**2. Polyphosphate mobilization during** *Rhipicephalus (Boophilus) microplus* **embryogenesis**

The tick *Rhipicephalus microplus* is a one-host tick that causes major losses to bovine herds, especially in tropical regions. In this scenario, major efforts have been made to develop immunoprophylatic tick control tools (Guerrero et al., 2006). Ticks are also vectors of parasites that cause hemoparasitic diseases, which are endemic in many cattle production areas (Sonenshine et al., 2006). *Rhipicephalus microplus* only has one host throughout all three life stages, usually a bovine animal, and a long feeding period (approximately 21 days). The adult female, after becoming completely engorged, drops off of the host and initiates oviposition approximately three days later. Being an oviparous creature, embryogenesis occurs in the absence of exogenous nutrients, and maternal nutrients are packaged in oocytes and mostly stored as yolk granules. Hatching occurs around 21 days after oviposition, and the emerging larvae can survive for several weeks before finding a host, using the remaining yolk as the only source of energy (Fagotto, 1990).

Early *R. microplus* embryonic stages are similar to those of *D. melanogaster* and mosquitoes (Bate & Arias, 1991; Monnerat et al., 2002). Tick embryogenesis is characterized by the formation of a non-cellular syncytium up to day 4 (Campos et al., 2006). After this, the embryo becomes a multicellular organism and starts organogenesis (Campos et al., 2006).

The function of polyphosphate as a phosphate reserve is well known in prokaryotes and also in eukaryote microorganisms (Kulaev & Vagabov, 1983; Kornberg, 1995; Kulaev, 2004). The cells of higher eukaryotes also carry polyphosphate, but in smaller amounts than found in microorganisms. Therefore, as well as being a source of phosphate, these biopolymers probably participate in regulatory processes (Kornberg et al., 1999). Total polyphosphate levels were quantified throughout *R. microplus* embryogenesis and the levels were found to be higher during embryo cellularization and segmentation, from the fifth to the seventh day of development, and declined after that until a plateau was reached. The free phosphate content rapidly decreased during syncytial blastoderm formation on the third day of development, and remained low until the twelfth day of embryogenesis, when it rapidly increased thereafter (Figure 2A). Exopolyphosphatase splits phosphate off from the end of a polyphosphate chain and represents one of the main enzyme types responsible for polyphosphate hydrolysis (Kulaev et al., 2004). The activity of exopolyphosphatase was analyzed during embryogenesis and its activity was in agreement with total polyphosphate mobilization (Figure 2B).

It is interesting to note that in *R. microplus* the decline in total polyphosphate content after the seventh day of embryogenesis did not reflect the increase in the free phosphate content, since this only occurs after the twelfth day, suggesting that polyphosphate also plays roles other than being a phosphate reserve for embryo development. In this case, an alternative source of phosphate could be derived from the dephosphorylation of vitellin, a major yolk protein that is gradually dephosphorylated throughout embryogenesis (Silveira et al., 2006). This source could mainly be used until segmentation of the embryo, on the seventh day of development, because there is no total polyphosphate mobilization during this period.

al., 2009; Kulakovskaya et al., 2010), thus, it would be intriguing to discover whether or not mitochondria have preserved polyphosphate functions such as the regulation of energy

**2. Polyphosphate mobilization during** *Rhipicephalus (Boophilus) microplus*

The tick *Rhipicephalus microplus* is a one-host tick that causes major losses to bovine herds, especially in tropical regions. In this scenario, major efforts have been made to develop immunoprophylatic tick control tools (Guerrero et al., 2006). Ticks are also vectors of parasites that cause hemoparasitic diseases, which are endemic in many cattle production areas (Sonenshine et al., 2006). *Rhipicephalus microplus* only has one host throughout all three life stages, usually a bovine animal, and a long feeding period (approximately 21 days). The adult female, after becoming completely engorged, drops off of the host and initiates oviposition approximately three days later. Being an oviparous creature, embryogenesis occurs in the absence of exogenous nutrients, and maternal nutrients are packaged in oocytes and mostly stored as yolk granules. Hatching occurs around 21 days after oviposition, and the emerging larvae can survive for several weeks before finding a host,

Early *R. microplus* embryonic stages are similar to those of *D. melanogaster* and mosquitoes (Bate & Arias, 1991; Monnerat et al., 2002). Tick embryogenesis is characterized by the formation of a non-cellular syncytium up to day 4 (Campos et al., 2006). After this, the embryo becomes a multicellular organism and starts organogenesis (Campos et al., 2006). The function of polyphosphate as a phosphate reserve is well known in prokaryotes and also in eukaryote microorganisms (Kulaev & Vagabov, 1983; Kornberg, 1995; Kulaev, 2004). The cells of higher eukaryotes also carry polyphosphate, but in smaller amounts than found in microorganisms. Therefore, as well as being a source of phosphate, these biopolymers probably participate in regulatory processes (Kornberg et al., 1999). Total polyphosphate levels were quantified throughout *R. microplus* embryogenesis and the levels were found to be higher during embryo cellularization and segmentation, from the fifth to the seventh day of development, and declined after that until a plateau was reached. The free phosphate content rapidly decreased during syncytial blastoderm formation on the third day of development, and remained low until the twelfth day of embryogenesis, when it rapidly increased thereafter (Figure 2A). Exopolyphosphatase splits phosphate off from the end of a polyphosphate chain and represents one of the main enzyme types responsible for polyphosphate hydrolysis (Kulaev et al., 2004). The activity of exopolyphosphatase was analyzed during embryogenesis and its activity was in agreement with total polyphosphate

It is interesting to note that in *R. microplus* the decline in total polyphosphate content after the seventh day of embryogenesis did not reflect the increase in the free phosphate content, since this only occurs after the twelfth day, suggesting that polyphosphate also plays roles other than being a phosphate reserve for embryo development. In this case, an alternative source of phosphate could be derived from the dephosphorylation of vitellin, a major yolk protein that is gradually dephosphorylated throughout embryogenesis (Silveira et al., 2006). This source could mainly be used until segmentation of the embryo, on the seventh day of development, because there is no total polyphosphate mobilization during this period.

metabolism and the participation in transport channel formation.

using the remaining yolk as the only source of energy (Fagotto, 1990).

**embryogenesis** 

mobilization (Figure 2B).

Fig. 2. Characterization of the total polyphosphate content during *R. microplus* embryogenesis. A) Total polyphosphate (▲) was extracted and quantified and free phosphate (■) was quantified in an egg homogenate on different days after oviposition. B) Total polyphosphate (▲) was extracted and quantified and exopolyphosphatase activity (●) was analyzed in an egg homogenate on different days after oviposition. Activity is expressed as units per milligram of total protein. The results represent the mean ± SD of three independent experiments, in triplicate.

Quantification of the major energy sources in the egg over the course of *R. microplus* embryogenesis suggests that lipids and carbohydrates are the main energy source used during early development of the embryo. The total lipid contents remained stable until the fifth day, dropped on the seventh day, and remained roughly unchanged until hatching (Figure 3A). The total sugar contents exhibited a similar pattern, although slightly delayed: the values remained stable until the seventh day, dropped on the ninth day and remained

Role of Inorganic Polyphosphate in the Energy Metabolism of Ticks 147

Fig. 4. Polyphosphate metabolism during *R. microplus* embryogenesis. Polyphosphate levels during embryogenesis in the mitochondrial fraction (●) and total polyphosphate (■) during embryogenesis. The results represent the mean ± SD of three independent experiments, in

Mitochondria from tick embryos in the segmentation stage (ninth day after oviposition) were isolated and respiration was measured using pyruvate as the substrate. The rate of oxygen consumption was 30 nmol/min/mg protein, and the respiratory control ratio (RCR) was 6.5. The process was KCN- and oligomycin-sensitive, his fraction exhibited an ATP hydrolyses azide sensitivity, a mitochondrial marker higher than 80%, and no activity of

State 3 State 4 RCR % F – ATPase

Once the mitochondria were characterized, mitochondria in eggs in the segmentation stage (ninth day after ovoposition) were isolated and exopolyphosphatase activity was measured in order to evaluate the regulation of its activity. The influence of NADH, phosphate, and ADP was investigated in concentrations ranging from 0.1 to 2.0 mM. The activity of exopolyphosphatase was stimulated by a factor of two by NADH, whereas its activity was completely inhibited by 2 mM phosphate and slightly stimulated by ADP (Figure 5A). The activity of exopolyphosphatase was also measured during mitochondrial respiration using pyruvate as the substrate and polyphosphate as the only phosphate source. During this assay, the addition of a small amounts of ADP (0.2 mM) induced state 3 (phosphorylating respiratory rate) followed by state 4 (non-phosphorylating respiratory rate), when all of the

Homogenate --- --- --- 49.50 % 2.9 ± 0.4

fraction 30.2 ± 3.2 4.6 ± 0.7 6.5 ± 0.4 83.45 % ---

azide sensitive

G6PDH (U/mg protein)

**2.1 Inorganic polyphosphate metabolism in tick mitochondria** 

glucose-6-phosphate dehydrogenase, a cytosol marker, was detected (Table 1).

triplicate.

Mitochondrial

Table 1. Mitochondrial characterization

ADP was converted to ATP.

constant until hatching (Figure 3B). This pattern suggests the utilization of lipids during the course of cellularization, a maternally driven process (Bate & Arias, 1993). On the other hand, carbohydrates would be the major energy source for the quick segmentation of the embryo, of zygotic nature (Nusslein-Volhard & Roth, 1989; Bate & Arias, 1993).

Fig. 3. Consumption of energetic sources. The major egg storage components of *R. microplus* were quantified on different days after oviposition. A) Lipid quantification, determined via the gravimetric method (Bligh & Dyer, 1959); B) total sugar concentration, measured using the method of Dubois (Dubois et al., 1956). The results represent the mean ± SD of three independent experiments, in triplicate.

Interestingly, mitochondrial polyphosphate utilization occurred during blastoderm formation and segmentation of the embryo, between the fifth and seventh days of development, and higher total polyphosphate utilization occurred after blastoderm formation and segmentation of the embryo, after the seventh day of development (Figure 4). Thus, mitochondrial polyphosphate levels seemed to correlate with the energy demand of the embryo during these developmental stages, during which the embryo utilized a large part of its reserve lipids and sugars.

constant until hatching (Figure 3B). This pattern suggests the utilization of lipids during the course of cellularization, a maternally driven process (Bate & Arias, 1993). On the other hand, carbohydrates would be the major energy source for the quick segmentation of the

Fig. 3. Consumption of energetic sources. The major egg storage components of *R. microplus* were quantified on different days after oviposition. A) Lipid quantification, determined via the gravimetric method (Bligh & Dyer, 1959); B) total sugar concentration, measured using the method of Dubois (Dubois et al., 1956). The results represent the mean ± SD of three

Interestingly, mitochondrial polyphosphate utilization occurred during blastoderm formation and segmentation of the embryo, between the fifth and seventh days of development, and higher total polyphosphate utilization occurred after blastoderm formation and segmentation of the embryo, after the seventh day of development (Figure 4). Thus, mitochondrial polyphosphate levels seemed to correlate with the energy demand of the embryo during these developmental stages, during which the embryo utilized a large

independent experiments, in triplicate.

part of its reserve lipids and sugars.

embryo, of zygotic nature (Nusslein-Volhard & Roth, 1989; Bate & Arias, 1993).

Fig. 4. Polyphosphate metabolism during *R. microplus* embryogenesis. Polyphosphate levels during embryogenesis in the mitochondrial fraction (●) and total polyphosphate (■) during embryogenesis. The results represent the mean ± SD of three independent experiments, in triplicate.
