oxygen supply and depends upon ATP cellular production, the high energetic cost of flying is related to the frequency of the flight muscles' contraction (Vishnudas & Vigoreaux, 2006). In vertebrate species, the existence of high-energetic molecules in the muscle (phosphocreatine) during its exercise has been well documented (Jubrias et al., 2001); however, in invertebrate species, the presence of phosphagen-kinases that catalyse the synthesis of these highenergetic phosphorylated molecules has not been widely distributed (Ellington & Hines, 1991). The insect flight muscle seems to lack such molecules, but some flying species are able to surpass such energy needs by the proximity of mitochondria to muscle myofibrils, thus facilitating the export of energy rich nucleotides – such as ATP – to myofibrils (Vishnudas &

Some other invertebrate phyla – such as crustaceans – are able to synthesise phosphagens differently from that of vertebrates, like phosphocreatine. Phosphoarginine – a phosphagen of L-arginine found in the tail muscle of shrimp and crabs as well as in the flight muscle of flying insects – is the chemical energy storage system of these tissues, and thus animals are able to rapidly produce ATP when it is required (Wegener, 1996; Kotlyar et al., 2000). The enzyme responsible for the synthesis of phosphoarginine from ATP and L-arginine in invertebrates is named 'arginine kinase' and it is also considered to be a major allergen

Since energetics are considered to be a key factor in limiting organisms' adaptation to extreme temperatures, several invertebrate species inhabiting marine polar environments are known to show a remarkable plasticity as regards their cellular system. Such adaptations may include an increasing number of mitochondria per cell as the temperature decreases as well as differences in the mitochondrial characteristics relating to the species' lifestyle, from motile species to sedentary ones (Peck, 2002). Studies in the mitochondrial function of the eurythermal polychaete *Arenicola marina* have concluded that invertebrates inhabiting higher latitudes – and consequently exposed to cold temperatures – showed higher oxygen consumption, mitochondrial densities and mitochondrial capacities when compared with those organisms living at lower latitudes with higher temperatures (Sommer & Portner, 1999; Peck, 2002). This adaptation of cold-acclimatised organisms is thought to occur in

Among other important environmental factors affecting the bioenergetic state of organisms, marine invertebrates face large daily fluctuations in the dissolved oxygen concentrations of water, as well as wide salinity changes between open ocean and coastal waters - where many species live at least during one specific stage of their life cycle - (Dall et al., 1990). Such variations can adversely affect some species whose physiological mechanisms usually do not allow them to cope with low oxygen levels (as oxyregulators) or to handle salinity changes (as osmoregulators). However, several species are able to swim or move from one place to other, searching for a suitable site to grow, reproduce and survive (Hochachka &

protein for shrimp-allergic individuals (Garcia-Orozco et al., 2007).

order to equate the level of metabolic activity present at warmer temperatures.

health problem around the world (Das et al., 2010).

Vigoreaux, 2006).

Somero, 2002; Abele et al., 2007). Nevertheless, other invertebrate species are highly adapted to live in extreme conditions such as those living in hypoxic or even anoxic environments, like the brine shrimp *Artemia franciscana* (Eads & Hand, 1999; 2003).

As has previously been stated, this chapter reviews the current state of knowledge of the mitochondrial function of invertebrate species. It asks two central questions: 1) How are invertebrates able to adapt to such diverse environmental conditions by using a common set of structures and mechanisms – their mitochondrial machinery – to fulfil their energy requirements along their entire life cycle? 2) Is it really important to understand the role of mitochondria in the life history of invertebrates? This chapter also includes original data on crustacean responses to the external factors affecting such mitochondrial functions as hypoxia, starvation and the energetically expensive molt cycle.
