The regenerative cells during the metamorphosis in the midgut of bees

Gustavo Ferreira Martinsa, Clovis Andrade Nevesb, Lucio Antonio Oliveira Camposb,

Jose Eduardo Serraob,*

aDepartamento de Biologia Animal, Universidade Federal de Vicosa, BrazilbDepartamento de Biologia Geral, Universidade Federal de Vicosa, 36570-000 Vicosa, MG, Brazil

Received 27 July 2005; revised 29 July 2005; accepted 30 July 2005

Abstract

The midgut epithelium of bees is formed by the digestive cells, responsible for enzyme secretion and nutrient absorption and for small

regenerative cells that are placed in nests scattered among the digestive cells. During metamorphosis, the larval midgut epithelium

degenerates and a new adult midgut epithelium is built during larval differentiation of regenerative cells. The present work focuses on the

midgut epithelial modifications during the post-embryonic development of the stingless bee Melipona quadrifasciata anthidioides worker

and the occurrence of regenerative cell proliferation during midgut metamorphosis in order to test the hypothesis that adult midgut epithelium

of worker bees results from regenerative cell proliferation during the pupal stage. Regenerative cell proliferation was detected during larval

lifespan. Larval aging is followed by an increase in the number and the size of the nests of regenerative cells. Larval epithelium degeneration

begins 2 days after the start of defecation process and in this period the nests of regenerative cells are in contact by means of cytoplasmic

extension which have many septate desmosomes and gap junctions. The BrdU immunoreactive regenerative cells were found in the prepupae

12 h after BrdU injection, suggesting that regenerative cell population increase during this larval period. Regenerative cell proliferation

results in the increase of the regenerative cell population and not in the formation of new digestive cells because the proliferation of

regenerative cells would not be enough to reestablish the nests of regenerative cells and at the same time form new adult digestive cells.

In this sense the hypothesis that digestive adult cells originate from regenerative cell proliferation during midgut metamorphosis in

M. quadrifasciata anthidioides was rejected.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Alimentary canal; BrdU; Development; Insect

1. Introduction

The midgut of bees has three cell types: digestive cells

that synthesize digestive enzymes and absorb the nutrients,

the endocrine cells producing hormone and the regenerative

cells responsible for the replacement of the former cell

types.

Digestive cells in newly hatched larvae form a

cylindrical or cubical epithelium acquiring granules,

vacuoles and apical protrusions with aging. In the prepupae

the midgut becomes narrowed and elongated, while in white

eyed pupae the midgut acquires a bottle shape and the

diameter increases. In pink eyed pupae the midgut diameter

0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2005.07.003

* Corresponding author. Tel.: C55 31 38991301; fax: C55 31 38992549.

E-mail address: jeserrao@ufv.br (J.E. Serrao).

becomes uniform and the digestive cell apex is cast out to

the lumen, but in black eyed pupae the midgut has

epithelium similar to the adult (Oertel, 1930; Snodgrass,

1956; Cruz-Landim and Mello, 1970; Neves et al., 2002).

The regenerative cells have little cytoplasm and the

nuclei have condensed chromatin. In the cytoplasm there are

few ribosome, mitochondria, endoplasmic reticulum and

Golgi apparatus profiles suggesting low metabolic activity

in these cells (Hecker et al., 1971; Cruz-Landim, 1985;

Cruz-Landim et al., 1996; Cruz-Landim, 1999). At

metamorphosis the larval midgut epithelium is ejected and

its reorganization occurs from regenerative cells. During the

regenerative differentiation cells elongate in direction to the

midgut lumen acquiring microvilli, followed by an increase

in the nuclear and cytoplasmic volume (Werner et al., 1991;

Cruz-Landim et al., 1996; Neves et al., 2003).

In Hymenoptera changes in the midgut begin in the

prepupa with the larval epithelium degenerating, leaving

Micron 37 (2006) 161–168

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G.F. Martins et al. / Micron 37 (2006) 161–168162

only the basement membrane and the regenerative cells. At

the same time, some cells proliferate and are scattered on

the basement membrane to build the new pupal epithelium

(Oertel, 1930; Snodgrass, 1956; Cruz-Landim and Mello,

1970; Gama and Cruz-Landim, 1984; Neves et al., 2002).

However, during the metamorphosis of bees, regenerative

cells in division had been rarely found (Oertel, 1930; Cruz-

Landim and Mello, 1970; Neves et al., 2003; Cruz-Landim

and Cavalcante, 2003).

Cell proliferation is easily recognized when there are

cells in mitosis with condensed chromosomes. An alterna-

tive procedure for cell proliferation detection is the use of

antibodies against a specific substance present in the cells

that are preparing to proliferate. 5-Bromo-2 0-deoxy-uridine

(BrdU) is an analogous of the thymidine nucleotide

incorporated to DNA during the replication process

(Griffiths et al., 2000; Voet et al., 2000). In this way,

antibody anti-BrdU can be used to identify cells that

incorporated BrdU providing evidence for cell proliferation

(Gratzner, 1982). BrdU has been used successfully in

insects for detection of cell proliferation (Kirschenbaum and

O’Shea, 1993; Zacharias et al., 1993; Stocker et al., 1995;

Loeb and Hakim, 1996; Schimidt-Capella and Hartfelder,

1998; Vitt and Hartfelder, 1998; Cayre et al., 2000). In this

work, we used this procedure to investigate the presence of

cell proliferation in the midgut of the stingless bee Melipona

quadrifasciata anthidioides during post-embryonic

development to test the hypothesis that new digestive cells

arise from regenerative cells due to mitosis.

2. Material and methods

2.1. Animals

Melipona quadrifasciata anthidioides were obtained

from nests maintained in the Federal University of Vicosa,

state of Minas Gerais, Brazil. The bees were analyzed in the

following developmental stage: 27 larvae of various sizes,

24 prepupae, 54 pupae (18 white eyed pupae, 18 brown eyed

pupae and 18 black eyed pupae) and eight adults (four nurse

and four forager workers). The greater number of immature

specimens was obtained because we injected BrdU in 30

specimens per stage and some them died after injection and

were discharged of the analyses. The 27 larvae were

represented by 23 feeding larvae, two at 2 days after the

beginning of defecation and two larvae at 5 days after

the beginning the defecation, because in larvae of bees the

passage from midgut to hindgut remain closed until the end

of larval stage.

2.2. Light microscopy

Individuals were cold immobilized for five minutes and

the midguts were dissected in saline solution for insect

(0.1 M NaCl, 20 mM KH2PO4 and 20 mM Na2HPO4) and

transferred to Zamboni’s solution (Stefanini et al., 1967)

during 6 h at 4 8C. For the larvae the midgut only was used,

while at the other developmental stages, both the foregut

and the midgut were analyzed.

The guts were dehydrated in a graded ethanol solution,

embedded in historesin JB-4 (Polysciences), cut as 1–2 mm

sections and stained with toluidine blue-borax.

2.3. Transmission electron microscopy

Midugts were dissected as described above, sectioned in

three pieces and transferred to 2.5% glutaraldehyde in 0.1 M

sodium cacodylate buffer for 4 h. The pieces were post-fixed

in 1% osmium tetroxide in the same buffer for 2 h, following

en bloc staining with 2% uranyl acetate overnight. The

midguts were dehydrated in graded aqueous acetone

solutions and embedded in Epon-Araldite resin. Ultrathin

sections were stained with lead citrate and analyzed in a

transmission electron microscope Zeiss EM 109.

2.4. Immunohistochemistry

The cell proliferation was studied using the Boehringer-

Mannheim detection kit (Cat. No. 1296 736).

Prepupae and pupae were injected with 5 ml BrdU in the

abdominal region with the aid of a microinjector NT-88NE

(Nikon). After the BrdU injection, guts were dissected at 4 h

intervals up to 24 h. After fixation in Zamboni’s solution for

2 h, the guts were submitted to the BrdU detection

according to manufacturer’s kit instructions. The pieces

were dehydrated in ethanol series, embedded in historesin,

cut as 15 mm sections, covered with VectaShield (Lab.

Vector) and analyzed by epifluorescence microscopy.

Some samples were submitted to indirect immunohis-

tochemistry using peroxidase conjugated antibody. After

fixation the pieces were washed in PBST following

incubation in 1% phenylhydrazine for 40 min at room

temperature. Subsequently, the samples were submitted to

the BrdU detection kit procedures. Then, incubation in anti-

mouse IgG peroxidase conjugated (1:300) was performed

for 24 h at 4 8C. Peroxidase was revealed with diamino-

benzidine. Then the pieces were submitted to standard

procedures for light microscopy, as described above, and the

sections eosin counterstained.

2.5. Controls

Three brains of prepupae and the foregut of pupae were

used as positive controls, because the occurrence of cell

proliferation in these organs during the metamorphosis of

bees is known. Negative controls were performed by

immunohistochemistry procedures, with omission of the

anti-BrdU antibody.

G.F. Martins et al. / Micron 37 (2006) 161–168 163

3. Results

In the M. quadrifasciata anthidioides larvae the midgut

epithelium is formed by a single layer of digestive cells that

are prismatic increasing in size with the larval development

and the nests of regenerative cells are located at the base of

the midgut epithelium (Fig. 1). In the young larvae the

regenerative cells are spherical or oval forming isolated

nests with one or two regenerative cells per nest. Some nests

are found lined by a digestive cell, but mostly they are found

between the basal limits of the digestive cells (Figs. 1 and 2).

Fig. 1–6. Light micrographs of the midgut epithelium of Melipona quadrifasciata a

digestive cell (dc). (2) Tangential section of young larva showing regenerative cell

section of old larva showing regenerative cell nest (rn) with cytoplasmic extensio

2 days after defecation showing digestive cells (dc) with nucleus (n) and regenera

the digestive cells. (5) Anterior midgut region of larva 2 days after defecation show

and without brush border in the cell apex being cast off (arrow). Notice regenerativ

of the anterior midgut region of larva 5 days after defecation showing the degene

formed by regenerative cells (rc) in differentiation process. Arrowheads, regenera

layer; n, digestive cell nucleus; pg, pollen grains; pm, peritrophic membrane. Ba

The number of the regenerative cells increase from 6 to 8 cells

per nest in defecating larvae. Besides, increasing in size, the

regenerative cells have a large nucleus and cytoplasmic

extensions (Fig. 3), which elongate until contact is made with

the extensions from cells in the neighboring nests in the old

larvae (Fig. 4).

In the defecating larvae, the digestive cells of the anterior

midgut region have nuclei with condensed chromatin and

the apical cytoplasm becomes vacuolated. Then, it is not

possible to recognize the brush border, but regenerative

cells in the differentiation process show strong staining by

nthidioides. (1) Young larva showing a regenerative cell nest (rn) lined by a

nests (rn) with different size and number of regenerative cells. (3) Tangential

ns (arrows). (4) Tangential section of the posterior midgut region of larva

tive cell nests (rn) forming a net by cytoplasmic extensions (arrows) among

ing digestive cells (dc) with vacuolated cytoplasm (v), pycnotic nucleus (n)

e cells (rc) in differentiation process strongly stained. (6) Tangential section

rated larval midgut epithelium (LE) onto the pupal midgut epithelium (PE)

tive cell nucleus; b, bacteria; bb, brush border; L, midgut lumen; m, muscle

rsZ10 mm.

G.F. Martins et al. / Micron 37 (2006) 161–168164

the toluidine blue (Fig. 5). Five days after the start of

defecation, the larval midgut epithelium is more disorga-

nized, with presence of two cell layers in the anterior midgut

(Fig. 6). The layer lining the midgut lumen is part of the old

larval epithelium in an advanced degenerative stage,

characterized by a pycnotic and fragmented nucleus and

vacuolated cytoplasm, while the basal layer is disorganized

with pleomorphic cells, which arise from the small

regenerative cells that are in the differentiation process

Fig. 7–13. Light micrographs of the midgut epithelium of M. quadrifasciata anth

digestive cells (dc) and basal regenerative cells (arrowheads) scattered among th

Midgut of prepupa showing digestive cells (dc) releasing vesicles (arrows). Notice

the midgut of young larva showing a regenerative cell in metaphase (arrowhead). (1

cells. (11) Midgut of black eyed pupa with cubic digestive cells (dc) and regene

Tangential section of the midgut epithelium of black eyed pupa showing regenera

worker showing regenerative cell nests with small inner cells (ic) and large cells wi

muscle layer; n, regenerative cells nucleus; N, digestive cell nucleus. BarsZ10 m

(Fig. 6). On the other hand, in the posterior midgut region

the epithelium is still single layered with viable digestive

cells, presenting a mid-basal nucleus and the regenerative

cells are larger than those found in young larvae. In this

midgut region the regenerative cells of different nests are in

contact (Fig. 4).

In the prepupae, that a new midgut epithelium arises

from the basal epithelium of the old larvae has already been

established, however, it is disorganized with cells of

idioides. (7) Midgut of prepupa showing the epithelium with newly formed

e digestive cells. Notice cell fragments (arrows) in the midgut lumen. (8)

the presence of epithelial infoldings (arrowhead). (9) Tangential section of

0) Midgut of white eyed pupa showing a regenerative cell nest (rn) with two

rative cell nests (rn) located in the center of the each villus (arrow). (12)

tive cell nests (rn) without cytoplasmic extensions. (13) Midgut of forager

th cytoplasmic extensions in the nest boundary (lc). bl, basement lamina; m,

m.

G.F. Martins et al. / Micron 37 (2006) 161–168 165

different length, creating a pseudostratified epithelium. The

regenerative cells are individualized and scattered in the

base of the epithelium and the midgut lumen is filled with

cell debris due to ejection of larval epithelium (Fig. 7). At a

more advanced period of the prepupae development the

midgut lumen releases vesicles from the digestive cell apex,

which are vacuolated and in some individuals can be

distinguished by a brush border and the epithelial surface

fold, forming the epithelial villi (Fig. 8).

In the larvae of different sizes a few mitosis profiles are

found in the regenerative cells (Fig. 9). In white eyed pupae

the midgut epithelium is more organized with tall cells, but

these are shorter than those found in prepupae. The

regenerative cells form small nests with few cells

(Fig. 10). In brown eyed and black eyed pupae the digestive

cells are lower than those from white eyed pupae. The

epithelial folds are more pronounced and the regenerative

cell nests have cells of different size without cytoplasm

extensions (Figs. 11 and 12). In old black eyed pupae, the

regenerative cell nests are more clearly defined and the

number of cells per nest increases. In the regenerative nests

of adult workers two cell types are found: cells at nest

boundaries that are large with cytoplasmic extensions, and

small central cells that form a concentric array (Fig. 13).

Ultrastructural analyses from tangential sections

(Fig. 14) shows that regions where cytoplasm prolongations

of different regenerative cells make contact, these cells are

separated by a narrow intercellular space (16–20 nm),

although in some regions there is a small opening of

Fig. 14–17. Transmission electron micrographs of tangential sections of the larva

cytoplasm prolongations from three regenerative cells (C1, C2 and C3) with nar

septate desmosome (arrowhead). m, mitochondria. BarZ1 mm. (15) Regenerative

0.1 mm. (16) Septate desmosome (arrowheads) and gap junctions (arrows) in the ce

gap junction in regenerative cell–cell contact from larval midgut. BarZ0.1 mm.

the intercellular space (Figs. 14 and 15). The cell–cell

contacts are characterized by septate desmosomes and gap

junctions (Figs. 16 and 17).

The BrdU immunoreactive cells are found in prepupae

12 h after the BrdU injection, with a positive reaction in the

newly formed digestive cells (Fig. 18) and regenerative cells

(Fig. 19). In prepupae, 24 h after BrdU injection, there are

many BrdU immunoreactive regenerative cells, as well as

newly formed digestive cells. In white eyed and brown eyed

pupae there are some BrdU immunoreactive cells, however,

these cells are not part of the midgut epithelium, adhering

externally to the midgut wall (Fig. 20). In the midgut

epithelium of black eyed pupae BrdU immunoreactive cells

are not found. In the positive controls many BrdU

immunoreactive cells are found in the brain of prepupae

and in the foregut of white eyed and brown eyed pupae.

4. Discussion

The midgut reorganization of bees durin metamorphosis

was studied by Oertel (1930); Snodgrass (1956); Cruz-

Landim and Mello (1970); Neves et al. (2002); Neves et al.

(2003); Cruz-Landim and Cavalcante (2003). However,

these studies did not look at the regenerative cell nest

reorganization.

The regenerative cell nests in adult of M. quadrifasciata

anthidioides are equidistant, as in Apis mellifera (Raes et al.,

1994). These nests are located in the deep regions of

l midgut epithelium of M. quadrifasciata anthidioides. (14) Contact of the

row intercellular space, small intercellular space enlargement (arrow) and

cell–cell contact with expansion of the intercellular space (arrow). BarZll–cell contact from regenerative cell. BarZ0.5 mm. (17) Detailed view of a

Fig. 18–20. Longitudinal section of the midgut of M. quadrifasciata

anthidioides. (18) Prepupa 12 h after BrdU injection showing BrdU

immunoreactive (arrow) and unlabelled (arrowhead) digestive cells.

Immunofluorescence. (19) Prepupa 24 h after BrdU injection showing

two BrdU immunmoreactive regenerative cell nuclei (arrowheads).

Immunofluorescence. (20) White eyed pupa 24 h after BrdU injection

showing BrdU immunoreactive nuclei (arrows) of cells close to the midgut

wall (mw). dc, digestive cell; m, muscle layer. BarsZ10 mm.

G.F. Martins et al. / Micron 37 (2006) 161–168166

the midgut villi, created by the circular muscle on the

midgut wall. However, in the larvae the regenerative cell

nests are randomly scattered among the digestive cells,

perhaps because the midgut villi are almost absent because

the muscle layer is less developed than in the adult

(Cruz-Landim and Mello, 1970).

Raes et al. (1994) have pointed out that regenerative cells

form crypts in the midgut epithelium of adult A. mellifera.

However, Cruz-Landim et al. (1996) describe the set of

regenerative cells of the bees as structures that form groups

or nests that are placed in the same level of the digestive

cells, and do not resemble crypts. Regenerative crypts are

found in Coleoptera where they are placed below the

basement line of the midgut wall (Chapman, 1998). So the

use of regenerative crypt to name the regenerative cell

clusters in bees is a misinterpretation of the morphological

localization of the regenerative cell nests at the level of the

midgut villi.

In M. quadrifasciata anthidioides the midgut size

increase during larval development resulting from digestive

cell growth (Cruz-Landim and Mello, 1970) and from the

increase of digestive cell number due to differentiation of

regenerative cells (Serrao and Cruz-Landim, 2000). In

Manduca sexta (Lepidoptera) from the first to fifth larval

instar the midgut surface increases 200-fold due to the

increase in size and number of the digestive cells (Baldwin

and Hakim, 1991). Werner et al. (1991) described

regenerative cells at different development stages among

the digestive cells for Gerris najas (Heteroptera). The

occurrence of regenerative cells during the differentiation

process was also found in the adult bees (Raes et al., 1994;

Cruz-Landim et al., 1996) and in caterpillars (Baldwin and

Hakim, 1991). Another mechanism that might be respon-

sible for the increase of midgut epithelial surface in M.

quadrifasciata anthidioides would be the increase in the

number of regenerative cell nests and/or in the size of the

nests, which play a role by filling the spaces between

the digestive cells during midgut growth.

In addition to the increasing the midgut epithelial

surface, the cytoplasm extensions of the larval regenerative

cells create contact among nests, suggesting the occurrence

of the cell synchronization during differentiation and

formation of the adult midgut epithelium. In Lepidoptera

the regenerative cells have gap junctions that play a role in

synchronization (Baldwin and Hakim, 1991). On the other

hand A. mellifera has not gap junctions in the cells placed in

the interior of the regenerative cell nests, but those cells in

the nest boundaries have fusomes (cytoplasm bridges) (Raes

et al., 1994), which probably play a similar role to the gap

junctions of Lepidoptera, by interconnecting the cytoplasm

of these cells and allowing synchronous differentiation.

Because we and Neves et al. (2002) have not found fusomes

in the regenerative cells and the finding of gap junctions in

the cytoplasm prolongations from regenerative cells of

adjacent nests may suggest that in M. quadrifasciata

anthidioides the synchronization of cell differentiation

occurs in a similar way to that proposed in Lepidoptera

for Baldwin and Hakim (1991)

Larvae of M. quadrifasciata anthidioides have regen-

erative cell proliferation detected by the presence of some

metaphases, as found in M. sexta (Baldwin and Hakim,

1991; Baldwin et al., 1993). However, the number of

mitoses found in the regenerative cells of M. quadrifasciata

anthidioides larvae seems to be too low to allow for the

increase in digestive cell number, suggesting that the midgut

is reorganized due to the differentiation of larval regen-

erative cells to replace the epithelial cells during metamor-

phosis as proposed by Judy and Gilbert (1970); Cruz-

Landim and Mello (1970); Serrao and Cruz-Landim (2000);

Cruz-Landim and Cavalcante (2003).

BrdU incorporation by regenerative cells suggests that in

the prepupae these cells proliferate for regenerative cell nest

formation in the adult bees, which become definitive in

brown eyed and black eyed pupae. This is an expected result

because most of the larval regenerative cells differentiate

into adult digestive cells (Serrao and Cruz-Landim, 1996,

G.F. Martins et al. / Micron 37 (2006) 161–168 167

2000; Neves et al., 2002). In this sense, regenerative cell

proliferation would occur to compensate for the use of

undifferentiated regenerative cells during the metamorpho-

sis and to guarantee the presence of these cells in the adult

bees, which also need regenerative cells for maintenance of

the midgut epithelium (Werner et al., 1991; Cruz-Landim

and Costa-Leonardo, 1996; Cruz-Landim et al., 1996,

1997).

The finding of BrdU immunoreactive newly formed

digestive cells in prepupae suggests that these cells originate

from regenerative cell division, because the immunoreac-

tivity found in these cells might be due to BrdU residues

incorporated by the regenerative cells during the S phase in

the prepupal stage. The presence of polyploid cells in the

insects has been found in salivary glands (Pavan and Da

Cunha, 1969) and Malpighian tublules of bees (Silva-de-

Moraes and Cruz-Landim, 1976). Midgut cells of Rhodnius

prolixus (Heteroptera) females become polyploid after a

blood meal (Billingsley, 1989), however, in bees the

influence of the feeding on the DNA content of the digestive

cells is not known, but due to the occurrence of BrdU

immunoreactive newly formed digestive cells in M.

quadrifasciata anthidioides prepupae does not negate the

hypothesis that these cells are polyploid. However, R.

prolixus is a hematophagous insect that ingests large

amounts of food in a short time (Billingsley, 1990), differing

from social bees that feed constantly, without food-deficient

periods.

In defecating larvae there are large numbers of

regenerative rather than digestive cells. So the population

of larval regenerative cells seems to be sufficient for the

reconstruction of the adult epithelium, because most of the

regenerative cells differentiate, but there still remain a small

population of regenerative cells that proliferate in the

prepupae, re-establishing the nests of regenerative cells in

the pupae and consequently in adult bees.

The BrdU immunoreactive cells in the foregut of

prepupae were found 4 h after the BrdU injection, while

in the midgut BrdU immunoreactive cells were found 12 h

after the BrdU pulse. Perhaps this difference is due to a

higher mitosis rate in the foregut. In both foregut and midgut

prepupae a pool of BrdU immunoreactive cells is present,

suggesting that mitoses predominate during this develop-

mental stage.

The hypothesis that adult digestive cells arise from

regenerative cell proliferation followed by their differen-

tiation during the metamorphosis in M. quadrifasciata

anthidioides midgut was rejected, because the regenerative

cell population in defecating larvae seems to be sufficient to

promote midgut epithelium renewal during the metamor-

phosis. Moreover, the number of mitoses detected seems

does not high to re-establish the regenerative cell nests and

to provide new regenerative cells. So, during the midgut

metamorphosis M. quadrifasciata anthidioides does not use

de novo epithelium formation, instead larval regenerative

cells differentiate into digestive cells. The reestablishment

of the regenerative cell nests is a consequence of cell

divisions in the prepupae, which play a role in digestive cell

renewal of adult bees.

Acknowledgements

We are grateful to Dr Silvia G. Pompolo and Dr Adilson

A. Zacaro (Federal University of Vicosa, Brazil) for

criticism and suggestions and to Dr L.A.O. Campos for

criticism and for providing the bees. This work was

financially supported by National Research Council

(CNPq) and Minas Gerais State Research Council

(FAPEMIG). G.F.M. is a graduate fellow of CNPq and

C.A.N., L.A.O.C and J.E.S. are staff members of UFV and

the two later are research fellow of CNPq.

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