**2. Amastigote form**

Amastigote means "without apparent flagellum". The flagellum in amastigotes is internal and non-functional [7, 20]. This phase is a response to the phagocytation by its host's defense cells, presenting itself in an intracellular form inside the phagolysosome [21].

After the blood-feeding, digestive enzymes, including trypsin, chymotrypsin, aminopeptidase, carboxypeptidase, and alpha-glycosidase degrade ingested infected cells and expose amastigote forms to the peritrophic membrane. The change in conditions from the mammalian host to the vector's gut, active membrane receptors that detect the change in the environment as the pH increases, from ~4.0 to 5.5 in the phagolysosome to ~6.8 to 7.4 in the midgut vector [14, 21, 22] and temperature decrease, stimulate the development of the parasite into promastigote form [10, 11].

An important response of the parasite to this environmental change is the modulation of enzyme activity in the midgut, assigning different roles to these molecules than that suggested for the mucin-like structures, which appear to protect the parasite surface against the proteolytic enzymes [14]. The secretion of chitinase and N-acetylglucosaminidase enzymes protects from the intense enzymatic activity resulting from digestion, allowing the escape of peritrophic membrane towards the intestinal wall of the vector [13].

Several intracellular signals are triggered and are directly related to the transition from amastigote to promastigote. Relative expression studies revealed increased expression of several genes related to: (calmodulin binding; Cyclic nucleotide biosynthetic process; GTPase activity; GTP binding; DNA association; Nucleosome activity; Nucleosome assembly; Synthesis-coupled proton transport ATP; Mitochondrial proton transporter ATP synthase; Intracellular signal transduction; Dinein complex; Protein complex; Protein heterodimerization activity; Protein polymerization; Proteolysis; Phosphorus-oxygen lyase activity; Calciumdependent cysteine-type endopeptidase] and a decrease in the expression of genes related to: (Antioxidant activity; Peroxiredoxin activity; Cysteine-type peptidase activity; DNA catabolic process; Triglyceride lipase activity) [12].

Transformation of amastigotes to promastigotes occurs within 12–18 h. These initially transformed promastigotes are termed procyclic and remain short, ovoid, and only slightly mobile [14].

## **3. Promastigote forms**

#### **3.1 Procyclic form**

A procyclic promastigote is similar to a cell in G1 or post-S phase that has inherited the new short flagellum [23]. Its morphological characteristic is a body length of 6.5–11.5 μm and the flagellum is shorter than the body length and can have variable body width [20]. The intense multiplication of these forms starts at approximately 18–24 h [14], the divisor promastigotes are found in rosettes with flagella directed towards the center. In promastigotes, the flagellum extends from the cell body, hits and moves the organism, emerging from the anterior end of the cell [7].

Membrane protein classes of the parasite enable the attachment of the procyclic promastigote to the midgut wall of the vector and compatibility between Leishmania species with the vector species. The main membrane glycoconjugates, including their unique and common structures, are lipophosphoglycans–LPG, glycophosphatidylinositol lipids–GIPLs, glycoprotein 63–gp63, secreted acid phosphatases–sAP, secreted proteophosphoglycans–sPPG [14].

The fact that significant differences in LPG-mediated binding were observed when different vector species were compared suggest that the molecules that serve as parasite attachment sites can vary between different species of sandflies.

Serum digestion products destroy incompatible Leishmania species, furthermore, studies suggest that inter- and intraspecies-specific polymorphisms in the LPG phosphoglycan domains may result in species- and strain-restricted intestinal binding and thus determine vector competence. Species- and strainspecific and may therefore provide the evolutionary pressure for structural LPG polymorphisms [14, 24]. Developmental-regulated modifications in LPG structure control the specificity of the midgut adhesion stage [25, 26]. Recent findings indicate that non-LPG-mediated fixation is used by some other species of Leishmania [27–31].

*Leishmaniasis: Molecular Aspects of Parasite Dimorphic Forms Life Cycle DOI: http://dx.doi.org/10.5772/intechopen.102370*

The gp63, also known as leishmanolysin, is a 63 kDa zinc metalloproteinase containing a GPI anchor and is expressed on the surface of promastigotes of several Leishmania species. It plays an important role in the annexation of the leishmaniasis protozoan and has stood out in several studies related to the understanding of the development and virulence of the parasite [31–36].

Gut-associated lectins or lectin-like molecules, which have been described for sandflies and presented as signaling sites conducive to parasite fixation [37–39].

Alternatively, lower affinity and less specific interactions, mediated by shared covering structures and/or flagellar proteins, may be sufficient for the parasite to resist the expulsive force it is exposed to in the vectors. Directing the anterior migration of unattached promastigotes to the thoracic midgut and stomodeal valve has generally been attributed to a glucose concentration gradient [14].

#### **3.2 Nectomonad form**

During 36–60 h, rapid multiplication continues, accompanied by the transformation of promastigotes into a long, slender, highly mobile form called nectomonads [14]. Nektós, comes from the Greek and means: "who swims". Its morphological characteristic is the body length greater than or equal to 12 μm with variable body width and flagellar length [20]. A nectomonad promastigote is like an S-phase cell [23]. The cell differentiation signals triggered, in comparison to the procyclic ones, a significant decrease in the expression of genes related to: Nucleosome activity and assembly; protein heterodimerization; DNA association; core; kinetochore; administration of calmodulin [12].

#### **3.3 Leptomonad form**

By 60–72 h, an enormous number of nectomonads are found bundled up in the anterior portion of the abdominal midgut, with many attached via their flagella to the microvilli of the epithelial cells. The anterior migration of promastigotes to the region of the cardia [middle thoracic intestine] and stomodeal valve proceeds until a large accumulation of parasites behind the valve is reached. A leptomonad promastigote is similar to a cell in the same stages of the cell cycle as a procyclic promastigote, but which has inherited the older, longer flagellum [23]. Leptos, comes from the Greek and means: "slender, thin, small". Its morphological characteristic is body length 6.5–11.5 μm, with flagellum greater than body length and variable body width [20]. Found lining the surface of the stomodeal valve and there can be differentiated haptomonad and metacyclic promastigotes [40].

#### **3.4 Haptomonad form**

It is the transformation of leptomonads into short, broad forms called haptomonads, which are occasionally seen to divide [7]. It comes from the Greek haptein, and means: "to hold, denoting contact or combination", the morphological characteristic of haptomonads is the discoid expansion of the tip of the flagellum, with body shape and variable flagellar length [20]. The haptomonad forms bind through hemidesmosomes to the thin cuticular layer called the intima of the stomodeal valve or to each other through the secretion of a viscous gel-like matrix that restricts its motility [17].

The main component of the gel secreted by promastigotes (PSG) is a high molecular weight glycoprotein called filamentous proteophosphoglycan [15]. The identification of PSG strengthened the hypothesis of vector valve blockage, because the gel-forming properties of the filamentous

proteophosphoglycan–fPPG may provide the physical obstruction necessary to cause regurgitation in the vector during repast [7].

The gelatinous nature of PSG, together with its high cell density, can cause local oxygen depletion, and anaerobiosis is also known to stimulate metacyclogenesis [16]. Furthermore, after differentiating leptomonad promastigotes in the middle of the PSG plug, metacyclic promastigotes can migrate to either pole, concentrating on the former in response to a chemotactic suggestion. The possibility of Leishmania responding to sugars or saliva released from the culture that could form a gradient in the midgut remains to be addressed [20].

#### **3.5 Metacyclic form**

The name "metacyclic" comes from the Greek Meta and means: "Between". They are morphologically classified as short, slender, body length less than or equal to 8 μm, body width less than or equal to 1 μm, and highly active with a flagellum at least twice the length of the cell body and are generally not seen in the division [7, 20].

When compared with the gene expression in the form of neptomonads, we can observe the regulation of several cellular activities, with the negative expression of genes related to rRNA processing and the small subunit process (SSU) [12].

Metacyclic promastigotes, originating from the foregut or behind the stomodeal valve to the esophagus, pharynx, and proboscis, are inoculated during the meal, where they initiate the infection in the mammalian host [14]. However, there are at least three known components that lead to infection by the leishmaniasis protozoa: the metacyclic promastigotes themselves, which are obviously essential for transmission; sand fly saliva; and the gel secreted by promastigotes–PSG. Sandfly saliva is a well-established disease exacerbation factor [41], at least for tegumentary leishmaniasis. This is due to the fact that it contains potent compounds with vasodilatory and anti-hemostatic properties [42]. Co-inoculation of saliva with parasites has been shown to worsen the disease in several studies, and this is due to the modulatory capacity of the immune response to contribute to parasite survival and replication [43–45]. Likewise, PSG has also been shown to contribute to the worsening of the disease, being directly related to the increase in the number of metacyclic promastigote parasites co-inoculated with saliva [27]. The presence of parasites in the salivary glands of sandflies has already been reported by some studies and, therefore, it has been proposed as a fact of great relevance for transmission [46, 47].
