The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease

Abstract

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas’ disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.

Silencing of RpATG8 impairs the biogenesis of maternal autophagosomes in vitellogenic oocytes, but does not interrupt follicular atresia in the insect vector Rhodnius prolixus

“…R. prolixus atretic oocytes present organelles with the typical morphology of autophagic vacuoles, acidified yolk organelles, and an increase in hydrolase activities (which are known to be involved in the degradation of the yolk) [22]. In D. maxima, it was shown that apoptosis and autophagy markers are stimulated [23], and that hydrolases are activated and target the yolk during atresia [24]. In Diptera and Lepidoptera, there are evidences suggesting that follicle cells and nurse cells, in each follicle, degenerate via apoptotic and autophagic mechanisms [12][13][14]25].…”

Investigation of three enzymes and their roles in the embryonic development of parthenogenetic Haemaphysalis longicornis

“…Cathepsin D is the major lysosomal aspartic protease and is widely distributed in the cells, where it regulates programmed cell death, autophagy and the degradation of yolk protein [18,25]. Our study showed that the expression of cathepsin D rapidly reached the highest level on day 3 and then gradually decreased to a low level in the later development stage.…”

“…In R. prolixus, acid phosphatase can hydrolyze polyphosphate (polyP) to abolish the inhibitory effect of polyP on cathepsin D, which could facilitate the degradation of vitellin by cathepsin D [18,19]. The same interaction of the two enzymes is found in D. maxima [25]. In future work, we will investigate the relationships between the three enzymes and identify their effects on vitellin and embryogenesis to reveal the reasons for the differences between the two reproductive mechanisms.…”

“…Many proteolytic enzymes accumulate in oocytes together with vitellin and regulate vitellin degradation to provide nutrients during embryonic development [14]. This physiological process has been observed in many insects, such as Bombyx mori [15][16][17], Rhodnius prolixus [18,19], Periplaneta americana [20], Blattella germanica [21], Culex quinquefasciatus [22], Musca domestica [23], Anticarsia gemmatalis [24], Dipetalogaster maxima [25], Spodoptera exigua [26] and Helicoverpa armigera [27]. However, related studies have only been reported in several tick species, including Ornithodoros moubata [28], Rhipicephalus microplus [14,[29][30][31][32][33] and bisexual H. longicornis [34].…”

Jaburetox, a urease‐derived peptide: Effects on enzymatic pathways of the cockroach Nauphoeta cinerea

“…The organs were homogenized in 20 mM sodium acetate buffer pH 4.0 containing 10 mM DTT, 10 mM Na 2 EDTA with protease inhibitors, and centrifuged at 14,000 × g for 5 min at 4°C. The ACP activity was measured following the hydrolysis of p ‐nitrophenyl phosphate ( p NPP) to p ‐nitrophenol ( p NP) as described previously (Leyria, Fruttero, Nazar, & Canavoso, 2015). The assays were conducted incubating 5 mM p NPP as substrate and 2.5 µg of protein homogenates as enzyme source for 30 min at 37°C.…”