3.2.3. Aerobic Metabolism

In principle, we must recognize that glucose is the main source of energy used by parasitic flatworms (both in adult and larval forms [81]), while glycogen formation is their main energy conservation strategy [82–87]. In fact, in the case of glycogen, it has been previously identified through histochemical techniques and later through transmission electron microscopy, where many glycogen granules are observed that can serve in cestodes as a source of energy in fasting situations as reported in *T. solium* tapeworms [88] and the metacestode (traditionally also known as *Cysticercus cellulosae*) [89].

Therefore, under aerobic conditions, these organisms will use the traditional pathways to obtain reducing power through the oxidation of glucose (glycolysis and the Krebs cycle) and the subsequent production of energy coupled to oxidative phosphorylation (OXPHOS) (Figure 4) [90]. In both trematodes [91] and cestodes [92], it has been possible to identify the genes encoding enzymes of each of these pathways at the genome level. However, transcriptomic analyses have shown a differential expression of these enzymes in the tissues of the parasite, as demonstrated in the metacestode of *Echinococcus granulosus* in which the

expression of enzymes from fermentative pathways associated with the germ layer and the gluconeogenic pathway associated with both the germinal layer and the protoscolex were detected [83]. Additionally, the expression can be influenced by experimental conditions. Fraga et al. [93] were able to successfully detect all the metabolites associated with the Krebs cycle in the metacestode of *T. crassiceps* under in vivo conditions; thus, it was inferred that this pathway is complete in the cysticercus. In this same parasite, we characterized the aerobic metabolism in the mitochondria of the tegument [62,94].

In the case of the metabolic pathways of lipids and proteins, there are great changes, which may be due to the adaptation to the conditions of a parasite related to what the host provides. In 2013, Tsai and a large team of collaborators reported the massive sequencing and comparison of four cestode genomes (*E. granulosus*, *Echinococcus multilocularis*, *Hymenolepis microstoma*, and *T. solium*) [92]. Basically, they reported a significant reduction in the metabolic capacity of these organisms, as well as the presence of specialized elements in the uptake of nutrients.

#### 3.2.4. Anaerobic Metabolism

Glycolysis can be considered a universal pathway by which many organisms can obtain energy. Its final product, pyruvate (Pyr), can be used in other alternative pathways known as fermentative pathways; they occur in the absence of O2 and allow the NADH generated during glycolysis to be oxidized to NAD+, a necessary substrate for, so that this path can continue (Figure 4).

A classic adaptation of anaerobic metabolism is lactate fermentation, in which pyruvate is reduced to lactate (Lac) by the enzyme lactate dehydrogenase (LDH) using electrons from NADH. It is now known that this pathway is also used in parasitic helminths. Direct evidence of its presence is the secretion of Lac into the medium, as has been reported in cestodes such as *Moniezia expansa* [95,96], *E. granulosus* [83], and *E. multilocularis* [86].

In addition to Lac secretion, the secretion of other reduced compounds such as succinate (Succ), acetate, and propionate (PPO) has been reported. This has been reported in *M. expansa* [32], and confirmed in *E. granulosus* [83], *T. crassiceps* [84], and *E. multilocularis* [86], where the main secreted product was succinate; these products are the result of a pathway known as malate dismutation (Figure 4).

Malate dismutation is the main anaerobic pathway present in parasitic platyhelminths [43,97–99], and has as its final products a reduced molecule and an oxidized (as occurs in the reactions called dismutation). During this process, phosphoenolpyruvate (PEP) produced during glycolysis is carboxylated to oxaloacetate (OAA) by PEP carboxykinase (PEPCK), producing ATP by substrate-level phosphorylation. OAA is reduced to malate (Mlt) through the cytosolic malate dehydrogenase (cMDH), which has NADH produced during glycolysis as another substrate. Subsequently, Mlt enters the mitochondria and, on the one pathway, through fumarase (also named fumarate hydratase, FH), it produces fumarate (Fum) and, by another, the mitochondrial malic enzyme (mME) oxidatively decarboxylates it to Pyr that can later generate acetate (Figure 4).

The fumarate produced is a substrate for the enzyme fumarate reductase (FDR) that reduces it to Succ; this is the main product of electron secretion in cestodes [95,96,100,101]. To not accumulate this metabolite and to maintain its redox balance, Succ is secreted into the surrounding medium, as reported in the culture medium of *T. crassiceps*, as well as in the cysticerci of *T. solium* removed from pig brains [84,96]. Additionally, it has been reported that Succ can generate PPO. Recently, two alternative pathways for propionate formation have been reported: (a) from succinyl-CoA to methylmalonyl-CoA that is decarboxylated to generate ATP and propionyl-CoA, which, in the presence of Succ, releases PPO and acetylates succinate; this process appears to occur under prolonged anaerobic conditions [102]; and (b) via Lac accumulation and its transformation to propionyl-CoA releasing PPO and regenerating CoA [103]. This contrasts with what Ritler et al. reported, after they could not detect propionate as a secretion product in *E. multilocularis* [86].

The other malate dismutation reaction is the one that produces acetate where, as mentioned, the pyruvate generated in the mitochondrial matrix, through the mME (and in addition NAD(P)H is generated) [98,101] and through the pyruvate dehydrogenase (PDH) complex, is oxidatively decarboxylated to generate acetyl-CoA and NADH. Finally, through the enzyme acetate-succinate-CoA transferase (ASCT), CoA is transferred to Succ, producing acetate and succinyl-CoA [104]. A search of available genomic databases indicates the presence of ASCT genes in the flukes *S. mansoni*, *P. westermani*, *C. sinensis*, and *F. hepatica* [105], as well as in the nematodes *Ostertagia ostertagi*, *Anisakis simplex*, and *Brugia malayi*. Although the presence of the gene encoding for ASCT in cestodes has not been reported, it is possible to suggest the presence of the enzyme (or an analogous pathway) since the presence of acetate has been reported as an end-product of anaerobic respiration in both *T. solium* [84] and *E. multilocularis* [86].

The NADH generated in the previous reactions transfers its electrons to the mitochondrial complex I NADH-rhodoquinone oxidoreductase which, contrary to what happens in aerobic conditions, reduces the rhodoquinone (RQ) instead of reducing the ubiquinone (UQ). This transfer of electrons is favorable because RQ has a redox potential of *E*m- = −63 mV, which is lower than that of UQ (*E*m- = +110 mV) [106,107]. The RQ donates electrons to the FDR to generate succinate from fumarate [108]. The measurement of FDR activity [39,109] is indicative of anaerobic metabolism [47,99], whereas the measurement of cytochrome *c* oxidase activity, of SDH, as well as the sensitivity of the electron transport chain (ETC) to different inhibitors (such as cyanide), are indicative of aerobic metabolism [62,110]. One point to highlight is that when NADH-rhodoquinone oxidoreductase participates, protons are translocated from the mitochondrial matrix to the intermembrane space, which, in turn, maintains a chemiosmotic gradient and generates ATP even in the absence of O2 (Figure 4).

In anaerobic metabolism [39], FRD performs the reverse reaction of succinate dehydrogenase (SDH) [40]. Both enzymes, SDH and FRD, are heterotetramers that share: (a) subunit 1 (Fp) that contains flavin-adenine dinucleotide (FAD); (b) subunit 2 (Ip) with three Fe-S centers; and (c) two subunits CybL and CybS, which maintain, on the one hand, binding to the inner mitochondrial membrane and, on the other, binding to the corresponding quinone [111,112]. In *A. suum*, there are isoforms in two of the four subunits as well; Fp and CybS are different between the aerobic larva and the anaerobic adult; no isoforms have been reported for the Ip and CybL subunits [113].

Considering the above, we can note that anaerobiosis-specific reactions are those catalyzed by FRD and ASCT. However, it is the presence of RQ that appears to be the only real difference between aerobic and anaerobic energy metabolism [114], as FRD expression has been described in cancer cells [115,116], while ASCT is an enzyme homologous to other transferases [105,117,118].

To recapitulate, in the cytosol of muscle cells under hypoxic conditions, lactate is produced by lactic acid fermentation. Unlike this, malate dismutation or malic fermentation has the following relevant aspects:


However, both aerobic and anaerobic metabolisms have the following aspects in common:


• Both are carried out in the mitochondrial compartments, which allows the formation of a proton gradient and therefore the synthesis of ATP.

Regardless of the type of energy metabolism, the redox balance is maintained. To keep it, organisms recycle their electron transporting coenzymes; thus, the number of reactions that produce NADH is equal to the reactions that consume it, or else, electrons are excreted in form to water, in aerobic organisms, and through succinate mainly in anaerobes [9].
