Journal of Insect Physiology 59 (2013) 1242–1249

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Journal of Insect Physiology

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Yolk hydrolases in the eggs of Anticarsia gemmatalis hubner(Lepidoptera: Noctuidae): A role for inorganic polyphosphatetowards yolk mobilization

0022-1910/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jinsphys.2013.09.008

⇑ Corresponding author at: Laboratório de Entomologia Médica, Programa deParasitologia e Biologia Celular, Instituto de Biofísica Carlos Chagas Filho (IBCCF),Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro (UFRJ),Cidade Universitária, Rio de Janeiro CEP 21941-590, RJ, Brazil. Tel.: +55 21 25626589; fax: +55 21 2280 8193.

E-mail address: ednildo@biof.ufrj.br (E.A. Machado).

Danielle M.P. Oliveira a,b, Fabio M. Gomes a,c, Danielle B. Carvalho a, Isabela Ramos d, Alan B. Carneiro d,Mario A.C. Silva-Neto d, Wanderley de Souza c,f, Ana P.C.A. Lima e, Kildare Miranda c, Ednildo A. Machado a,f,⇑a Laboratório de Entomologia Médica, Programa de Parasitologia e Biologia Celular, Instituto de Biofísica Carlos Chagas Filho (IBCCF), Centro de Ciências da Saúde (CCS), UniversidadeFederal do Rio de Janeiro (UFRJ), Cidade Universitária, Rio de Janeiro CEP 21941-590, RJ, Brazilb Departamento de Bioquímica, Instituto de Química, Centro de Ciências da Matemática e Natureza, UFRJ, Cidade Universitária, Rio de Janeiro CEP 21941-909, RJ, Brazilc Laboratório de Ultraestrutura Celular Hertha Meyer, IBCCF, CCS, UFRJ, Cidade Universitária, Rio de Janeiro CEP 21941-590, RJ, Brazild Instituto de Bioquímica Médica, CCS, UFRJ, Cidade Universitária, Rio de Janeiro CEP 21941-590, RJ, Brazile Laboratório de Bioquímica e Biologia Molecular de Proteases, Programa de Imunobiologia, IBCCF, CCS, UFRJ, Cidade Universitária, Rio de Janeiro CEP 21941-590, RJ, Brazilf Instituto Nacional de Metrologia Normalização e Qualidade Industrial-RJ (INMETRO), DIPRO-Diretoria de Programas Xerém, Duque de Caxias CEP 25250-020, RJ, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 February 2013Received in revised form 25 September 2013Accepted 27 September 2013Available online 15 October 2013

Keywords:Pest insectAcid phosphatasePolyphosphateProteaseYolk degradation

Despite being the main insect pest on soybean crops in the Americas, very few studies have approachedthe general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies withembryo formation and yolk mobilization in this species. In the present work, we identified an acid phos-phatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochem-istry and cell biology experiments. By testing several candidate substrates, this enzyme proved chieflyactive with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphoryla-tion of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous shortchain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of phys-iological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesiclesclearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. AsPolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays witha cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominentin Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease.We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A.gemmatalis.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

As oviparous animals, insect must allocate in their eggs suffi-cient nutrients to sustain embryogenesis, insect fat body synthe-sizes massive amounts of proteins that are posteriorly secreted tothe hemolymph and delivered to the ovaries where they will beincorporated into the developing oocytes by receptor-mediatedendocytosis (Raikhel and Dhadialla, 1992). Once inside the oocyte,yolk proteins accumulate in organelles called yolk granules

(Snigirevskaya et al., 1997) wherein they are stored together withseveral hydrolases, such as acid phosphatase and proteases(Nussenzveig et al., 1992; Ribolla et al., 1993; Giorgi et al., 1999;Zhao et al., 2005; Oliveira et al., 2008). Subpopulations of yolk gran-ules of various sizes, densities, and contents have been described inseveral oviparous models (Wallace, 1985; Fausto et al., 2001), andhave been linked with the triggering of yolk degradation by hydro-lases (Liu and Nordin, 1998; Cho et al., 1999; Fialho et al., 2002).

Acid phosphatases (AP) (EC 3.1.3.2) are typical lysosomal en-zymes that catalyze the hydrolysis of orthophosphoric monoestersfrom a wide range of substrates. They are also one of the best stud-ied hydrolases stored in animals’ eggs. The presence of AP in yolkgranules and its role in yolk mobilization was first described inthe axolotl Ambystoma mexicanum (Lemansky and Aldoroty,1977), and was later described in the yolk granules of severalinsects (Steinert and Hanocq, 1979; Kawamoto et al., 2000; Fialho

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et al., 2002). Nevertheless, the range of substrates of AP remainscontroversial. While several yolk proteins are strongly dephospho-rylated by AP during embryo development (Wimmer et al., 1998;Silveira et al., 2006; Oliveira and Machado, 2006), lysosomal APtypically hydrolyzes a broad range of substrates. For instance,in vitro assays have shown that egg AP from the kissing bugRhodnius prolixus (rpAP) dephosphorylate inorganic polyphosphate(PolyP), which are polymers of phosphate residues that inhibit anegg aspartic protease in R. prolixus (Gomes et al., 2010). Curiously,rpAP are initially stored in small vesicles in separation from themain population of yolk granule – a pattern also observed amongother invertebrate models (Ribolla et al., 2001) – and depend onCa2+-mediated fusion to be transferred into yolk granules (Ramoset al., 2007). A general model suggests that, upon fusion, rpAPhydrolyzes yolk granule PolyP, liberating aspartic protease activity,which in turn triggers yolk mobilization (Gomes et al., 2010).

In the present report, we analyzed the presence and physiolog-ical function of an AP found in the eggs of the velvet bean caterpil-lar Anticarsia gemmatalis (Hübner) – the major insect soybean pestin the Americas (Kogan and Turnipseed, 1987). Despite its econom-ical importance, little is known about the general biology of Anti-carsia and there are no published aspects of its reproductive andembryonic biology. Here, we characterized an acid phosphatasemainly present in a population of small vesicles inside eggs of A.gemmatalis (agAP). Inhibitor profile suggests it is a typical lyso-somal acid phosphatase; also able to dephosphorylate phosphoty-rosine and short chain PolyP. We also detected significant PolyPstorage inside the yolk granules of Anticarsia eggs, and evidencedthe inhibition profile of an egg cysteine protease by PolyP. To-gether, our data suggest that agAP is involved in yolk mobilizationby hydrolysis of both yolk proteins and PolyP during animaldevelopment.

2. Materials and methods

2.1. Chemicals

Aprotinin, ammonium molybdate, bovine serum albumin, caf-feine, levamisole, tetramisole, sodium orthovanadate, E-64, adeno-sine triphosphate (ATP), inorganic pyrophosphate (PPi),pentasodium tripolyphosphate (PolyP-3), sodium phosphate glass:type 25 (PolyP-25) and type >75 (PolyP-75), b-glycerophosphate,p-nitrophenyl phosphate (pNPP), PMSF, Pepstatin A, phosphotyro-sine, phosphothreonine and phosphoserine were all purchasedfrom Sigma Chemical Company (St. Louis, MO, USA). Dithiothreitol(DTT) was purchased from Calbiochem-Novabiochem (La Jolla, CA,USA). Ultrafree-MC centrifugal filter unit was purchased from Mil-lipore Co. (Billerica, MA, USA). Molecular mass standards were pur-chased from Promega Co. (Madison, WI, USA). SuperSignal WestPico Chemiluminescent Substrate was purchased from Pierce–Thermo Fisher Scientific (Rockford, IL, USA). Mouse monoclonalanti-phosphotyrosine PY-99 and goat anti-mouse IgG-HorseradishPeroxidase were purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA). All other chemicals were of analytical grade.

2.2. Insects, eggs and protein estimation

A colony of A. gemmatalis was established from eggs obtainedfrom Embrapa Soja, Londrina, PR, Brazil. This colony was main-tained under controlled conditions (25 ± 3 �C, 70 ± 10% relativehumidity and 14:10 (L:D) photoperiod) and fed on the artificialdiet as described by Hoffmann-Campo et al. (1985). Eggs were col-lected either daily (up to 24 h after oviposition) or freshly (up to1 h after oviposition), depending on experimental needs.

2.3. Determination of phosphatase activity

Phosphatase activity was colorimetrically assayed by measur-ing after the release of p-nitrophenol (pNP) from pNPP hydrolysisas described elsewhere (Oliveira and Machado, 2006). Briefly, reac-tions were performed at 37 �C by adding either egg extract or agAP(0.24–0.32 lg of protein) in a reaction medium (10 mM DTT,10 mM EDTA, 4 mM pNPP, 0.1 M sodium acetate buffer pH 4.0 or5.5). After 60 min, reactions were stopped by the addition of 1 NNaOH; release of pNP was measured with a microplate reader(Thermomax, Molecular Devices) as a function of absorbance at405 nm.

2.4. Enrichment of acid phosphatase

A pool of 24 h-old-eggs was collected and homogenized in buf-fer A (10 mM DTT, 10 mM EDTA, 0.1 M sodium acetate buffer pH5.5) followed by 3 washing steps (centrifugation at 20,000g,10 min, 4� C). After concentration in a Millipore Ultrafree-MC-30centrifugal filter unit (1400g, 4 h, 4 �C), samples were applied toa Shimadzu HPLC coupled Superose 6HR gel filtration column pre-viously equilibrated with buffer B (0.15 M NaCl, 0.1 M sodium ace-tate buffer pH 5.5). Elution was performed in buffer B for 60 minusing a flow rate of 0.5 mL/min; protein in collected fractionswas estimated by milliabsorbance (mAbs) detected at 280 nm.Fractions with higher pNPPase activity were pooled, labeled agAP,and concentrated with a SpeedVac machine (Thermo Savant).

2.5. Determination of acid phosphatase activity againstphosphorylated substrates

Potential biological substrates were evaluated by adding 7 lLagAP (0.24–0.32 lg) in a reaction medium (10 mM DTT, 10 mMEDTA, 0.1 M sodium acetate pH 4.0) containing several phosphor-ylated substrates at a final concentration of 4 mM of Pi residues.After 60 min of reaction at 37 �C, release of Pi was colorimetricallymeasured as previously described (Fiske and Subbarow, 1925).

2.6. Cytochemical localization of acid phosphatase activity

Yolk granule suspensions were obtained by gently rupturing of24-h-old eggs in 0.4 M sucrose, 10 mM Hepes pH 7.2. Sampleswere washed (1 min, 10,000g, room temperature centrifugation),and fixed at room temperature for 30 min in 0.4 M sucrose,10 mM Hepes pH 7.2, 0.5% glutaraldehyde, 0.5% formaldehyde.After washing in 0.4 M sucrose, 10 mM Hepes pH 7.2, sampleswere resuspended and incubated for 1 h at 37 �C in acid phospha-tase reaction medium (1 mM sodium b-glycerophosphate, 2 mMCeCl3, 0.1 M sucrose, 0.1 M sodium acetate pH 4.0) (Hulstaertet al., 1983). Controls were carried out without the substrate orin the presence of 10 mM Na+ K+ tartrate. Samples were washedtwice in 0.1 M sodium acetate pH 4.0, once in 0.1 M sodium caco-dylate pH 7.2 and posteriorly fixed for 2 h at room temperature by2.5% glutaraldehyde, 4% formaldehyde, 0.1 M sodium cacodylatepH 7.2. Samples were then washed in cacodylate buffer, post-fixedin 1% OsO4 for 1 h at room temperature, dehydrated in ethanol ser-ies and embedded in a Polybed 812 resin. Ultrathin sections wereobserved in a JEOL 1200 EX transmission electron microscope,operating at 80 kV. For X-ray microanalysis, X-rays were collectedfor 150 s using a Si (Li) detector with a Norvar window in a0–10 keV energy range with a resolution of 10 eV/channel.Analysis was performed using a Noran Voyager III analyzer.

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2.7. Western blotting against phosphotyrosine residues

Freshly-laid eggs were homogenized in 20 mM Hepes pH 7.5and centrifuged twice for 10 min at 18,000g at 4 �C. Supernatantswere centrifuged at 10,000g for 2 h at 4 �C in a Millipore Ultra-free-MC-5 centrifugal filter unit and retained samples were resus-pended in 20 mM Hepes pH 7.5, and labeled ‘‘yolk protein’’.Following, 40 lg of ‘‘yolk protein’’ was incubated at 37 �C in a reac-tion medium (P8340 protease inhibitor cocktail, 2.5 mM DTT,2.5 mM EDTA, 25 mM sodium acetate pH 4.0) containing 0.32 lgagAP protein. When specified, 10 mM Na+ K+ tartrate was used asagAP inhibitor. Following, 12.5% SDS–PAGE was performed andthe proteins were transferred to a nitrocellulose membrane thatwas blocked for 90 min with blocking buffer (0.05% TBS-Tween20, 3% BSA). The membrane was then incubated overnight in block-ing buffer containing 1:1000 PY-99 (raised against phosphotyro-sine). Membrane was washed and revealed using a SuperSignalWest Pico (Pierce) after incubation of 1:2000 anti-mouse peroxi-dase-conjugated IgG. All incubation steps were performed at roomtemperature.

2.8. Polyphosphate detection by fluorescence microscopy

PolyP detection was performed as described (Gomes et al.,2008). Briefly, yolk granule suspensions were obtained by gentlyrupturing of 24-h-old eggs in 20 mM Hepes pH 7.2. Samples werewashed (1 min, 10,000 g, room temperature) and incubated in20 mM Hepes pH 7.2, 50 lg/mL DAPI for 20 min at room tempera-ture. Following, samples were mounted on glass slides and ob-served using a DAPI-PolyP optimized filter set (excitation:350 nm, emission: >500 nm) and observed in an upright epifluo-rescence microscope (Axioplan, Carl Zeiss).

Fig. 1. Characterization of yolk proteolysis and acid phosphatase activity during embryoand immediately frozen at different intervals of time; extracts were prepared at neutralNumbers on the left indicate molecular weight standards (lane P). Lanes 0 to 62: Degrafigure). (B) Pools of eggs were collected at different intervals of time after oviposition andat 37 �C for 60 min with 0.05 lg/lL of protein. Each point represented the mean ± S.E.,collected and prepared as described before. The homogenate was concentrated and the rewere tested for pNPPase activity (black line) and optical density at 280 nm is shown as

2.9. Determination of acid phosphatase activity against endogenousPolyP

Endogenous PolyP levels were measured as described (Ruizet al., 2001). Briefly, 24-h-old eggs were homogenized in distilledwater at 4 �C. For long-chain PolyP, egg homogenate was addedto lysis buffer (6 M Guanidine Thiocyanate, 50 mM Tris–HCl pH7.0) and powdered glass was used to bind PolyP. After DNase andRNase treatment, bound PolyP was eluted at 95 �C in 50 mMTris–HCl pH 8.0. For short chain PolyP, egg homogenate was addedto 0.5 N HClO4 followed by neutralization using KOH and KHCO3.Both long chain and short chain PolyP levels were determined asthe amount of Pi released upon treatment with an excess of recom-binant exopolyphosphatase from Saccharomyces cerevisiae (scPPX).Aliquots of short chain or long chain polyP were incubated for15 min at 37 �C in reaction medium (60 mM Tris–HCl pH 7.5,6 mM MgCl2) containing purified scPPX and the malachite greenassay was used for Pi quantification (Gomes et al., 2010).

Following, the ability of agAP to hydrolyze endogenous PolyPwas evaluated by addition of 1.5 lg of agAP in a reaction medium(10 mM DTT, 10 mM EDTA, 0.1 M sodium acetate buffer pH 4.0)containing either short chain or long chain PolyP obtained fromA. gemmatalis eggs. Incubation was held for 60 min at 37 �C. Afterthat, PolyP levels were determined by the scPPX assay.

2.10. Profile of yolk proteins during embryogenesis and determinationof protease activity

Freshly-laid eggs were collected and left to develop for differenttimes before homogenization in 20 mM Hepes pH 7.5supplemented with P8340 protease inhibitor cocktail. After

genesis. (A) Proteolysis of yolk proteins during embryogenesis. Eggs were collectedpH with protease inhibitors. Proteins were observed after SDS–PAGE as described.

dation of yolk at different times (in hours) after egg laying (indicated above in thewere prepared in sodium acetate buffer 0.1 M pH 4.0. Samples were tested for pNPPperformed in triplicate. (C) AgAP chromatography elution profile. 24-h eggs weretained sample was chromatographed in a Superose 6HR gel filtration unit. Fractionsa gray line (mAbs at 280 nm).

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centrifugation (10,000g, 10 min, 4 �C), supernatants were submit-ted to 12.5% SDS–PAGE and stained with Coomassie blue.

Egg homogenates were prepared using 24- and 48-h-old egg ex-tracts in 50 mM sodium acetate pH 5.0 followed by two washingsteps (10,000g, 10 min, 4 �C). Protease assays were performed atroom temperature in a reaction medium (0.2 M NaCl, 5 mM EDTA,2.5 mM DTT, sodium acetate buffer 50 mM pH 5.0) containing5 lM z-Phe-Arg-AMC. Steady-state velocities were obtained by lin-ear regression of the hydrolysis curve (Lima et al., 2001) as fol-lowed in an Fmax fluorescence microplate reader (molecularDevices) using 380/440 nm as excitation/emission wavelengthsfor 30 min. When expressed, the influence of PolyP was deter-mined by the addition of PolyP-3, -25 or -75 into the reactionmedium.

Table 1.Influence of different inhibitors on egg-derived acid phosphatase of A. gemmatalis.

Inhibitor Specific activitya Relativeb

Control (no inhibitor) 724.80 ± 1.46 100Levamisole 10 mM 813.60 ± 19.38 112.2Tetramisole 10 mM 997.90 ± 115.30 137.7NaF 10 mM 47.23 ± 0.05 6.5NaF 1 mM 677.70 ± 17.22 93.5Na+/K+ Tartrate 10 mM 0.00 ± 0.00 0Na+/K+ Tartrate 1 mM 56.78 ± 3.13 7.8Ammonium Molybdate 1 mM 0.00 ± 0.00 0Ammonium Molybdate 0.1 mM 157.70 ± 4.80 21.8Ammonium Molybdate 0.05 mM 280.70 ± 14.41 38.7Pi 10 mM 157.70 ± 0.43 21.8Pi 1 mM 601.60 ± 10.85 83.0Caffeine 5 mM 819.80 ± 10.69 113.1Caffeine 0.5 mM 758.00 ± 39.35 104.6Control (no DTT or EDTA) 600.10 ± 4.10 100CuSO4 1 mM 432.10 ± 1.08 72.0CuSO4 0.1 mM 584.10 ± 23.91 97.3

Fig. 2. Phosphotyrosine as substrate for AgAP. (A) A fresh egg homogenate obtainedat neutral pH in the presence of a cocktail protease inhibitor was incubated at acidicpH with AgAP for different intervals of time at 37 �C. The control group wasincubated in the presence of phosphatase inhibitor (10 mM Na+ K+ tartrate). Afterincubations, samples were submitted to Western blotting against anti-phosphoty-rosine antibodies. Molecular weight standards (kDa) are represented on the left sideand the arrow on the right indicates a dephosphorylated polypeptide aftertreatment. A 50 kDa protein stained with Ponceau Red (below the western blottingdata) was showed to normalize loading of samples (arrowhead). (B) The differentphosphoaminoacids (phosphotyrosine, phosphoserine and phosphothreonine) wereincubated with AgAP (0.24–0.32 lg), at final concentration of 4 mM. Results areexpressed as nmol Pi �mg�1 ptn �min�1. Each bar represents mean ± S.E. of twoindependent experiments, performed in triplicates.

3. Results

3.1. Yolk proteolysis and acid phosphatase during A. gemmatalisembryogenesis

As yolk granule hydrolases are strongly associated with yolkmobilization, we wondered whether yolk mobilization was corre-lated with hydrolase activity during Anticarsia development. Fromhomogenates prepared from eggs at different times after oviposi-tion, we observed a smooth mobilization of major storage proteinsaround 80, 30, and 10 kDa (Fig. 1A). The first evidences of yolkmobilization were observed 20–40 h after oviposition – the sameperiod where an increase of acid phosphatase activity was also de-tected in egg extracts (Fig. 1B) – suggesting a correlation betweenthese two events.

In order to further characterize the acid phosphatase present inthe eggs of A. gemmatalis, we proceeded to obtain an enriched frac-tion of acid phosphatases from 24-h-old egg homogenates by HPLCchromatography. After gel filtration, a major peak with PNPPaseactivity with an approximate molecular mass of 45 kDa was en-riched by a factor of forty times (Fig. 1C). Samples eluting adja-cently to this major fraction were pooled, labeled as agAP, andfurther characterized. AgAP maximum activity was observed atpH 4.0 at 37 �C (data not shown) resulting in a km and Vmax of0.32 mM and 6.13 nM pNP/s, respectively. Tyrosine dephosphoryl-ation of a major 80-kDa yolk protein was observed in vitro, sug-gesting that agAP targets yolk proteins during embryodevelopment (Fig. 2A). Under the improved conditions, agAP wasshown to preferentially hydrolyze phosphotyrosine (Ptyr)(300.5 nmols Pi �mg ptn�1 �min�1) as compared against phos-phoserine and phosphothreonine (20. 00 and 0.901 nmols Pi �mgptn�1 �min�1, respectively) (Fig. 2B).

Like other egg phosphatases (Fialho et al., 2002), agAP wasstrongly modulated by classical inhibitors of lysosomal acid phos-phatases such as Na+/K+ tartrate and NaF (Table 1). The assayedinhibitors are used to classify these enzymes, at millimolar andsubmillimolar concentration levels. Ammonium molybdate and so-dium orthovanadate (modulators of tyrosine phosphatases) alsoinhibited agAP. Other phosphatase modulators such as CuSO4 orPi were moderately effective against agAP activity. The alkalinephosphatase inhibitors (levamisole and tetramisole) or phospho-esterase inhibitor (caffeine) had no effect on agAP, confirmingthe acidic nature of agAP.

Vanadate 0.1 mM 27.04 ± 1.35 4.5Vanadate 0.05 mM 50.68 ± 9.12 8.4

AgAP was assayed against pNPP in the presence of different phosphohydrolaseinhibitors. Control represents activity without inhibitor. Control without DTT orEDTA was used at the assays with CuSO4 or vanadate. Each point indicates themean ± S.E. of three independent experiments, performed in duplicate.

a Means nmol pNP �mg ptn�1 �min�1.b Means relative activity (percent) to the control.

3.2. Cytochemical localization of agAP

In order to evaluate the subcellular compartmentalization ofagAP in yolk granules suspensions, the b-glycerophosphate-CeCl3

assays for cytochemical detection of acid phosphatase activitywas used (Hulstaert et al., 1983). After 24 h of oviposition, acid

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phosphatase activity was mainly restricted to a smaller populationof vesicles sized 200–600 nm separated from larger yolk granules(Fig. 3A–D). Tracing of cerium by X-ray microanalysis was usedto confirm CePO4 precipitation by endogenous agAP (Fig. 3E). Dur-ing the assay, acid phosphatase is stained electron-dense by thedeposition of insoluble CePO4 from usage of CeCl3 as a releasedphosphate capture agent. Negative controls were performed byavoiding addition of phosphatase substrate to the reaction med-ium, and no precipitates were found under those conditions (datanot shown).

3.3. AgAP as an egg PolyPase and the presence of PolyP in the eggs of A.gemmatalis

PolyP is an ubiquitous biological polymer that plays a role in theregulation of several physiological processes. While specific PolyP-ases have been described from a few eukaryotic models (Lichkoet al., 2006; Tammenkoski et al., 2008), reports suggested that gen-eral phosphatases could also hydrolyze PolyP, thus regulating cel-lular levels of the polymer. Although to a smaller extent thanphosphotyrosine, agAP was able to hydrolyze PolyP-3, suggestingthat under favorable conditions, agAP is a short chain PolyPase

Fig. 3. Cytochemical localization of AgAP. The yolk organelles of eggs were suspendesubstrate. (A) Control (with no substrate); (B–D): cytochemical localization of AgAP inWhite arrows: small labeled vesicles; arrowhead: non-labeled vesicle adjacent to an elemicroanalysis of one small labeled vesicle. Arrows are indicating the presence of phosaccumulation).

(Fig. 4A). No PolyPase activity for PolyP-75 was observed, andb-glycerophosphate, PPi, and ATP were only hydrolyzed at tracelevels. Accordingly, in vitro assays suggest that agAP was able tohydrolyze endogenous short chain PolyP, but endogenous longchain levels remained unaltered (Fig. 4B).

We then tested whether PolyP stores could be detected in yolkgranules suspensions by DAPI-PolyP assay, as PolyP is able to shiftDAPI fluorescence emission to a higher wavelength (525–550 nm)that can be detected after blocking the typical blue fluorescence(450 nm) from stained nuclei. Similar to acid phosphatase activity,PolyP signals were mainly observed in small vesicles (Fig. 4D). Nev-ertheless, weaker signals were also frequently observed in largeryolk granules.

3.4. Influence of PolyP towards yolk mobilization

Yolk mobilization of insect eggs is performed by activation ofeither cysteine or aspartic protease during embryo development.In egg extracts of Anticarsia, no aspartic protease activity was de-tected 24- or 48-h after oviposition (data not shown). On the otherhand, hydrolysis of the fluorogenic substrate z-phe-arg-AMC wascompletely abolished by the cysteine protease inhibitor E-64, sug-

d and assayed to detect phosphatase activity in situ using b-glycerophosphate assmall vesicles inside egg ooplasm. Bars: 500 nm (except D, which has bars-1 lm).ctron dense precipitate (black arrow). m: mitochondria; YG: yolk granule. (E) X-rayphorous and cerium, in addition to a higher count of sulfur (indicative of protein

Fig. 4. Presence of polyphosphate yolk granules and AgAP as polyPase. (A) Determination of substrate preference by AgAP. Different phosphomolecules were tested assubstrate for AgAP after incubations at 37 �C, as described before. All substrates were at final concentrations of 4 mM, except for polyP-3 (1.3 mM), PPi (2 mM) and polyP-75(52 lM). Results are expressed as nmol Pi �mg ptn�1 �min�1. Each bar represents mean ± S.E. of two independent experiments, performed in triplicates. (B) polyPaseactivity was measured adding endogenous SC and LC polyP to the reaction medium of AgAP containing an excess of the enzyme (EXP). No AgAP was added to the controlreaction (CT). Assays were conducted at 37 �C for 60 min after which PolyP levels were determined by treatment with scPPX as above. Control was 1.00 as reference.Average ± SEM is presented and asterisk on (B) designates significant difference (P < 0.05) as detected by One Way ANOVA using Tukey as post-test (n = 3). To localizepolyphosphate, a suspension of yolk granules (20 mM Hepes pH 7.2) was prepared from 24-h eggs. Image shows the phase contrast micrograph (C) corresponding to thefluorescence image of DAPI labeling (D). Bars: 10 lm.

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gesting that a cysteine protease is the main active acid protease atthis development stage (Fig. 5A).

It has been suggested that inhibition of an aspartic-like proteaseby PolyP is a control mechanism hindering yolk mobilization dur-ing the early development of R. prolixus. In that sense, activation ofacid phosphatases would be a triggering mechanism, as yolk mobi-lization would follow hydrolysis of PolyP and derepression of theaspartic protease. As there is interplay between acid yolk hydro-lases (proteases and phosphatases) as described in several insectmodels (Purcell et al., 1988; Yamamoto and Takahashi, 1993;Oliveira et al., 2008), we tested whether a similar mechanism could

Fig. 5. Determination of acid protease activity and PolyP-3 influence on in vitro proteasefluorogenic substrate z-Phe-Arg-AMC at 25 �C, pH 5.0 and substrate hydrolysis was monit(30 lM) was added to the same reaction. The product formation was expressed as specificactivity (± SEM) of seven different samples. (B) 24-h egg extract was prepared at acidicdifferent concentrations of polyP-3 (black circles); polyP-25 (black squares) and polyP-7polyP), which was 1.00 as reference. Each point represents the average activity (±SEM)

be observed in Anticarsia. Accordingly, 10 lM of PolyP-3 abolishedcysteine protease activity of the 24-h eggs, (Fig. 5B). Other polymersizes did not show significant modulation at the testedconcentrations.

4. Discussion

Velvet bean caterpillar A. gemmatalis infestations in soybeancrops are usually controlled with insecticides, usually combinedwith the application of nucleopolyhedrovirus (Negreiro et al.,

activity. (A) Samples were prepared from 24-h and 48-h eggs and assayed with theored continuously for 3 min. Aprotinin (10 lg/ mL) was added and after 3 min, E-64activity (AMC concentration � lg ptn�1 � s�1). Each column represents the average

pH (5.0) and protease activity was assayed with z-Phe-Arg-AMC in the presence of5 (black triangles). Relative activity was determined compared to control (withoutof four different samples.

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2004; Guedes et al., 2012). Nevertheless, Anticarsia defoliationkeeps negatively impacting annual crops production, indicatingthe need for improved control techniques. Also, resistant popula-tions were reported among several pest insects and appearanceof resistance has been modeled for A. gemmatalis (Negreiro et al.,2004). In searching for specific control strategies, the insect repro-ductive and embryonic physiology is regarded as potential sourcefor new control methods. However, there are few studies on gen-eral Anticarsia biology, thus most strategies proposed are basedon information derived from other lepidopteran models.

In the present report, we have focused on yolk mobilization inA. gemmatalis eggs and a related AP (agAP). In insect eggs, AP arestored as yolk granule hydrolases and have been described in sev-eral models, such as the house fly Musca domestica (Ribolla et al.,1993), R. prolixus (Fialho et al., 2002), the cockroach Periplanetaamericana (Oliveira et al., 2008), and the tick Rhipicephalus (Booph-ilus) microplus (Silveira et al., 2006). We observed that agAP pre-sented an inhibition profile typical of egg AP, and a majorenzymatic activity with phosphotyrosine; dephosphorylation oftyrosine phosphorylated yolk protein by agAP was confirmed byWestern blot analysis.

We also analyzed compartmentalization of agAP before theonset of yolk mobilization, where, agAP activity is initially con-centrated in small vesicles separated from yolk granules subpop-ulations. Similar organization was observed in P. americanaovaries (Oliveira and Machado, 2006), and embryos of crusta-ceans (Perona and Vallejo, 1985) and amphibians (Komazakiand Hiruma, 1999). In R. prolixus, rpAP activity is posteriorlytransferred into larger yolk granules by Ca2+-mediated fusiontaking place during early embryogenesis and preceding yolkmobilization (Ramos et al., 2007). It has been suggested thatthe fusion of subpopulations of vesicles allows the assembly ofa yolk mobilization system at the yolk granules, composed ofacid hydrolases, proton pumps and yolk proteins that progres-sively nurture embryonic anabolism. In that sense, compartmen-talization of AP into yolk granules by vesicle fusion couldrepresent one regulation step of a general model for yolk mobi-lization during embryogenesis of invertebrates (Oliveira et al.,2008; Motta et al., 2009; Gomes et al., 2010). As future perspec-tives, it would be interesting to evaluate to what extent agAPactivity is transported into larger yolk granules at the onset ofyolk mobilization or by Ca2+-induced events.

While removal of tyrosine phosphate by AP was observed in P.americana and R. (B.) microplus eggs (Oliveira et al., 2008; Silveiraet al., 2006), the physiological range of egg phosphatases sub-strates had remained poorly explored. Lysosomal APs are hydro-lases with a broad range of substrates, thus – although yolkgranules compartmentalization would suggest preferential hydro-lysis of yolk protein – other targets should be investigated. In fact,in R. prolixus eggs it has been reported that rpAP fails to efficientlyhydrolyze yolk proteins (Fialho et al., 2005), but was shown to cat-alyze hydrolysis of PolyP in vitro (Gomes et al., 2010). In the pres-ent study, agAP efficiently released phosphate fromphosphotyrosine amino acids and yolk proteins. Also, stronghydrolysis of short chain PolyP was observed with either endoge-nous or exogenous PolyP. Previously, we have suggested that PolyPwas a physiological inhibitor of an aspartic proteinase of YG of R.prolixus, and that mobilization of yolk proteins would depend ofPolyP degradation mediated by rpAP (Gomes et al., 2010). Curi-ously, several authors have evidenced a temporal link betweenphosphatase and protease activation that has remained poorly ex-plained. Here, we observed that PolyP-3 might play a role in theinhibition of a cysteine protease activity on Anticarsia egg extracts.Based on the profile of hydrolysis of exogenous substrates, wedetermined that a cysteine protease could be the main yolk gran-ule acid protease. In that sense, its inhibition by short chain PolyP

can be accounted for a regulation mechanism similar to what hasbeen described in Rhodnius.

Acknowledgments

We express our gratitude to Hatisaburo Masuda and Pedro Lag-erblad Oliveira for kindly providing laboratory supplies and facili-ties and to Heloísa S. L. Coelho for excellent technical assistance.We also acknowledge Flavio Moscardi for kindly providing the in-sects and Eduardo Fox for proof reading and scientific advice. Thiswork was supported by Grants from Conselho Nacional de Desen-volvimento Científico e Tecnológico (CNPq); Fundação de Amparo àPesquisa Carlos Chagas Filho (FAPERJ); Programa de apoio aoDesenvolvimento Científico e Tecnológico (PADCT), Centro de pes-quisa da Petrobrás (CENPES), Instituto Nacional de EntomologiaMolecular (INCT).

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