**3. The Invertebrates mitochondrial genome**

The study of the mitochondrial genome has provided enormous amounts of information from which it has become feasible to infer the origin of species by using comparative and evolutionary genomics (Jiang et al., 2009) in order to understand the ancient phylogenetic relationships among species, to comprehend population genetics (Boore et al., 1995; Boore, 1999), and to recognise the mechanisms coordinating the nuclear and mitochondrial genomes so as to synthesise a large number of functional proteins located in this organelle.

To date, the mtDNA of several invertebrates has been sequenced and characterised, including ascidians (Yokobori et al., 1999), echinoderms (Jacobs et al., 1988; Asakawa et al., 1995), insects (Clary & Wolstenholme, 1985), nematodes (Okimoto et al., 1992), molluscs (Yu & Li, 2011; Cheng et al., 2011), and various crustacean species such as shrimp and crabs (Staton et al., 1997; Shen et al., 2007; Peregrino-Uriarte et al., 2009). Several reports have shown that the mitochondrial genome of invertebrate species varies, and ranges between 12 and 20 kbp. This may be due to contrasting ecological habitats or it may be a response to different selective pressures (Table 1).


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

The study of the mitochondrial genome has provided enormous amounts of information from which it has become feasible to infer the origin of species by using comparative and evolutionary genomics (Jiang et al., 2009) in order to understand the ancient phylogenetic relationships among species, to comprehend population genetics (Boore et al., 1995; Boore, 1999), and to recognise the mechanisms coordinating the nuclear and mitochondrial genomes so as to synthesise a large number of functional proteins located in this organelle. To date, the mtDNA of several invertebrates has been sequenced and characterised, including ascidians (Yokobori et al., 1999), echinoderms (Jacobs et al., 1988; Asakawa et al., 1995), insects (Clary & Wolstenholme, 1985), nematodes (Okimoto et al., 1992), molluscs (Yu & Li, 2011; Cheng et al., 2011), and various crustacean species such as shrimp and crabs (Staton et al., 1997; Shen et al., 2007; Peregrino-Uriarte et al., 2009). Several reports have shown that the mitochondrial genome of invertebrate species varies, and ranges between 12 and 20 kbp. This may be due to contrasting ecological habitats or it may be a response to

**size (bp)** 

**Cnidaria** *Hydra oligactis* 16,314 NC\_010214 Kayal & Lavrov,

*Aurelia aurita* 16,937 NC\_008446 Shao et al., 2006

*Clonorchis sinensis* 13,877 JF729304 Cai et al., 2011

19,381 NC\_015640 ---

**Porifera** *Plakinastrella sp.* 19,790 NC\_010217 Lavrov et al.,

**GenBank Acc. No.** 

20,088 NC\_010171 Belinky et al.,

17,427 NC\_010769 Rosengarten et

14,803 NC\_014578 Mwinyi et al.,

13,647 NC\_014768 Liu et al., 2011

12,672 NC\_010484 Suga et al., 2008

13,888 NC\_006892 Steinauer et al.,

**References** 

2008

2008

2008

2010

2005

al., 2008

environments.

**3. The Invertebrates mitochondrial genome** 

different selective pressures (Table 1).

**Platyhelminthes** *Symsagittifera* 

**Rotifera** *Brachionus* 

**Acanthocephala** *Leptorhynchoides* 

**Phylum Species mtDNA** 

*Negombata magnifica* 

*Aphrocallistes vastus* 

*Fungiacyathus stephanus* 

*roscoffensis* 

*Taenia taeniaeformis* 

*plicatilis* 

*thecatus* 


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

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

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 nucleotides fragment (Boore & Brown, 1995).

Invertebrates Mitochondrial Function and Energetic Challenges 193

Although, to date, the mitochondrial genes expression mechanisms are not fully understood, and the evolutionary processes by which the mitogenome suffers a rearrangement are not clear. It is proposed that a new order in genes' arrangements must preserve or facilitate those signals or mechanisms required for the transcription and processing of RNAs to

The mitochondrial DNA from animal cells is known to be easily affected, since it is not protected by DNA-binding proteins or histones such as nuclear DNA. Several studies have found that mtDNA can be affected by aging, hypoxia and random events of mutation or insertion/deletion (rates of mutation for mitochondrial genomes are known to be much higher than those in the nuclear DNA) that can produce increased oxidative stress and high levels of ROS in this organelle. Defective proteins which result from altered mtDNA molecules cause defective mitochondrial function, as an impaired respiratory chain and increased electron leaks so as to finally generate larger amounts of ROS (Wei et al., 1998). Insects' mitogenomes are known to be affected at the transcriptional level by chemicals, since the mtDNA copy number has been shown to increase to meet the bioenergetic demands of the organism, as observed in the fly *D. melanogaster* when exposed to tetracycline. Treatment with this antibiotic causes an energetic deficiency, promoting an up-

accomplish the mitochondrial function in animal species (Boore, 1999).

regulation of the mtDNA copy number (Moraes, 2001; Ballard & Melvin, 2007).

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

stage (Hochachka & Somero, 2002).

(Nestel et al., 2003).

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

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

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

In 2006, the description of the mtDNA of the moon jellyfish (*Aurelia aurita*) was reported. It was surprising to find that mitochondria of this organism contain a linear genome, which became the first non-circular genome described in a Metazoan. Besides its linearity, its organisation involves two additional sequences of 324 and 969 nucleotides, the last (ORF969) encodes a putative family B-DNA polymerase, tentatively identified as *dnab,* which was previously only reported in algae mtDNAs (Shao et al., 2006). Subsequently, the linear mitogenome of Cnidarians of the genus *Hydra* was also reported, although it was found that it is fragmented as two linear mitochondrial "chromosomes" (mt1 and mt2) where all genes are unidirectionally-oriented (Voigt et al., 2008).

In addition, the invertebrate's mitochondrial genetic code differs from the universal/standard genetic code, and it is suggested that this is species-specific since several studies have identified some changes in animal mitochondrial code, as shown by Table 2 (taken from Watanabe, 2010). As observed in this table, invertebrate mtDNAs are largely represented by different changeable codons – depending upon the species. This is the case for the AUA codon which usually codes Ile in the standard genetic code but in the mitochondria of some species of Nematoda, Mollusca, Platyhelminthes and Vertebrata it encodes a Met (Himeno et al., 1987; Bessho et al., 1992). Also, in several species, the start codon differs from the AUG but still codifies a methionine, and in most of the species the stop codon is an incomplete codon, such as UA or U (Watanabe, 2010).


Table 2. The relationships between the genetic codes of animal mitochondria. Modified from: Watanabe, 2010. Bold letter: non-universal codon; Term: termination codon.

In 2006, the description of the mtDNA of the moon jellyfish (*Aurelia aurita*) was reported. It was surprising to find that mitochondria of this organism contain a linear genome, which became the first non-circular genome described in a Metazoan. Besides its linearity, its organisation involves two additional sequences of 324 and 969 nucleotides, the last (ORF969) encodes a putative family B-DNA polymerase, tentatively identified as *dnab,* which was previously only reported in algae mtDNAs (Shao et al., 2006). Subsequently, the linear mitogenome of Cnidarians of the genus *Hydra* was also reported, although it was found that it is fragmented as two linear mitochondrial "chromosomes" (mt1 and mt2)

In addition, the invertebrate's mitochondrial genetic code differs from the universal/standard genetic code, and it is suggested that this is species-specific since several studies have identified some changes in animal mitochondrial code, as shown by Table 2 (taken from Watanabe, 2010). As observed in this table, invertebrate mtDNAs are largely represented by different changeable codons – depending upon the species. This is the case for the AUA codon which usually codes Ile in the standard genetic code but in the mitochondria of some species of Nematoda, Mollusca, Platyhelminthes and Vertebrata it encodes a Met (Himeno et al., 1987; Bessho et al., 1992). Also, in several species, the start codon differs from the AUG but still codifies a methionine, and in most of the species the

> **AAA (Lys)**

**AGA (Arg)** 

**Met** Lys **Term Term** 

**Met** Lys **Gly Gly** 

Ile **Asn Ser Ser** 

**Met** Lys **Ser Ser** 

**Met** Lys **Ser Ser** 

Ile **Asn Ser Ser** 

Ile Lys Arg Arg

**AGG (Arg)** 

where all genes are unidirectionally-oriented (Voigt et al., 2008).

stop codon is an incomplete codon, such as UA or U (Watanabe, 2010).

**Arthropods Met** Lys **Ser Ser**  Most (shrimp, daphnia) **Met** Lys **Ser Ser**  Insect (Drosophila) **Met** Lys **Ser** -

**Platyhelminthes Met Asn Ser Ser** 

Rhabditophora (Planaria) Ile Lys Arg Arg

Table 2. The relationships between the genetic codes of animal mitochondria. Modified from: Watanabe, 2010. Bold letter: non-universal codon; Term: termination codon.

**AUA (Ile)** 

**Codon** 

**(Universal code)** 

asymmetron)

**Molluscs** (squid, octopus, Liolophura, Mesogastropoda)

starfish)

ascaris)

Trematoda)

hydrozoa)

**Vertebrates** (human, bovine, rat, mouse, chicken, frog)

**Prochordates** (ascidian,

**Echinoderms** (sea urchin,

**Nematodes** (nematodes,

Most (Echinostomida,

**Coelenterates** (jellyfish, coral, sea anemone,

Although, to date, the mitochondrial genes expression mechanisms are not fully understood, and the evolutionary processes by which the mitogenome suffers a rearrangement are not clear. It is proposed that a new order in genes' arrangements must preserve or facilitate those signals or mechanisms required for the transcription and processing of RNAs to accomplish the mitochondrial function in animal species (Boore, 1999).

The mitochondrial DNA from animal cells is known to be easily affected, since it is not protected by DNA-binding proteins or histones such as nuclear DNA. Several studies have found that mtDNA can be affected by aging, hypoxia and random events of mutation or insertion/deletion (rates of mutation for mitochondrial genomes are known to be much higher than those in the nuclear DNA) that can produce increased oxidative stress and high levels of ROS in this organelle. Defective proteins which result from altered mtDNA molecules cause defective mitochondrial function, as an impaired respiratory chain and increased electron leaks so as to finally generate larger amounts of ROS (Wei et al., 1998).

Insects' mitogenomes are known to be affected at the transcriptional level by chemicals, since the mtDNA copy number has been shown to increase to meet the bioenergetic demands of the organism, as observed in the fly *D. melanogaster* when exposed to tetracycline. Treatment with this antibiotic causes an energetic deficiency, promoting an upregulation of the mtDNA copy number (Moraes, 2001; Ballard & Melvin, 2007).
