Micron 41 (2010) 123–129

Circulating hemocytes from larvae of Melipona scutellaris (Hymenoptera,Apidae, Meliponini): Cell types and their role in phagocytosis

Isabel Marques Rodrigues Amaral a,*, Joao Felipe Moreira Neto a, Gustavo Borges Pereira b,Mariani Borges Franco c, Marcelo Emılio Beletti c, Warwick Estevam Kerr a,Ana Maria Bonetti a, Carlos Ueira-Vieira a

a Laboratorio de Genetica, Instituto de Genetica e Bioquımica, Universidade Federal de Uberlandia, Campus Umuarama, Sala 2E33, Uberlandia-MG, CEP: 3840-902, Brazilb Departamento de Biologia Celular e Molecular e Bioagentes Patogenicos, Faculdade de Medicina de Ribeirao Preto, USP, Ribeirao Preto, SP, Brazilc Instituto de Ciencias Biomedicas, Laboratorio de Histologia, Universidade Federal de Uberlandia-MG, Brazil

A R T I C L E I N F O

Article history:

Received 29 July 2009

Received in revised form 1 October 2009

Accepted 3 October 2009

Keywords:

Stingless bee

Hemocytes

Phagocytosis

Cellular immunity

A B S T R A C T

Infection in insects stimulates a complex defensive response. Recognition of pathogens may be

accomplished by plasma or hemocyte proteins that bind specifically to bacterial or fungal

polysaccharides. Several morphologically distinct hemocyte cell types cooperate in the immune

response. Hemocytes attach to invading organisms and then isolate them by phagocytosis, by trapping

them in hemocyte aggregates called nodules, or by forming an organized multicellular capsule around

large parasites. In the current investigation the cellular in the hemolymph third instar larvae of M.

scutellaris has been characterized by means of light microscopy analysis and phagocytosis assays were

performed in vivo by injection of 0.5 mm fluorescence beads in order to identify the hemocyte types

involved in phagocytosis. Four morphotypes of circulating hemocytes were found in 3rd instar larvae:

prohemocytes, plasmatocytes, granulocytes and oenocytoids. The results presented plasmatocytes and

granulocytes involved in phagocytic response of foreign particles in 3rd instar larvae of M. scutellaris.

� 2009 Elsevier Ltd. All rights reserved.

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1. Introduction

Pathogens represent a real and present danger. Although bothplants and animals are protected by physical barriers that block theentry of many potential pathogens, they have to be able torecognize when microorganisms have breached these barriers, andto respond rapidly to infection by deploying a range of defensivestrategies. These functions are assumed by the innate immunesystem, which identifies, contains and kills invading pathogens(Kavanagh and Reeves, 2004).

Invertebrates avoid the infection establishment through anefficient immune response. This response consists in cellularreactions such as phagocytosis, encapsulation and nodule forma-tion (Lavine and Strand, 2002) and, humoral reactions includingserine proteases cascades participating in melanization andcoagulation, and production of killing factors such as antimicrobialpeptides (AMPs) and reactive oxygen and nitrogen species(Hultmark, 2003; Cherry and Silverman, 2006; Wang andLigoxygakis, 2006).

* Corresponding author. Tel.: +55 34 9181 4670; fax: +55 34 3218 2203x24.

E-mail address: Isabel_mra@yahoo.com.br (I.M.R. Amaral).

0968-4328/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2009.10.003

The cellular response is mediated by hemocytes, which areversatile cells whose numbers and types are species specific anddiffer with the insect’s age (Gupta, 1979; Gotz and Boman, 1985).The several types of insect hemocytes are traditionally identifiedusing morphological, histochemical and functional characteristics(Gupta, 1985; Brehelin and Zachary, 1986; Kurihara et al., 1992).The most common types of insect hemocytes reported in theliterature are prohemocytes, granulocytes, plasmatocytes, spher-ulocytes, and oenocytoids. Hemocytes recognize a variety offoreign targets either by direct interaction of their surfacereceptors with molecules from the invading organism, or,indirectly, by recognition of humoral receptors. Inter- andintracellular signaling events must then coordinate effectorsresponses such as phagocytosis or encapsulation (Lavine andStrand, 2002).

The meliponiculture, stingless bee’s rational creation, is one ofthe essential economic factors in some northern and northeasterncommunities in Brazil (Campos, 2003); moreover, stingless beesare important pollinators in tropical regions (Kerr et al., 1996;Heard, 1999) and, along with other pollinators, of great concern inconservation (Kearns et al., 1998). Few studies on stingless beehemocytes are found in the literature. Cruz-Landim and Cunha(1971) evaluated the hemocytes in immature stages of Melipona

Fig. 1. Scheme representing the larval development and the five phases of the Melipona scutellaris 3rd larvae instars.

I.M.R. Amaral et al. / Micron 41 (2010) 123–129124

quadrifasciata anthidioides and also made histological andhistochemical observations of hemocytes in M. quadrifasciata

worker bees (Cunha and Cruz-Landim, 1972). Cruz-Landim(1996b) in another study analyzed the ultrastructure of thehemocytes present in the dorsal thoracic region of larvae and pre-pupae of M. quadrifasciata found in this area only plasmatocytes,granulocytes, and adipohemocytes.

This study focuses on cellular immune response in the stinglessbee Melipona scutellaris. The goals of the presented work is tocharacterize the cellular population in the hemolymph of Melipona

scutellaris larvae by light and phase-contrast microscopy analysisand to identify the hemocytes involved in phagocytosis byConfocal scanning. Our results shows that four well definedhemocyte types are present in the hemolymph of M. scutellaris

larvae. Fluorescent beads were found only in Plasmatocytes andgranulocytes after phagocytosis assay suggesting that cells may beinvolved in phagocytosis.

2. Materials and methods

2.1. Stingless bee melipona scutellaris

The 3rd larval instar was separated and analyzed according tofood amount and consistency, and the presence or absence of fecesin the brood combs (Fig. 1).

2.2. Hemolymph collection

The hemolymph was collected from larvae of different phases:larvae 3-1 (L3-1), larvae 3-2 (L3-2), larvae 3-3 (L3-3), predefecatinglarvae (LPD) and defecating larvae (LD) by puncturing the softcuticle with fine forceps sterilized in 70% ethanol. Two microlitersof hemolymph were collected from the resulting bubble ofhemolymph, transferred to 0.5 mL microcentrifuge tube with18 mL anticoagulant buffer (0.098 M NaOH, 0.186 M NaCl, 0.017 MEDTA and 0.041 M citric acid, pH 4.5) and used for total hemocytecount. For morphological characterization by light microscopy,5.0 mL of hemolymph was collected.

2.3. Total hemocyte count

The hemocytes were counted under a light microscope at 40�magnification using 10 mL of the diluted hemolymph solution to animproved Neubauer hemocytometer (Fisher Scientific).

2.4. Differential hemocyte count and cells characterization by phase-

contrast microscopy

Briefly, for the differential hemocyte count the hemolymph wasmixed 1:1 with phosphate-buffered saline (PBS) on a glass slideand dried in room temperature for 20 min. During this time thehemocytes adhered to the glass. Cells were then fixed in methanol

for 10 min. After natural air-drying of the fixative, hemocytes werestained with Giemsa-Rosenfeld for 15 min and slides were rapidlywashed with bidistilled water. For differential counting weanalyzed one hundred cells per slide.

In assays involving cells morphological characterization byphase-contrast 10 mL of hemolymph were placed in cell cultureplates, 24-well plate (NuncTM) containing 990 mL of Grace’s InsectMedium (Gibco). The cells were identified by light microscope orphase-contrast microscope at 40�magnification and images wereacquired with a photo camera.

2.5. Phagocytosis assays

Fluorescent red carboxylated latex beads (0.5 mm of diameter-Sigma) were used according to manufacturer’s instructions, andcovalently bonded to BSA (bovine serum albumin). Before theconjugation of proteins, beads were washed twice in 0.1 M MES (2-N-morpholino-ethanesulfonic acid) buffer, pH 4.7 (isoeletric pointBSA). They were then resuspended in 0.1 M MES, pH 4.7 and weremixed with the BSA 5% and incubated overnight. They were thenwashed five times with 0.1 M MES, pH 4.7 to remove BSAuncoupled and resuspended in 1 mL of PBS.

For slides production were collect 10 mL of 3rd instar larvalhemolymph (L3-3 phase) and mixed with 100 mL of anticoagulantbuffer. Cells were centrifuged at 3000 rpm for 5 min andresuspended in 100 mL of Grace’s Insect Medium (Gibco) with0.5 mL of bead and incubated at room temperature for 30 min toremove non-internalized particles from the cells. To analyze thehemocyte phagocytosis, 20 mL of cell suspension were transferredto glass slide and observed by LSM 510 META confocal microscope(ZEISS).

2.6. Statistical analysis

Statistical analyses were conducted using StatView software forWindows version 4.57 (Abacus Concepts, Inc., Copyright 1992–1996). We used analysis of variance (ANOVA) and Student’s t-testto investigate possible differences between the means of eachfactor studied.

3. Results

Bees of the genus Melipona have three larval instars inaccordance with the Dyar rule (Dyar, 1890; Dias et al., 2001). InMelipona, the 3rd larval instar is divided into five phases (Fig. 1)characterized by the food quantity, consistency and the presenceor absence of feces in the brood combs: L3-1, large quantity ofliquid food, larvae with curved body and pearly color; L3-2, slightless food with viscous consistency, the larvae continue curved withpearly color; L3-3, few food in the brood combs with solidconsistency, the larvae have pearly bright color; LPD: brood combswithout food where the defecation process has not started, the

Fig. 2. Changes in total hemocyte count (THC) (A), body weight (B), and THCg (C)

during the last larval instar of M. scutellaris. Each data point is the means � SD of at

least 10 individual. Different letters indicate significant differences between groups

(ANOVA, p < 0.01).

I.M.R. Amaral et al. / Micron 41 (2010) 123–129 125

larvae are in the form of a comma and still have bright pearl color;LD, brood combs without food and the larvae started thedefecation, have whitish color, straight with the head directedtoward top.

In this work we choose the 3rd instar of development for theinnate immune system analysis, because the larvae are incontinuous contact with the food and the microbiota present init (Gilliam et al., 1990; Cruz-Landim, 1996a).

3.1. Total hemocyte count and body weight

Total hemocyte count (THC) is a measure of the concentrationof hemocytes within the hemolymph (cells/mL). The THC betweenthe phases of M. scutellaris last larval instar not showed differencesin the hemocytes total numbers (Fig. 2A). Larval body weightshowed significant increase between phases, ranging from 40 to155.6 mg, reaching a maximum peak in LPD (155.6 mg), followedof significant reduction in LD (104 mg) (Fig. 2B) (ANOVA, p < 0.01).

Since body weight and hemolymph volume changed drama-tically during the last larval instar, an estimate of the number of allhemocytes in each 3rd larval phases (THCg) was calculated fromthe THC (cells/mL) multiplied with the bodyweight (mg) (Beetzet al., 2008). The THCg graph (Fig. 2C) shows a similar profile tobody weight, however, with two groups statistically different(ANOVA, p < 0.01). The L3-1 and L3-2 phase were significantlylower than L3-3, LPD and LD phases regarding the number ofhemocytes.

3.2. Hemocytes morphorlogical characterization

Four types of hemocytes were found and identified in thehemolymph of 38 instar larval of M. scutellaris: prohemocytes,plasmatocytes, granulocytes and oenocytoids.

3.2.1. Prohemocytes

These cells (Fig. 3A) are the smallest hemocytes encountered inthe hemolymph. After Giemsa staining, the cytoplasm displaysblue color (basophilic) and the nucleus, dark purple it is ametachromatic staining that also indicates intensive basophily.They are oval in shape (8.69 mm � 8.64 mm in diameter). The largeand centrally located nucleus nearly fills the cell so that thecytoplasm occupies a narrow area around the nucleus.

3.2.2. Plasmatocytes

The plasmatocytes (Fig. 3B) are highly polymorphic cells; theyshow rounded, oval, spindle-shaped or sometimes irregular form.Plasmatocytes are also variable in size. When spherical in shape,they present average 11.81 � 2.45 mm ranging from 10.0 to 15.0 mmin diameter. When oval, they are average 11.81 � 2.45 mm, rangingfrom 10.0 to 15.0 mm in diameter and wide average of10.68 � 1.95 mm, ranging from 6.29 to 12.25 mm. The nuclei canbe spherical or oval. After Giemsa staining, the cytoplasm displaysblue color (basophilic) and the nucleus, dark purple like found inprohemocytes. Found multiple plasmatocytes containing one or morevacuoles with unstained content (Fig. 3C, arrows). Cells withintermediate features between prohemocytes and plasmatocyteswere also observed.

3.2.3. Granulocytes

These hemocytes (Fig. 3D) are easily recognized by itsnumerous vacuoles present in the cytoplasm. Granulocytes arevariable in shape and size. They can be large or small sphericalhemocytes with size average of 19.12 � 4.11 mm (15.25–27.61 mm)in diameter or oval shape with average 19.12 � 4.11 mm (15.25–27.61 mm) in diameter and 18.09 � 3.90 (13.25–24.75 mm) in wide.The nucleus is acidophilic and generally occupies a central position.We also observed a cell with cytoplasm disruption and presence ofgranules (Fig. 3E). Possibly this effect was due to a frustratedphagocytosis or it might mean cell death by a process of fragmenta-tion. Cells with intermediate features between plasmatocytes andgranulocytes were also observed.

3.2.4. Oenocytoids

These cells (Fig. 3F) are the larger hemocytes encountered in thehemolymph. Oenocytoids can be spherical with average20.25 � 2.77 mm (ranging from 16.44 to 28.0 mm) or oval withaverage 20.25 � 2.77 mm (ranging from 16.44 to 28.0 mm) indiameter and 20.61 � 2.73 (ranging from 16.79 to 26.48 mm) inwide. And present nuclei with the same general shape of the cell. AfterGiemsa staining the oenocytoids exhibit a moderate acidophilia, andreveal a homogeneous cytoplasm containing acidophilic granulation.

We also observed in LD phase an agglomeration of hemocytes(Fig. 3F) as described previously by Gregoire and Florkin (1950)and Gregoire and Goffinet (1979). All hemocyte types presentedsuggestive features of mitotic process (Fig. 3H–L), mainly in LDphase, which is the end of 3rd instar larvae of M. scutellaris,followed by metamorphosis to the pupal stage.

3.3. Differential hemocyte count and cells characterization in vivo by

phase-contrast microscopy

The differential hemocyte count (DHC) represents the count ofindividual hemocyte types within the total hemocyte population.By means of the DHC (Fig. 4) it was verified that the number of

Fig. 3. Morphology of different types of M. scutellaris hemocytes. (A) Prohemocyte, (B and C) plasmatocytes, (D) granulocyte, (E) cytoplasm disruption and presence of

granules, (F) oenocytoid, (G) agglomeration of hemocytes, predominantly in LD. (H) granulocyte on interphase show heterochromatinic regions. All hemocyte types

presenting features suggestive of mitotic figures: (I) metaphase, (J) anaphase, (K) telophase and (L) cytokinesis. Arrows in 3C indicate vacuoles with unstained content.

Fig. 4. Differential hemocyte counts (DHC) of 3rd instar larvae of Melipona scutellaris. (Ph) Prohemocytes, (Pl) plasmatocytes, (Gr) granulocytes and (Oe) oenocytoids. Different

letters indicate significant differences between groups (ANOVA, p < 0.05).

I.M.R. Amaral et al. / Micron 41 (2010) 123–129126

Fig. 5. Hemocytes morphology in vivo by phase-contrast microscopy. (A) Prohemocytes, (B) plasmatocytes, (C) prohemocytes, plasmatocytes and prohemocytes

intermediary, (D) granulocytes, (E) oenocytoids, (F) all hemocytes population: Ph: Prohemocytes. Ph-I: prohemocytes intermediary. PI: plasmatocytes. Gr: granulocytes. Oe:

oenocytoids.

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prohemocytes at the end of the 3rd instar (LD) increasedsignificantly. Plasmatocytes were significantly greater in the L3-3 phase than in previous phases, and remained steady until the endof the 3rd instar larval. The analyses showed that granulocytes andoenocytoids diminish significantly during the 3rd larval instar(ANOVA, p < 0.05).

The cells morphology in phase-contrast microscopy (Fig. 5)were similar with the ones stained with Giemsa, showing that theprocess of fixation with methanol and staining with Giemsa do notcause morphologic modifications. Granulocytes had been the mostrefractory cells, followed by oenocytoids and less refractory, theprohemocytes. Cells with intermediate features between prohe-mocytes and plasmatocytes were also observed.

3.4. Phagocytosis assays

In assay adding pure bead in the hemolymph of M. scutellaris,was not observed phagocytic activity. Thus, bovine serum albumin(BSA) was covalently bonded to beads to leave them antigenic.

We observed only plasmatocytes and granulocytes of M.

scutellaris hemocytes were able to phagocytize fluorescent micro-spheres after 30 min incubation in the medium (Fig. 6). Our resultsdid not demonstrate prohemocytes and oenocytoids with beadsinternalized, suggesting that these hemocytes population do notphagocytosis perform.

Fig. 6. Phagocytosis assay of M. scutellaris hemocytes with fluorescent beads (arrow tip).

with beads phagocytized (arrow tip), the arrow shows a prohemocytes not held phago

4. Discussion

Stingless bee M. scutellaris, have four developmental stages:embryo, larva, pupa and adult. The measurements of larvae headcapsule using the rule of Dyar (1890) showed that the larvaldevelopment of M. scutellaris has three instars (Dias et al., 2001). Inthis work we propose the division of the 3rd larval instar ofMelipona in five phases: L3-1, L3-2, L3-3, LPD and LD, which canwell be characterized by the food consistency and the presence orabsence of feces in brood combs. The larval instars division indefined phases enables better analysis of the physiologicalprocesses as innate immune system analysis and caste or sexdetermination.

There is a gradual increase in body weight (mg) during the 3rdlarval instar, with maximum in LPD phase, followed by significantdecline in LD phase. This is expected because in the LPD larvae arein the end of the feeding process therefore it reaches maximumweight. The decrease in LD mass can be explained by the beginningof the defecation process.

In insects, immune defense is characterized by a wide variety ofadaptive responses involving opsonisation, phagocytosis, melani-zation, encapsulation and coagulation; each of these varying inspeed and specificity among different taxa (Schmid-Hempel,2005). Each taxon follows a strategy under environmental selectivepressure modulating cellular and molecular mechanisms involved

(A) Plasmatocytes after phagocytosis showing bead in cytoplasm, (B) plasmatocytes

cytosis, (C) granulocytes with beads phagocytized (arrow tip).

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in host defense against pathogens (Rolffand and Siva-Jothy, 2003).Hemocytes are the primary mediators of cell-mediated immunityin insects.

A recent review attempted to overcome the diversity ofterminology used by different authors simplifying the classifica-tion scheme into four major blood cell types in invertebrates:prohemocytes, hyaline hemocytes (plasmatocytes or monocytes),granular hemocytes (granulocytes) and eleocytes (hemocytes withinclusions). Moreover the author claimed that oenocytoids appearto be a further type of circulating blood cell exclusive to arthropods(Hartenstein, 2006).

In the present study, M. scutellaris 3rd instar larvae present fourmorphotypes of circulating hemocytes, all easily distinguishable insmears, after Giemsa staining, namely: prohemocytes, plasmato-cytes, granulocytes and oenocytoids. The present paper reports, forthe first time, a study focused on stingless bee Melipona scutellaris

(Meliponinae) hemocytes. Our approach has description for the firsttime the cells types present in the Melipona scutellaris hemolymph.

Studies of hemocytes differentiation in several insects rated theprohemocytes as stem cells, which are characterized by the abilityof differentiating in other cellular types (Gupta, 1979, 1985, 1991).In Drosophila, one group of prohemocytes in the head regiondifferentiates into plasmatocytes, while another group of prohe-mocytes in the anterior midgut region differentiates into crystalcells (Rehorn et al., 1996; Lebetsky et al., 2000). The Lepidoptera,Bombyx mori showed approximately 43% of the prohemocytesdifferentiates in plasmatocytes, spherulocytes and granulocytes.These findings demonstrate that B. mori prohemocytes can self-renew and are pluripotent, suggesting that they have featurescharacteristic of stem cells (Yamashita and Iwabuchi, 2001).

Our results demonstrate low number of prohemocytes in thehemolymph during the M. scutellaris 3rd larval instar, except in LDphase, which is the last larval phase. Insect hemocytes play a majorrole in developmental processes where they disassociate andrebuild metamorphosing tissues while undergoing physiologicalchanges themselves. Some authors already had described that thehemocytes play a crucial role in the dramatic tissue remodelingthat takes place during metamorphosis (Jones, 1970; Akai and Sato,1971; Feir, 1979; Ratcliffe et al., 1985; Kohlmaier and Edgar, 2008).Gupta (1979, 1985) verified that the prohemocytes showed highmitotic index in post-embryonic development where the hemo-cytes quantification was just 5% of the total cells. In M. scutellaris

we found intermediate prohemocytes differentiating in plasma-tocytes (Fig. 5C) suggesting that prohemocytes of M. scutellaris canbe little differentiated cells that can differentiate in plasmatocyteswhen necessary. The beehives are in constant contact withmicroorganisms. The primary sources of microbiological contam-ination are the pollen, the digestive treatment of the bees, the dustand the flowers. The microorganisms found in the beehives aremainly bacteria and yeasts. The larvae can be sterile initially, but asthey ingest the larval food (rich in pollen and nectar) they acquirethe microbiota present in it (Snowdon and Cliver, 1996; Gilliam,1997).

Initial defenses of invertebrates include the physical barriers ofthe integument or gut, clotting responses by hemolymph, and theproduction of various cytotoxic molecules at the site of wounding.Microorganisms that invades these barriers and enter thehemocoel must contend with additional cytotoxic molecules aswell as an array of different hemocytes (Gillespie et al., 1997). Thistask probably must be made by the hemolymph cells primarilyplasmatocytes and granulocytes, which as we saw in this work hadcarried phagocytosis.

In larval stage Lepidoptera, granulocytes and plasmatocytes arethe only hemocyte types capable of adhering to foreign surfaces,and together usually comprise more than 50% of the hemocytes incirculation (Lackie, 1988; Ratcliffe, 1993; Strand and Pech, 1995).

Plasmatocytes represent the majority of cells in hematopoietictissues in adults and larvae of Carabus Lefebvre (Giglio et al., 2008).Those cells can be best compared with macrophages of vertebrates.They are generally recognized as phagocytotic cells (macrophages)involved in the removal of apoptotic cells during development aswell as in the ingestion or encapsulation of pathogens (Evans et al.,2003; Hartenstein, 2006).

Granulocytes are involved in developmental and metabolicfunctions as well as in immune functions, including woundhealing, blood clotting, phagocytosis, encapsulation of pathogensand tecidual remodelling (Hartenstein, 2006; Nardi et al., 2001). InM. scutellaris, the Granulocytes showed more intense staining ofthe granules in the last phase of the 3rd larval instar (LD). Thissame staining was also found in the last stage of Manduca sexta

larvae (Beetz et al., 2004). Some authors affirm the role ofgranulocytes in tissue remodeling, for the metamorphosis (Nardiand Miklasz, 1989; Kurata et al., 1991, 1992; Rheuben, 1992;Murray et al., 1995; Kiger et al., 2001; Nardi et al., 2001).

Oenocytoids gradually disappears during all phases of the lastM. scutellaris larval instar. This behavior already was observed inManduca sexta larvae. The oenocytoids reduction in the last larvalinstar of Manduca sexta was directly related to the hormone 20-hydroxyecdysone production, which stimulates the synthesis ofproteins involved in metamorphosis. High levels of this hormoneinhibit the cellular division in oenocytoids thus promoting theapoptosis of these cells (Beetz, 2002).

This work identified prohemocytes, plasmatocytes, granulo-cytes and oenocytoids in the Melipona scutellaris hemolymphcells. The hemocytes characterization provides relevant informa-tion to prevent and eliminate infections in stingless bees andcontributes to understanding the role of hemocytes in the beeimmunity.

Acknowledgments

This work was supported by Conselho Nacional de Desenvolvi-mento Cientıfico e Tecnologico (CNPq), Coordenacao de Aper-feicoamento de Pessoal de Nıvel Superior (CAPES) and Fundacao deAmparo a Pesquisa do Estado de Minas Gerais (FAPEMIG). Wewould like to thank Rodrigo Redondo and Fausto Capparelli forcritical comments and early review of the manuscript.

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