**2. Ecological relationships: Classical definitions and approaches**

#### **2.1 Epibiosis**

Epibiosis is a facultative association of two organisms: the epibiont, which colonizes the surface of live substrates, and the basibiont, which hosts the epibionts [145]. Some species of epibiotic communities show preferences for specific location sites on the host [76]. According to Wahl and Mark [146], when the effects associated with epibiosis are neutral or positive for a basibiont species and beneficial for an epibiont species, selection should favor the evolution of the epibiotic relationship, which tends to increase specificity through evolutionary history. Although many epibiont ciliates are not harmful to their basibionts, some studies have shown that the epibionts can cause deleterious effects on their hosts [147–149].

Historically, studies involving epibiont ciliates focus on the following interests: new records and checklists [27, 28], descriptions of new taxa using morphological and molecular data [150], possible deleterious effects on hosts [149, 151], distribution and preferred sites of epibiont populations and communities [152], spatial and temporal distribution of the epibiotic relationship [153], laboratory rearing and experimentation studies [154–156], and even investigations into extrinsic and intrinsic factors involved in the kinetics of epibiont ciliate populations [157, 158].

#### **2.2 Mutualism**

Mutualism is a relationship with high metabolic dependence, where both organisms, ciliate and their hosts, obtain benefits [159, 160]. In the phylum Ciliophora, this type of relationship is seen, mainly in the subclass Trichostomatia, which includes the ciliates of the digestive tract of herbivorous mammals [161]. The symbiont ciliates represent approximately 2,600 of the described organisms, of which around 1000 species belong to the subclass Trichostomatia [2]. This subclass comprises ciliated protists, mostly mutualists of the digestive tract of

several vertebrate hosts, with only one species showing parasitism in humans, *Balantidium coli* [2, 162, 163]. The subclass Trichostomatia is divided into three orders: Vestibuliferida, Entodiniomorphida, and Macropodiniida.

Ruminant ciliates and the host have a fundamental symbiosis relationship for the digestion and absorption of large amounts of plant material by the ruminant [164, 165]. On the one hand, the host provides an ideal environment for the survival of the symbiotic microbiota. The rumen is a strictly anaerobic environment, with temperatures ranging from 38 to 41° C, redox potential around 250 to 450 mV (millivolts), osmolarity ranging from 260 to 340 mOsm (millivolts), and pH levels between 5.0 and 7.5. Maintaining these characteristics is essential for microbial enzymatic activity to occur. In return, symbionts provide energy, protein, and vitamins to the host [166]. In energy terms, about 50–70% of the energy obtained by the host comes from the absorption of volatile fatty acids (VGAs) (eg. acetate, butyrate, and propionate), which are absorbed after the breakdown and fermentation of plant fiber by ruminal microorganisms [165]. Ciliates also represent a great source of protein for the ruminant (about 2 to 5%). Still, the ruminal microbiota also synthesizes B and K vitamins in sufficient quantities for the maintenance and growth of the animal. Due to the important participation in the physiology of the ruminant, the evolutionary dynamics of ruminal ciliates has been suggested as closely associated with the radiation of their hosts [167–169].

### **2.3 Commensalism and parasitism**

Commensalism occurs when the symbiont inhabits in the host with no evident benefit or harm [170].

Parasitism, which is less common in ciliates, involves the parasites that usually cause disease being pathogens. They may be localized or spread throughout a host, defined as the independent and dominant member of the symbiotic pair. Here, the parasite inhabits on or inside the host to obtain resources and to harm it [171].
