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Egg and fourth instar larvae gut of Aedes aegypti as a source of stem cells
Accepted Manuscript Title: Egg and fourth instar larvae gut of Aedes aegypti as a source of stem cells Author: Lara C. Mario J´essica Borghesi Wilson T. Crivellari-Damasceno Phelipe O. Favaron Ana Claudia O. Carreira Sonia E.A.L. Will Durvanei A. Maria Maria A. Miglino PII: DOI: Reference:
S0040-8166(16)30020-9 http://dx.doi.org/doi:10.1016/j.tice.2016.05.004 YTICE 1005
To appear in:
Tissue and Cell
Received date: Revised date: Accepted date:
28-1-2016 20-4-2016 14-5-2016
Please cite this article as: Mario, Lara C., Borghesi, J´essica, Crivellari-Damasceno, Wilson T., Favaron, Phelipe O., Carreira, Ana Claudia O., Will, Sonia E.A.L., Maria, Durvanei A., Miglino, Maria A., Egg and fourth instar larvae gut of Aedes aegypti as a source of stem cells.Tissue and Cell http://dx.doi.org/10.1016/j.tice.2016.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Egg and fourth instar larvae gut of Aedes aegypti as a source of stem cells
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Lara C. Marioa,*, Jéssica Borghesia, Wilson T. Crivellari-Damasceno a,b, Phelipe O.
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Favarona, Ana Claudia O. Carreirac, Sonia E. A. L. Willb, Durvanei A. Mariaa,b, Maria
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A. Miglinoa
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a Departament
of Surgery, School of Veterinary Medicine and Animal Science, University of Sao Paulo,
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Sao Paulo, SP, Brazil
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b Laboratory c Center
of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, SP, Brazil
for Molecular and Cellular Therapy (NUCEL) and Center for Cellular and Molecular Therapy
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(NETCEM), School of Medicine, University of Sao Paulo, Sao Paulo, SP, Brazil
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* Corresponding author:
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Email: [email protected]
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Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, 05508-270, São Paulo-SP, Brasil.
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Tel/Fax: 55 11 30917690
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Highlights
Due the epidemiological importance of dengue, many investments in new research in genetic engineering and molecular biology have been conducted. Through characterization of stem cells of Aedes aegypti, we evaluated the morphology, expression of markers, and cell cycle phases. Studies with these cells may represent an important tool for biotechnology, resulting in innovative information for public health.
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Abstract
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According to the World Health Organization, 2015 registered more than 1.206.172 cases of Dengue in the
27
Americas. Recently, the Aedes aegypti has been not only related to Dengue, but also with cases of Zika
28
virus and Chikungunya. Due to its epidemiological importance, this study characterized the morphology of
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the embryonated eggs of A. aegypti and provided a protocol to culture stem cells from eggs and digestive
30
tract of fourth instar larvae in order to examine cell biology and expression of markers in these vectors.
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Cells were isolated and cultured in DMEM-High at 28 °C, and their morphology, cell cycle and
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immunophenotyping were examined. Morphologically, embryos were at the end of the embryonic period
33
and showed: head, thorax, and abdomen with eight abdominal segments. The embryonic tissues expressed
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markers related to cell proliferation (PCNA), pluripotency (Sox2 and OCT3/4), neural cells (Nestin),
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mesenchymal cells (Vimentin and Stro-1), and endosomal cells (GM130 and RAB5). In culture, cells from
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both tissues (eggs and larvae gut) were composed by a heterogeneous population. The cells had a globoid
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shape and small size.. Cell cycle analysis on passage 1 (P1) showed 27.5% ±2.0% of cell debris, 68% of
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cells on G0-G1 phase, 30.2% on S phase, 1.9% ± 0.5% on G2-M phase. In addition, cells on passage 2
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showed: 10% of cell debris, 92.4% of cells on G0-G1 phase, 6.8% on S phase, 0.6% on G2-M phase.
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Embryonated eggs expressed markers involved with pluripotency (Sox2 and Oct 3/4), mesenchymal cells
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(vimentin and Stro-1), neural cells (Nestin), and cellular death by apoptosis (Caspase 3). Specific
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endosomal markers for insect cells (GM130 and RAB5) were also highly expressed. In cell culture of Aedes
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aegypti larvae gut the same labeling pattern was observed, with a small decrease in the expression of
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mesenchymal (vimentin and Stro-1) and neural (Nestin) markers. In summary, we were able to establish a
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protocol to culture embryonated eggs and larvae gut of Aedes aegypti, describing the characteristics of
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undifferentiated cells, as well as the cell cycle and expression of markers, which can be used for
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biotechnology studies for the biological control of this vector.
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Keywords: Dengue; Cell culture; Morphology; Emerging diseases
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1. Introduction
52
Dengue is an emerging disease that is spreading rapidly worldwide, with about
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100 million people infected each year, which about 20,000 die (Brasil, 2009). According
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to the World Health Organization, in 2015 it was registered approximately 1.206.172
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cases of Dengue in the Americas. Brazil by itself contributes to 85% of the total number
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of cases in the Americas. In addition, there are approximately 2.5 billion people at risk
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worldwide (Americas, 2015).
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The most important vector of dengue fever is Aedes aegypti and the infection is
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caused by a virus of the genus Flavivirus, transmitted by the female to the host during
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blood feeding (Gluber et al., 2007). This virus has four serotypes DENV1, DENV2,
61
DENV3 and DENV4. The simultaneous circulation of different serotypes increase
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successive infections contributing to the emergence of new and more severe clinical
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forms, such as dengue hemorrhagic disease, which usually occurs after the first infection
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with a different serotype (Pessoa et al., 2005; Guzman, 2009).
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Other diseases transmitted by the same mosquito include Chikungunya and Zika
66
virus. Chikungunya has resembling characteristics to those found in dengue, but the
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transmitter arbovirus belongs to the Togaviridae family (Kuniholm et al., 2006). In 2014,
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according to the Pan American Health Organization, 33 countries in the Americas have
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reported incidence of this disease totaling 570.972 suspected cases (Organização Pan-
70
Americana, 2014). Zika fever, caused by virus Zika (ZIKV), an arbovirus of the genus
71
Flavivirus (Flaviviridae family), shows symptoms similar to those found in dengue as
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fever, diarrhea, pain and a rash throughout the body. Recent focus of arbovirus found in
3 73
different regions worldwide, demonstrated that this virus has the potential to spread into
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various territories where the Aedes vector is located (Organização Pan-Americana, 2015).
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The study of insect cells began around 1915, but it was only in 1959 that it was
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possible to cultivate cell prolongations and thereby understand morphogenesis in insects
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(Grace, 1959). Today, research on insect cell lineages are used to investigate mechanisms
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of infections control, biotechnologies through the development of transgenic insects, and
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model of cell lines for mammals. The cell types most frequently studied in insects are
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intestinal stem cells of medium intestine, used to understanding the control mechanisms
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and renewal of intestinal cells, (Loeb et al., 1996; 1999; 2003; Micchelli and Perrimon,
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2006; Ohlstein and Spradling, 2006; Baton and Ranford-Cartwright, 2007; Hakim et al.,
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2009; Nardi et al., 2010; Navascués et al., 2012) and ovarian follicle, studied to determine
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the development of insects and formation of recombinant proteins and bio-pesticides
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(Silva et al.,1998; Swevers and Iatrou, 2003; Swevers et al., 2005; Belles and Piulachs,
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2014; Jia et al., 2014).
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The insect cell culture has been demonstrated by the establishment of more than
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500 cell lineages, representing an important tool for applied biotechnology area (Segura
89
et al., 2012). In this sense, many mechanisms for infection control are being tested
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including new tissue self-renewal models whereas insects have parallel parameters with
91
mammalian system, which make them useful models for the study of cellular maintenance
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in other taxons (Navascues et al., 2012; Sahai-Hernandez et al., 2012).
93
Considering the epidemiological significance of dengue investments on new
94
research in genetic engineering and molecular biology have been conducted. Thus, this
95
study aimed to establish the first protocol to culture undifferentiated cells from Aedes
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embryonated egg and larvae gut at fourth instars. In addition, we evaluated the cell cycle
97
phases on different passages and the expression of markers related mainly to pluripotency,
98
mesenchymal stem cells and apoptosis. Our data represent an important tool for
99
biotechnology, resulting in innovative information of great importance and applicability
100
for public health and to establish new methods of control of this species.
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2. Material and Methods
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Aedes aegypti eggs in the end of embryonic period, 60 hours after oviposition,
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approximately and fourth instar larvae were used in this experiment. The project was
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approved by Bioethics Committee of the School of Veterinary Medicine and Animal
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Science of the University of Sao Paulo (Protocol number 2949/2013).
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2.1. Collection of material
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Eggs and larvae of Aedes aegypti were collected in the insect breeding at the
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Maranhão State University (UEMA) in Caxias city, Maranhão, and from the Culicidae
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facility Parasitology Department to ICB/BMP, University of São Paulo, São Paulo-SP.
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After collection, the material was kept at room temperature for cell culture.
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2.2. Microscopic characteristics of the embryo
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Embryonated eggs of Aedes aegypti were fixed in 10% formaldehyde solution
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Then, they were dehydrated in a series of ethanol (70% to 100%), diaphanized in xylene
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twice for 5 minutes each, and embedded in paraffin (Histosec® - Merck, lot number
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K91225309). The material was subjected to microtomy in order to obtain 3 µm thick
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sections using an automatic microtome (Leica RM2165). The sections were placed on
122
slides and dried in an oven at 60 °C, and posteriorly stained with hematoxylin and eosin
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(HE). Morphological characteristics were photodocumented using a Nikon Eclipse 80i
124
microscope (Nis Elements system).
125 126
2.3. Scanning electron microscopy
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The embryonated eggs of Aedes aegypti were fixed in glutaraldehyde solution 2%
129
and washed three times in succession with sodium cacodylate buffer (pH 7.2) for 15
130
minutes. Then, the material was impregnated with osmium 1% for 1 hour, followed by
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washes with buffer solution of sodium cacodylate for three times during 15 minutes.
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Dehydration was then carried out in an increasing concentration series of ethanol (15%;
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30%; 50%; 70%; 90%; 100%), for 15 minutes. The material was dried at the critical point
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using carbon dioxide as a liquid of transitional fluid. The structures were mounted on
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stubs, and then was used the sputter system to make the metal cover with gold. The images
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were obtained using a scanning electron microscope FEI Quanta 250.
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2.4. Transmission electron microscopy
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The samples to be used for ultrastructural analysis were fixed in 3%
140
glutaraldehyde. After fixation, the material was treated with 1% osmium tetroxide (4%
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osmium tetroxide w/w solution in water, Polysciences, Inc., USA) for one hour.
142
Following further washes in phosphate buffer, the fragments were dehydrated using a
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series of increasing ethanol concentrations (70% to 100%) and immersed in propylene
144
oxide (Propylene oxide EM Grade, Polysciences, Inc., USA). The samples were
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maintained
146
Polysciences, Inc., USA) and Spurr resin (Spurr’s kit – Electron Microscopy Sciences,
147
Co., USA) with stirring for a period of 12 hours and then in pure resin for four hours.
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After this period, the samples were placed in moulds filled with pure resin and placed in
149
an oven at 60ºC for 72 hours to allow resin polymerisation. The samples were
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subsequently cut with a Leica Ultracut UCT ultramicrotome to obtain 1-µm thin sections,
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which were subsequently hot stained with a 1% aqueous sodium borate solution in
152
distilled water containing 0.25% toluidine blue for light microscopy analysis. Next,
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ultrathin sections (approximately 60 nm thick) were placed on copper screens and
154
contrasted with 2% uranyl acetate in distilled water for five minutes and 0.5% lead citrate
155
in distilled water for 10 minutes. Ultrastructures were finally analysed and photographed
156
with a Morgagni 268D microscope.
in
a 1:1 mixture of propylene
oxide (Propylene
oxide
EM
Grade,
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2.5. Immunohistochemistry
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Immunohistochemical reactions were performed in sections of Aedes aegypti embryos
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for: Stro-1 (1:200, mouse monoclonal Santa Cruz Biotechnology, antibody sc-47733, Inc,
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Europe); PCNA (1:200, mouse monoclonal Santa Cruz Biotechnology, antibody sc-56,
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Inc, Europe); Oct 3/4 (1:100, rabbit, policlonal Abcam antibody ab-137427); Vimentin
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(1:100, mouse monoclonal anti-human Santa Cruz Biotechnology, antibody sc-73259,
165
Inc, Europe); Stro-1 (1:100, Mouse monoclonal sc-7733, Santa Cruz Biotechnology, Inc,
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Europe); Nestin (1:100, mouse monoclonal, Santa Cruz, antibody sc-33677 Inc, Europe);
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Sox2 (1:100, goat polyclonal, Santa Cruz, antibody sc-17320 Inc, Europe); GM130
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(rabbit polyclonal, Abcam, antibody ab- 30637, Cambridge, MA, USA); RAB5 (rabbit
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polyclonal, Abcam, antibody ab-31261, Cambridge, MA, USA). The negative control of
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the immunohistochemical reactions was performed using IgG (Goat anti-Mouse IgG - AP
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308F, Chemical International, Temecula, California, USA).
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Sections were deparaffinized in xylol and subsequently blocked in peroxidase
173
(H2O2). Then they were treated with citrate buffer 0.1M and irradiated in a microwave
174
oven. Subsequently, they were balanced with the Dako Protein Block kit (X 0909, Dako
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Cytomation, Carpinteria, CA, USA). The incubation with primary antibodies were
176
performed overnight at 4 °C. After this, sections were washed in Phosphate Buffered
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Saline (PBS) and incubated with secondary conjugated antibody using the peroxidase Kit
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Dako LSAB (K 0690, Dako Cytomation, Carpinteria, CA, USA) followed by
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streptoavidin
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Cytomation, Carpinteria, CA, USA), followed by counterstaining with hematoxylin.
from the same kit. The reaction was visualized from DAB (Dako
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2.5. Aedes aegypti eggs and intestine cell culture
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The Aedes aegypti eggs were placed in a pestle, washed twice with Tween 20
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solution (LGC Biotechnology, code: 13-1316.05) at 20% and 10%. Then, the material
186
was washed four times using PBS solution for 5 minutes, and incubated for 5 minutes in
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antibiotic solution containing gentamicin (Sigma Life Science, code: G1272 - 10 mL),
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penicillin and streptomycin (Gibco by Life Technologies, code: 15140-122 Pen 10.000
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units/mL and Strep 10.000 µg/mL), and amphotericin B (Sigma Life Science, code:
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A2942- 20 mL, 20µg/mL), then washed in PBS. Thereafter, 2mL of Dulbecco´s Modified
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Eagle Medium (DMEM-High Glucose) was added and eggs were macerated with a pestle.
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Then, they were cultured in a 25cm2 culture flask with DMEM-High supplemented with
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10% Fetal Bovine Serum (FBS) and 1% antibiotic solution (gentamicin, streptomycin,
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penicillin and amphotericin B) in an incubator at 28°C ± 2°C. In order to promote the
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expansion, the cells were centrifuged at 100 g for 5 minutes, and the pellet was
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resuspended in culture medium and divided in the plates. The cells were cultured in the
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first, second and fourth passages.
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Intestine cell culture was from the fourth instar larvae. With the aid of a Leica
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S6E stereomicroscope, the larvae were placed on a slide. The abdomen was opening in
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order to remove the digestive tract. Then, the digestive tract was passed through a cell
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sieve (Greiner bio-one, Easystrainer- pore 40 microns, code: 542.040), and washed three
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times with antibiotic solution (Penicillin-Streptomycin and PBS in the ratio 1:100). After
203
treatment, the intestines were placed in 30 mm culture plates with 2 mL DMEM-High,
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containing fragments of the material to promote cell proliferation. Then, the plate was
205
placed in an oven at 28 ± 2°C and the cells from the fourth and sixth passages were used
206
in the experiments.
207 208
Cellular morphology was examined and photographed every 24 hours using an inverted microscope Nikon Eclipse TS-100.
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2.6. Analysis of the cell cycle phases
211 212
For the cell cycle, 106 cells were centrifuged at 100 g for 5 minutes in 1 mL FACS
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Flow buffer (PBS, EDTA 2 mM, SFB 2%). The supernatant was discarded and the pellet
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re-suspended in 100 µL of FACS buffer. Then was added 10 µL of Triton X-100 and 5
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uL of 1.8µg/mL propidium iodide (Sigma, P4170, 1 mg/mL). Samples were analyzed
216
using a BD FACS Calibur flow cytometer and analyzed by software Modfit LT version
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2.9 program.
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2.7. Immunophenotyping by flow cytometry
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For each sample were used 106 cells. The material was centrifuged at 200 g for 10
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minutes. After centrifugation, the pellet was re-suspended in 1 mL of Phosphate Buffered
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Saline and centrifuged again at 200 g for 10 minutes. Thereafter, the pellet was re-
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suspended in FACsFlow buffer, then transferred to cytometry tubes, and incubated with
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the following antibodies: Oct 3/4 (1:100, rabbit policlonal, Abcam antibody ab-137427
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Cambridge, MA, USA); Vimentin (1:100, mouse monoclonal anti-human Santa Cruz
227
Biotechnology, antibody sc-73259, Inc, Europe); Caspase 3 (1:100, mouse monoclonal,
228
Santa Cruz, antibody, sc-271759 Inc, Europe); Stro-1 (1:100, Mouse monoclonal sc-
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7733, Santa Cruz Biotechnology, Inc, Europe); Nestin (1:100, mouse monoclonal, Santa
230
Cruz, antibody sc-33677 Inc, Europe); Sox2 (1:100, goat polyclonal, Santa Cruz,
231
antibody sc-17320 Inc, Europe); HSP70 (mouse monoclonal, Abcam antibody ab-2787
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Cambridge, MA, USA); GM130 (rabbit polyclonal, Abcam, antibody ab- 30637
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Cambridge, MA, USA); RAB5 (rabbit polyclonal, Abcam, antibody ab-31261
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Cambridge, MA, USA). After 30 minutes, the samples were centrifuged at 200 g for 10
235
minutes. Unbound antibody was washed 2 times using PBS. Then, the pellet was
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incubated with secondary antibody (1:500 goat anti-mouse Life Technologies A11017,
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Alexa Fluor 488 or 1:500 donkey anti-rabbit Abcam150061) for 30 minutes, centrifuged,
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washed again using PBS and fixed in paraformaldehyde 4% for analysis.
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The analysis of cellular markers expression was performed in a FACS Calibur flow cytometer and interpreted using the WinMDI 2.9 program.
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3. Results
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3.1. Morphological analysis of Aedes aegypti embryonic tissue
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At the end of the incubation period the eggs were, on average, 1 mm length and
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had a flattened shape (Fig. 1A). The histological examination of the embryos in the Aedes
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aegypti eggs showed that they were divided into: head, thorax and abdomen, with eight
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abdominal segments (Fig. 1B and C). The digestive tract morphology of fourth instar
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larvae Aedes aegypti (Fig. D) showed the presence of the anterior, middle and posterior
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intestine. In the anterior region, gastric blinds were observed, and from the end of the
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midgut to the beginning of the posterior intestine the presence of malpighian tubule was
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observed (Fig.1 E and F).
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3.2. Immunohistochemistry
256 257
After morphological analysis, immunohistochemistry tests were carried out on
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embryonated eggs of Aedes aegypti at the end of the embryonic period. Related to the
259
expression of cell proliferation markers, the PCNA marker was positive. However, for
260
the pluripotency markers Oct 3/4 was less expressed when compared to Sox2.
261
Mesenchymal markers as Vimentin and Stro-1 were also tested, which were positively
262
expressed in these cells. A positive expression was observed for nerve marker Nestin.
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The specific markers of Drosophila tested on Aedes aegypti, GM130 and RAB5, were
264
positive for Golgi complex and endosomal cells, respectively. To confirm these last two
265
markers a positive control was done on adult cells of Drosophila spp. (Fig.2).
266 267
3.3. Cell isolation and characterization
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The culture of embryonated eggs was performed using approximately 100-150 eggs with fourth instar Aedes aegypti larvae in which only the intestines were used.
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The cells obtained from cellular maceration of embryonated eggs were classified
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accordingly to their form, being small and globoid, and with colony formation. The insect
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cells grew rapidly after 24 hours of culture, and needed to be expanded to a second
274
passage (Fig. 3 A and B). The gut cells were small and globular, maintaining this format
275
until the sixth passage (P6) (Fig. 3 C and D).
276
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3.4. Cell Morphology by flow cytometry
278 279
The quantitative analysis of Aedes aegypti cell types showed the presence of cells
280
with granularities and different sizes at culture expressed first passage (P1) and P2. This
281
assay occurred concomitantly with the analysis of the cell cycle phases. In P1, it was
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observed that the different cell populations were uniform in size but with different
283
densities (Fig. 4A). Analyzing the graphs in P2 can be observed a similarity in cell
284
population (Fig. 4B).
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3.5. Analysis of the cell cycle phases
288 289
The phases of the cell cycle had, on average, 27.5% ± 2.0% cellular debris in P1, 68%
290
G0-G1 of the rest, where the cells were at the beginning of cell growth, 30.2% in S-phase
291
and 1.9% ± 0.5% in G2-M phase, indicating that the cells were in preparation for cell
292
division. Then samples were analyzed after the P2 in cell culture, and the following values
293
were obtained: 10% of cell debris and of the cells 92.4% were in the G0-G1 phase, 6.8%
294
in S-phase, and 0.6% in G2-M phase (Fig.4C).
295 296
3.6. Immunophenotyping by flow cytometry
297 298
Immunophenotyping was performed by flow cytometry of Aedes aegypti
299
embryonated eggs in P2 and P4, and from the digestive tube of fourth instar larvae, at P4
300
and P6, to determine cell markers and cell characterization. The markers expressed by the
301
cells of Aedes aegypti embryonated eggs in P2 were: vimentin, Caspase 3, Stro-1, Nestin,
302
Sox2, HSP70, GM130 e RAB5 (Fig. 5A). InP4 the following markers were expressed:
303
Oct 3/4, Vimentin, Caspase 3, Stro-1, Nestin, Sox-2, HSP70, GM130 e RAB5 (Fig. 5A).
304
In the digestive tract, the markers: Caspase 3, Sox2, HSP70, GM130 and RAB5 (Fig. 5B)
305
were differentially expressed. In P4 of cell culture from fourth instar larvae intestine the
306
markers expressed were: Caspase 3, Sox2, HSP70, GM130 and RAB5 (Fig. 5B).
307 308 309 310
4. Discussion
10 311
The embryonic development of Aedes aegypti generally occurs 62 hours after
312
fertilization. At this stage the embryo has all features present in the first instar larvae
313
(Vital et al., 2010). Even without any water contact at ideal temperatures (12 °C to 35
314
°C), the egg keeps viable on average for a year (Farnesi et al., 2009). In the present study,
315
the embryos were at the end of the incubation period (after 62 hours), and at this stage
316
were divided into head, thorax and abdomen, in which eight abdominal segments were
317
identified, as described by Vital et al. (2010). The present study corroborates with the
318
morphological characteristics of Aedes aegypti intestines described by Clark et al. (2005),
319
Bernick et al. (2007).
320
Cell culture with insects encountered major problems with contaminants. This
321
data adds to the information presented by Lynn (1996), once the author reports that the
322
small size of cells tends to hinder their handling, in addition to the environment where the
323
insects live be predisposed to cell contamination, due to dirt accumulation and preexisting
324
microorganisms. The intestine is the largest organ in insect larvae due to its high need of
325
feeding during the immature period of its development, therefore the larvae ingest large
326
amounts of contaminants along with food (Lehane and Billingdley, 1996). A recent study
327
showed that the medium intestine lumen of Aedes is highly colonized by bacteria
328
(Gusmão et al., 2010), which complicates cell culture of the digestive tract.
329
Even so, the success of cell culture using insects was demonstrated for several
330
established cell lines. The development of a new cell line, into a new species, requires
331
adaptations to existing protocols (Lynn, 2001). Many studies have been made with insect
332
cells. Drosophila cell lines are largely used as experimental models for various
333
organisms. The two most studied cell lines are stem cells of ovarian follicles and intestinal
334
stem cells. A recent study with Drosophila middle posterior intestine cells aimed to create
335
a useful model for maintenance of adult intestinal stem cells (Roth, 2001; Navascués et
336
al., 2012; Panthak et al., 2012). There are several cell lines being studied in Drosophila.
337
Bello et al. (2008) studied neural stem cells in studies of neurogenesis in the brain of
338
larvae and proliferation of neuroblasts. In adult insects the medium intestine is
339
maintained/renewed by pluripotent stem cells. The digestive tract of vertebrates and
340
invertebrates show extensive similarity in their cell development and genetic control
341
(Ohlstein and Spradling, 2006).
342
Using immunohistochemistry, a positive staining to marker of cellular
343
proliferative activity, PCNA, was observed. This demonstrates the proliferative potential
11 344
of embryonated eggs, as observed by Vital et al. (2010). For cell pluripotency markers,
345
the Oct 3/4 showed low labeling where as Sox2 expression was higher. When these
346
markers are expressed in cells whose objective includes a cell treatment it is required
347
great attention due to the potential of these cells on differentiating into some types of
348
tissues, thus they have great chance of becoming genetically unstable (Abreu et al., 2008;
349
Carrion et al., 2009; Yarak and Okamoto, 2010).
350
The Stro-1 and vimentin mesenchymal cell markers were identified. When
351
isolated, these cells are largely used in regenerative medicine, due to its high cellular
352
differentiation capacity (Nikolić et al., 2013). In vertebrates, they can differentiate into
353
three cell types: osteogenic, adipogenic and chondrogenic (Bydlowski et al., 2009), but
354
in invertebrates only adipogenic is seen.
355
The specific insect markers, GM130 and RAB5, were expressed by the embryos.
356
These data are consistent with results reported by Wang et al. (2014) in which GM130 is
357
strongly expressed in Drosophila eyes, and Zschätzsch et al. (2014) where RAB5 was
358
expressed in areas where primary neurons are found.
359
In cell culture of Aedes aegypti several different cell types were seen. In a study
360
of Aedes aegypti cell lines larvae (Sudeep et al., 2009) the authors described three cell
361
types: epithelial (92%), fibroblast (7%) and giant cells (1%).
362
The data regarding the phases of cell cycle in cultures demonstrated that cells
363
underwent their natural processes in interphase and cell division. The cell cycle is
364
required to produce cellular copies responsible for maintaining dead cells of a tissue, and
365
also for embryonic development, in which the individual is formed from a single cell
366
through the processes of cell multiplication and differentiation (Albert, 2002).
367
The pluripotency Oct 3/4 and Sox2 markers were expressed in cell cultures.
368
According to Zang and Cui (2014), these markers are expressed in embryonic cells. The
369
mesenchymal cell markers, represented by Stro-1 and Vimentin, showed positive staining
370
in egg cultures but were not expressed in intestine cultures. These data are an addition to
371
those reported by Loeb et al. (2003) in Heliothis virescens intestine cell culture. The
372
following cell types were found: monolayer epithelium, goblet cells, columnar epithelial
373
cells and a few secretory stem cells which are responsible for replacement of intestinal
374
cells in adults. The apoptosis marker, caspase3, was present in all cell culture passages,
375
and its expression decreased in the sixth passage culture of intestine cells. Death by
376
apoptosis serves to maintain homeostasis in multicellular organisms (Meça et al., 2010;
377
Bernick, et al., 2008), as well as in embryonic tissues, when these proteins undergo self-
12 378
degenerating regression (Hengartner, 2000). Thus, as embryo cells are differentiating
379
over time it is common to find some cells in apoptosis.
380
The two specific markers used for Aedes aegypti cells had positive staining. The
381
first, GM130, a marker that expresses protein belonging to the Retremer group complex
382
involved in retrograde transport of proteins from endosomes to the Trans-Golgi network
383
of the Golgi apparatus. The Retremer complex is required for recycling rhodopsin. This,
384
in turn, is a protein coupled receptor that acts as the light sensor of the insect compound
385
eyes photoreceptors. The Drosophila has on average 800 hexagonal units, and each one
386
has about eight sites expressing rhodopsin (Wang et al., 2014). The second specific
387
marker, which is also present in endosomes, showed strong labeling in culture..
388
Zschätzsch et al. (2014) showed that at the beginning of endosome recycling the factor
389
EGFR is present (Epidermal Growth Factor Receptor) which is responsible for location
390
of primary neurons in Drosophila. The endocytosis regulates spatial distribution of EGFR
391
signaling in several types of epithelial cells in vitro and also of neurons in vivo
392
(Zschätzsch et al., 2014).
393
Many studies involving insect cell lines are being carried out, especially to
394
mosquitoes of medical importance such as Aedes aegypti. New studies of insect cells
395
infected with arboviruses are being conducted to improve results in the fight against these
396
diseases (Bello et al., 2008; Lawrie et al., 2004; Sudeep et al., 2009). For the first time, a
397
cell culture protocol was established for embryonated egg cells and digestive tube of
398
fourth instar Aedes aegypti larvae without contaminants. This allows to characterize
399
undifferentiated cells with great potential for cell proliferation. The morphological
400
description of the obtained cell of Aedes aegypti may contribute to the development of
401
new biotechnologies with the potential for combating diseases transmitted by Aedes
402
aegypti.
403 404
Acknowledgements
405 406
The authors acknowledge FAPESP (Support Foundation of São Paulo Research), for
407
funding this research [grant number 2013/07649-2]; researchers: Dr. Connie McManus,
408
Dr. Juliana Guimarães, Dr. Margareth de Lara Capurro-Guimalhães, Ms. Danilo de
409
Oliveira Carvalho, Dra. Valeria Cristina Soares Pinheiro, Dr. Vera Margarete Scarpassa,
410
Ms. Gersonval Leandro Monte, Dr. Ronildo Baiatone Alencar, Dr. Ormezinda Celeste
411
Fernandes Christ, Ms. Silmara Mundim, Dr. Maria Marta Antoniazzi, Dr. Claudia Regina
13 412
Stricagnolo; the technicians Dr. Rose Eli Grassi Rici; Ronaldo Augustine; Claudio
413
Arroyo, and to all who directly or indirectly contributed to the realization of this research.
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Roth, S., 2001. Drosophila oogenesis: coordinating germ line and soma. Curr. Biol. 2, 779-781.
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Wang, S., Tan, K. L., Agosto, M. A., Xiong, B., Yamamoto, S., Sandoval, H., Manish Jaiswal, M., Bayat, V., Zhang, K., Charng, W., David, G., Duraine, L., Venkatachalam, K., Wensel, T. G., Bellen, H. J., 2014. Theretromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration. Plos Biol. 12, 1001-1023.
Sahai-Hernandez, P., Castanieto, A., Nystul, T., 2012. G. Drosophila models of epithelial stem cells and their niches. Wiley Interdiscip Rev Dev Biol. 1, 447-57. Segura, N., Santamaría, E., Cabrera, O., Bello, F., 2012. Establishment and characterisation of a new cell line derived from Culex quinquefasciatus (Diptera: Culicidae). Mem. Inst. Oswaldo Cruz. 107, 89-95. Silva, H. H. G., Silva, I. G., Lira, K. S., 1998. Metodologia de criação de Aedes (Stegomyia) aegypti (Linnaeus, 1762) (Díptera Culicidae) em condições laboratoriais. Rev. Patol. Trop. 27, 53-63. Sudeep, A. B., Parashar, D., Jadi, R.S., Basu, A., Mokashi, C., Arankalle, V.V.A., Mishra, A. C., 2009. Establishment and characterization of a new Aedesa egypti (L.) (Diptera: Culicidae) cell line with special emphasis on virus susceptibility. In Vitro Cell Dev Biol Anim. 45, 491-49. Swevers, L., Iatrou, K., 2003. The ecdysone regulatory cascade and ovarian development in lepidopteran insects: insights from the silkmoth paradigm. Insect Biochem Molec Biol. 33, 1285-1297.
Vital,W., Rezende, G. L., Abreu, L., Moraes, J., Lemos, F. J.A., Silva Vaz Jr, I., Logullo, C., 2010. Retração banda Germ como um marco no metabolismo da glicose durante o Aedes aegypti embriogênese. BMC Dev Biol.10, 10-25.
Zhang, S., Cui, W., 2014. Sox2, a key factor in the regulation of pluripotency and neural differentiation. World J Stem Cells. 6, 305-311. Zschätzsch, M., Oliva, C., Langen, M., De Geest, N., Ozel, M. N., Williamson, W. R., Lemon, W. C., Soldano, A., Munck, S., Hiesinger, P. R., Sanchez-Soriano, N., Hassan, B. A., 2014. Regulation of branching dynamics by axon-intrinsic asymmetries in tyrosine kinase receptor signaling. eLife. 3, 1-23. Yarak, S., Okamoto, O. K., 2010. Células-tronco derivadas de tecido adiposo humano: desafios atuais e perspectivas clínicas. An. Bras. dermatol. 85, 647-656.
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Legends
604 605 606
Fig.1: Morphological analysis of embryonated eggs and of larval gut of Aedes aegypti.
607
In A, electron microscopy of an Aedes aegypti embryonated egg showing its flattened
608
shape (O). In B, photomicrograph of Aedes aegypti embryonated eggs, we observe
609
embryonic divisions: head (H), esophageal ganglia (*), thorax, abdomen and final portion
610
of the embryo (seta). In C, thoracic and abdominal segments (S) and intestinal cells
611
(arrowhead). In D, E and F dissection of the intestine of fourth instar of Aedes aegypti
612
larvae. Images were obtained using a stereomicroscope (Leica Mod. S6E). In D, superior
613
portion of the intestine with gastric cecum (Gc) and foregut (Fg). In E, midgut (Mg),
614
hindgut (Hg) and Malpighian tubules (Mt). In F, overview of the digestive tract, showing
615
the foregut (Fg), midgut (Mg), Malpighian tubules (Mt) and hindgut (Hp).
616 617
Fig. 2: Immunohistochemistry of Aedes aegypti embryo at the end of embryonic period.
618
In A, PCNA expression, nuclear marker for cell proliferation (arrow) in the thoracic
619
region (TR), abdominal region (AR) and posterior region (PR) from larvae. In B, positive
620
staining for Stro-1, endothelial marker expressed in the abdominal region. In C, there was
621
poor marking to Oct3/4, nuclear marker the abdominal segments, in Aedes aegypti
622
embryo (*). In D, strong labeling for Vimentin (arrow), cytoplasmic marker used to detect
623
proteins in the head region (HR), thoracic region (TR), abdominal region (AR). In E, there
624
was strong marking for Sox2 (arrow), nuclear marker in the head region (HR).In F, poor
625
marking to Nestin (*), a cytoplasmic marker expressed in the region of the head (HR),
626
thoracic region (TR) and abdomen region (AR), especially in the abdominal segments
627
(*).
18
628 629
Fig. 3: Immunohistochemistry of Aedes aegypti embryo at the end of embryonic period.
630
In A, RAB5 expression, cytoplasmic marker (arrow) for endosomal cells in Aedes aegypti
631
in the head region (HR), thoracic region (TR) and abdominal region (AR). In B, GM130
632
expression (arrow), cytoplasmic marker responsible for the Golgi Complex proteins, in
633
Aedes aegypti in the head region (HR), thoracic region (TR) and abdominal region (AR).
634
In C, positive control of GM130 marker, (arrow), in Drosophila spp showing the
19 635
compound eyes (CE) in the head. In D, positive control of RAB5 marker showing the
636
compound eyes (CE) in the head (arrow), in Drosophila spp.
637 638
Fig.4: Cell Culture of embryonated eggs and larval gut of Aedes aegypti and transmission
639
electron microscopy of the progenitor cells from embryonated eggs of Aedes aegypti . In
640
A embryonated eggs (arrow) in P2, magnification: 10x. In B, intestine cells (arrow) in
641
P2, magnification: 20x. In C and D embryonated eggs in P2, the cells had an ovoid shape
642
with nuclei (N) located in the periphery, dispersed and evident chromatin (Cr), and central
643
nucleolus (Nc). The plasmatic membrane (Pm) delimited the cytoplasm (Ct), which
644
showed several mitochondria (Mt), droplet vesicles (Dv), and lysosomes (Ly).
645
20
646 647
Fig.5: Cell cycle analysis by flow cytometry of Aedes aegypti embryonated eggs. In A,
648
the cell granularity analysis of culture in P1 (first passage). In B, the cell granularity
649
analysis of culture in P2 (second passage). In C, analysis of cell cycle phases cell debris
650
(sub-G1) G0/G1, S and G2M in cell culture from embryonated eggs in P1 and P2.
651 652
Fig.6: Analysis of Aedes aegypti embryonated eggs and larval gut markers by flow
653
citometry. In A, comparison of markers by flow cytometry of embryonated eggs from
21 654
Aedes aegypti in first and second passages. In B, comparison of markers by flow
655
cytometry of embryonated eggs from Aedes aegypti in fourth and sixth passages.
656 657 658
S0040-8166(16)30020-9 http://dx.doi.org/doi:10.1016/j.tice.2016.05.004 YTICE 1005
To appear in:
Tissue and Cell
Received date: Revised date: Accepted date:
28-1-2016 20-4-2016 14-5-2016
Please cite this article as: Mario, Lara C., Borghesi, J´essica, Crivellari-Damasceno, Wilson T., Favaron, Phelipe O., Carreira, Ana Claudia O., Will, Sonia E.A.L., Maria, Durvanei A., Miglino, Maria A., Egg and fourth instar larvae gut of Aedes aegypti as a source of stem cells.Tissue and Cell http://dx.doi.org/10.1016/j.tice.2016.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Egg and fourth instar larvae gut of Aedes aegypti as a source of stem cells
2 3
Lara C. Marioa,*, Jéssica Borghesia, Wilson T. Crivellari-Damasceno a,b, Phelipe O.
4
Favarona, Ana Claudia O. Carreirac, Sonia E. A. L. Willb, Durvanei A. Mariaa,b, Maria
5
A. Miglinoa
6 7
a Departament
of Surgery, School of Veterinary Medicine and Animal Science, University of Sao Paulo,
8
Sao Paulo, SP, Brazil
9 10
b Laboratory c Center
of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, SP, Brazil
for Molecular and Cellular Therapy (NUCEL) and Center for Cellular and Molecular Therapy
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(NETCEM), School of Medicine, University of Sao Paulo, Sao Paulo, SP, Brazil
12
* Corresponding author:
13
Email: [email protected]
14
Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, 05508-270, São Paulo-SP, Brasil.
15
Tel/Fax: 55 11 30917690
16 17 18 19 20 21 22 23
Highlights
Due the epidemiological importance of dengue, many investments in new research in genetic engineering and molecular biology have been conducted. Through characterization of stem cells of Aedes aegypti, we evaluated the morphology, expression of markers, and cell cycle phases. Studies with these cells may represent an important tool for biotechnology, resulting in innovative information for public health.
24 25
Abstract
26
According to the World Health Organization, 2015 registered more than 1.206.172 cases of Dengue in the
27
Americas. Recently, the Aedes aegypti has been not only related to Dengue, but also with cases of Zika
28
virus and Chikungunya. Due to its epidemiological importance, this study characterized the morphology of
29
the embryonated eggs of A. aegypti and provided a protocol to culture stem cells from eggs and digestive
30
tract of fourth instar larvae in order to examine cell biology and expression of markers in these vectors.
31
Cells were isolated and cultured in DMEM-High at 28 °C, and their morphology, cell cycle and
32
immunophenotyping were examined. Morphologically, embryos were at the end of the embryonic period
33
and showed: head, thorax, and abdomen with eight abdominal segments. The embryonic tissues expressed
34
markers related to cell proliferation (PCNA), pluripotency (Sox2 and OCT3/4), neural cells (Nestin),
35
mesenchymal cells (Vimentin and Stro-1), and endosomal cells (GM130 and RAB5). In culture, cells from
36
both tissues (eggs and larvae gut) were composed by a heterogeneous population. The cells had a globoid
2 37
shape and small size.. Cell cycle analysis on passage 1 (P1) showed 27.5% ±2.0% of cell debris, 68% of
38
cells on G0-G1 phase, 30.2% on S phase, 1.9% ± 0.5% on G2-M phase. In addition, cells on passage 2
39
showed: 10% of cell debris, 92.4% of cells on G0-G1 phase, 6.8% on S phase, 0.6% on G2-M phase.
40
Embryonated eggs expressed markers involved with pluripotency (Sox2 and Oct 3/4), mesenchymal cells
41
(vimentin and Stro-1), neural cells (Nestin), and cellular death by apoptosis (Caspase 3). Specific
42
endosomal markers for insect cells (GM130 and RAB5) were also highly expressed. In cell culture of Aedes
43
aegypti larvae gut the same labeling pattern was observed, with a small decrease in the expression of
44
mesenchymal (vimentin and Stro-1) and neural (Nestin) markers. In summary, we were able to establish a
45
protocol to culture embryonated eggs and larvae gut of Aedes aegypti, describing the characteristics of
46
undifferentiated cells, as well as the cell cycle and expression of markers, which can be used for
47
biotechnology studies for the biological control of this vector.
48 49
Keywords: Dengue; Cell culture; Morphology; Emerging diseases
50 51
1. Introduction
52
Dengue is an emerging disease that is spreading rapidly worldwide, with about
53
100 million people infected each year, which about 20,000 die (Brasil, 2009). According
54
to the World Health Organization, in 2015 it was registered approximately 1.206.172
55
cases of Dengue in the Americas. Brazil by itself contributes to 85% of the total number
56
of cases in the Americas. In addition, there are approximately 2.5 billion people at risk
57
worldwide (Americas, 2015).
58
The most important vector of dengue fever is Aedes aegypti and the infection is
59
caused by a virus of the genus Flavivirus, transmitted by the female to the host during
60
blood feeding (Gluber et al., 2007). This virus has four serotypes DENV1, DENV2,
61
DENV3 and DENV4. The simultaneous circulation of different serotypes increase
62
successive infections contributing to the emergence of new and more severe clinical
63
forms, such as dengue hemorrhagic disease, which usually occurs after the first infection
64
with a different serotype (Pessoa et al., 2005; Guzman, 2009).
65
Other diseases transmitted by the same mosquito include Chikungunya and Zika
66
virus. Chikungunya has resembling characteristics to those found in dengue, but the
67
transmitter arbovirus belongs to the Togaviridae family (Kuniholm et al., 2006). In 2014,
68
according to the Pan American Health Organization, 33 countries in the Americas have
69
reported incidence of this disease totaling 570.972 suspected cases (Organização Pan-
70
Americana, 2014). Zika fever, caused by virus Zika (ZIKV), an arbovirus of the genus
71
Flavivirus (Flaviviridae family), shows symptoms similar to those found in dengue as
72
fever, diarrhea, pain and a rash throughout the body. Recent focus of arbovirus found in
3 73
different regions worldwide, demonstrated that this virus has the potential to spread into
74
various territories where the Aedes vector is located (Organização Pan-Americana, 2015).
75
The study of insect cells began around 1915, but it was only in 1959 that it was
76
possible to cultivate cell prolongations and thereby understand morphogenesis in insects
77
(Grace, 1959). Today, research on insect cell lineages are used to investigate mechanisms
78
of infections control, biotechnologies through the development of transgenic insects, and
79
model of cell lines for mammals. The cell types most frequently studied in insects are
80
intestinal stem cells of medium intestine, used to understanding the control mechanisms
81
and renewal of intestinal cells, (Loeb et al., 1996; 1999; 2003; Micchelli and Perrimon,
82
2006; Ohlstein and Spradling, 2006; Baton and Ranford-Cartwright, 2007; Hakim et al.,
83
2009; Nardi et al., 2010; Navascués et al., 2012) and ovarian follicle, studied to determine
84
the development of insects and formation of recombinant proteins and bio-pesticides
85
(Silva et al.,1998; Swevers and Iatrou, 2003; Swevers et al., 2005; Belles and Piulachs,
86
2014; Jia et al., 2014).
87
The insect cell culture has been demonstrated by the establishment of more than
88
500 cell lineages, representing an important tool for applied biotechnology area (Segura
89
et al., 2012). In this sense, many mechanisms for infection control are being tested
90
including new tissue self-renewal models whereas insects have parallel parameters with
91
mammalian system, which make them useful models for the study of cellular maintenance
92
in other taxons (Navascues et al., 2012; Sahai-Hernandez et al., 2012).
93
Considering the epidemiological significance of dengue investments on new
94
research in genetic engineering and molecular biology have been conducted. Thus, this
95
study aimed to establish the first protocol to culture undifferentiated cells from Aedes
96
embryonated egg and larvae gut at fourth instars. In addition, we evaluated the cell cycle
97
phases on different passages and the expression of markers related mainly to pluripotency,
98
mesenchymal stem cells and apoptosis. Our data represent an important tool for
99
biotechnology, resulting in innovative information of great importance and applicability
100
for public health and to establish new methods of control of this species.
101 102
2. Material and Methods
103
Aedes aegypti eggs in the end of embryonic period, 60 hours after oviposition,
104
approximately and fourth instar larvae were used in this experiment. The project was
105
approved by Bioethics Committee of the School of Veterinary Medicine and Animal
106
Science of the University of Sao Paulo (Protocol number 2949/2013).
4 107 108
2.1. Collection of material
109 110
Eggs and larvae of Aedes aegypti were collected in the insect breeding at the
111
Maranhão State University (UEMA) in Caxias city, Maranhão, and from the Culicidae
112
facility Parasitology Department to ICB/BMP, University of São Paulo, São Paulo-SP.
113
After collection, the material was kept at room temperature for cell culture.
114 115
2.2. Microscopic characteristics of the embryo
116 117
Embryonated eggs of Aedes aegypti were fixed in 10% formaldehyde solution
118
Then, they were dehydrated in a series of ethanol (70% to 100%), diaphanized in xylene
119
twice for 5 minutes each, and embedded in paraffin (Histosec® - Merck, lot number
120
K91225309). The material was subjected to microtomy in order to obtain 3 µm thick
121
sections using an automatic microtome (Leica RM2165). The sections were placed on
122
slides and dried in an oven at 60 °C, and posteriorly stained with hematoxylin and eosin
123
(HE). Morphological characteristics were photodocumented using a Nikon Eclipse 80i
124
microscope (Nis Elements system).
125 126
2.3. Scanning electron microscopy
127 128
The embryonated eggs of Aedes aegypti were fixed in glutaraldehyde solution 2%
129
and washed three times in succession with sodium cacodylate buffer (pH 7.2) for 15
130
minutes. Then, the material was impregnated with osmium 1% for 1 hour, followed by
131
washes with buffer solution of sodium cacodylate for three times during 15 minutes.
132
Dehydration was then carried out in an increasing concentration series of ethanol (15%;
133
30%; 50%; 70%; 90%; 100%), for 15 minutes. The material was dried at the critical point
134
using carbon dioxide as a liquid of transitional fluid. The structures were mounted on
135
stubs, and then was used the sputter system to make the metal cover with gold. The images
136
were obtained using a scanning electron microscope FEI Quanta 250.
137 138
2.4. Transmission electron microscopy
139
The samples to be used for ultrastructural analysis were fixed in 3%
140
glutaraldehyde. After fixation, the material was treated with 1% osmium tetroxide (4%
5 141
osmium tetroxide w/w solution in water, Polysciences, Inc., USA) for one hour.
142
Following further washes in phosphate buffer, the fragments were dehydrated using a
143
series of increasing ethanol concentrations (70% to 100%) and immersed in propylene
144
oxide (Propylene oxide EM Grade, Polysciences, Inc., USA). The samples were
145
maintained
146
Polysciences, Inc., USA) and Spurr resin (Spurr’s kit – Electron Microscopy Sciences,
147
Co., USA) with stirring for a period of 12 hours and then in pure resin for four hours.
148
After this period, the samples were placed in moulds filled with pure resin and placed in
149
an oven at 60ºC for 72 hours to allow resin polymerisation. The samples were
150
subsequently cut with a Leica Ultracut UCT ultramicrotome to obtain 1-µm thin sections,
151
which were subsequently hot stained with a 1% aqueous sodium borate solution in
152
distilled water containing 0.25% toluidine blue for light microscopy analysis. Next,
153
ultrathin sections (approximately 60 nm thick) were placed on copper screens and
154
contrasted with 2% uranyl acetate in distilled water for five minutes and 0.5% lead citrate
155
in distilled water for 10 minutes. Ultrastructures were finally analysed and photographed
156
with a Morgagni 268D microscope.
in
a 1:1 mixture of propylene
oxide (Propylene
oxide
EM
Grade,
157 158
2.5. Immunohistochemistry
159 160
Immunohistochemical reactions were performed in sections of Aedes aegypti embryos
161
for: Stro-1 (1:200, mouse monoclonal Santa Cruz Biotechnology, antibody sc-47733, Inc,
162
Europe); PCNA (1:200, mouse monoclonal Santa Cruz Biotechnology, antibody sc-56,
163
Inc, Europe); Oct 3/4 (1:100, rabbit, policlonal Abcam antibody ab-137427); Vimentin
164
(1:100, mouse monoclonal anti-human Santa Cruz Biotechnology, antibody sc-73259,
165
Inc, Europe); Stro-1 (1:100, Mouse monoclonal sc-7733, Santa Cruz Biotechnology, Inc,
166
Europe); Nestin (1:100, mouse monoclonal, Santa Cruz, antibody sc-33677 Inc, Europe);
167
Sox2 (1:100, goat polyclonal, Santa Cruz, antibody sc-17320 Inc, Europe); GM130
168
(rabbit polyclonal, Abcam, antibody ab- 30637, Cambridge, MA, USA); RAB5 (rabbit
169
polyclonal, Abcam, antibody ab-31261, Cambridge, MA, USA). The negative control of
170
the immunohistochemical reactions was performed using IgG (Goat anti-Mouse IgG - AP
171
308F, Chemical International, Temecula, California, USA).
172
Sections were deparaffinized in xylol and subsequently blocked in peroxidase
173
(H2O2). Then they were treated with citrate buffer 0.1M and irradiated in a microwave
174
oven. Subsequently, they were balanced with the Dako Protein Block kit (X 0909, Dako
6 175
Cytomation, Carpinteria, CA, USA). The incubation with primary antibodies were
176
performed overnight at 4 °C. After this, sections were washed in Phosphate Buffered
177
Saline (PBS) and incubated with secondary conjugated antibody using the peroxidase Kit
178
Dako LSAB (K 0690, Dako Cytomation, Carpinteria, CA, USA) followed by
179
streptoavidin
180
Cytomation, Carpinteria, CA, USA), followed by counterstaining with hematoxylin.
from the same kit. The reaction was visualized from DAB (Dako
181 182
2.5. Aedes aegypti eggs and intestine cell culture
183 184
The Aedes aegypti eggs were placed in a pestle, washed twice with Tween 20
185
solution (LGC Biotechnology, code: 13-1316.05) at 20% and 10%. Then, the material
186
was washed four times using PBS solution for 5 minutes, and incubated for 5 minutes in
187
antibiotic solution containing gentamicin (Sigma Life Science, code: G1272 - 10 mL),
188
penicillin and streptomycin (Gibco by Life Technologies, code: 15140-122 Pen 10.000
189
units/mL and Strep 10.000 µg/mL), and amphotericin B (Sigma Life Science, code:
190
A2942- 20 mL, 20µg/mL), then washed in PBS. Thereafter, 2mL of Dulbecco´s Modified
191
Eagle Medium (DMEM-High Glucose) was added and eggs were macerated with a pestle.
192
Then, they were cultured in a 25cm2 culture flask with DMEM-High supplemented with
193
10% Fetal Bovine Serum (FBS) and 1% antibiotic solution (gentamicin, streptomycin,
194
penicillin and amphotericin B) in an incubator at 28°C ± 2°C. In order to promote the
195
expansion, the cells were centrifuged at 100 g for 5 minutes, and the pellet was
196
resuspended in culture medium and divided in the plates. The cells were cultured in the
197
first, second and fourth passages.
198
Intestine cell culture was from the fourth instar larvae. With the aid of a Leica
199
S6E stereomicroscope, the larvae were placed on a slide. The abdomen was opening in
200
order to remove the digestive tract. Then, the digestive tract was passed through a cell
201
sieve (Greiner bio-one, Easystrainer- pore 40 microns, code: 542.040), and washed three
202
times with antibiotic solution (Penicillin-Streptomycin and PBS in the ratio 1:100). After
203
treatment, the intestines were placed in 30 mm culture plates with 2 mL DMEM-High,
204
containing fragments of the material to promote cell proliferation. Then, the plate was
205
placed in an oven at 28 ± 2°C and the cells from the fourth and sixth passages were used
206
in the experiments.
207 208
Cellular morphology was examined and photographed every 24 hours using an inverted microscope Nikon Eclipse TS-100.
7 209 210
2.6. Analysis of the cell cycle phases
211 212
For the cell cycle, 106 cells were centrifuged at 100 g for 5 minutes in 1 mL FACS
213
Flow buffer (PBS, EDTA 2 mM, SFB 2%). The supernatant was discarded and the pellet
214
re-suspended in 100 µL of FACS buffer. Then was added 10 µL of Triton X-100 and 5
215
uL of 1.8µg/mL propidium iodide (Sigma, P4170, 1 mg/mL). Samples were analyzed
216
using a BD FACS Calibur flow cytometer and analyzed by software Modfit LT version
217
2.9 program.
218 219
2.7. Immunophenotyping by flow cytometry
220 221
For each sample were used 106 cells. The material was centrifuged at 200 g for 10
222
minutes. After centrifugation, the pellet was re-suspended in 1 mL of Phosphate Buffered
223
Saline and centrifuged again at 200 g for 10 minutes. Thereafter, the pellet was re-
224
suspended in FACsFlow buffer, then transferred to cytometry tubes, and incubated with
225
the following antibodies: Oct 3/4 (1:100, rabbit policlonal, Abcam antibody ab-137427
226
Cambridge, MA, USA); Vimentin (1:100, mouse monoclonal anti-human Santa Cruz
227
Biotechnology, antibody sc-73259, Inc, Europe); Caspase 3 (1:100, mouse monoclonal,
228
Santa Cruz, antibody, sc-271759 Inc, Europe); Stro-1 (1:100, Mouse monoclonal sc-
229
7733, Santa Cruz Biotechnology, Inc, Europe); Nestin (1:100, mouse monoclonal, Santa
230
Cruz, antibody sc-33677 Inc, Europe); Sox2 (1:100, goat polyclonal, Santa Cruz,
231
antibody sc-17320 Inc, Europe); HSP70 (mouse monoclonal, Abcam antibody ab-2787
232
Cambridge, MA, USA); GM130 (rabbit polyclonal, Abcam, antibody ab- 30637
233
Cambridge, MA, USA); RAB5 (rabbit polyclonal, Abcam, antibody ab-31261
234
Cambridge, MA, USA). After 30 minutes, the samples were centrifuged at 200 g for 10
235
minutes. Unbound antibody was washed 2 times using PBS. Then, the pellet was
236
incubated with secondary antibody (1:500 goat anti-mouse Life Technologies A11017,
237
Alexa Fluor 488 or 1:500 donkey anti-rabbit Abcam150061) for 30 minutes, centrifuged,
238
washed again using PBS and fixed in paraformaldehyde 4% for analysis.
239 240
The analysis of cellular markers expression was performed in a FACS Calibur flow cytometer and interpreted using the WinMDI 2.9 program.
241 242
3. Results
8 243 244
3.1. Morphological analysis of Aedes aegypti embryonic tissue
245 246
At the end of the incubation period the eggs were, on average, 1 mm length and
247
had a flattened shape (Fig. 1A). The histological examination of the embryos in the Aedes
248
aegypti eggs showed that they were divided into: head, thorax and abdomen, with eight
249
abdominal segments (Fig. 1B and C). The digestive tract morphology of fourth instar
250
larvae Aedes aegypti (Fig. D) showed the presence of the anterior, middle and posterior
251
intestine. In the anterior region, gastric blinds were observed, and from the end of the
252
midgut to the beginning of the posterior intestine the presence of malpighian tubule was
253
observed (Fig.1 E and F).
254 255
3.2. Immunohistochemistry
256 257
After morphological analysis, immunohistochemistry tests were carried out on
258
embryonated eggs of Aedes aegypti at the end of the embryonic period. Related to the
259
expression of cell proliferation markers, the PCNA marker was positive. However, for
260
the pluripotency markers Oct 3/4 was less expressed when compared to Sox2.
261
Mesenchymal markers as Vimentin and Stro-1 were also tested, which were positively
262
expressed in these cells. A positive expression was observed for nerve marker Nestin.
263
The specific markers of Drosophila tested on Aedes aegypti, GM130 and RAB5, were
264
positive for Golgi complex and endosomal cells, respectively. To confirm these last two
265
markers a positive control was done on adult cells of Drosophila spp. (Fig.2).
266 267
3.3. Cell isolation and characterization
268 269 270
The culture of embryonated eggs was performed using approximately 100-150 eggs with fourth instar Aedes aegypti larvae in which only the intestines were used.
271
The cells obtained from cellular maceration of embryonated eggs were classified
272
accordingly to their form, being small and globoid, and with colony formation. The insect
273
cells grew rapidly after 24 hours of culture, and needed to be expanded to a second
274
passage (Fig. 3 A and B). The gut cells were small and globular, maintaining this format
275
until the sixth passage (P6) (Fig. 3 C and D).
276
9 277
3.4. Cell Morphology by flow cytometry
278 279
The quantitative analysis of Aedes aegypti cell types showed the presence of cells
280
with granularities and different sizes at culture expressed first passage (P1) and P2. This
281
assay occurred concomitantly with the analysis of the cell cycle phases. In P1, it was
282
observed that the different cell populations were uniform in size but with different
283
densities (Fig. 4A). Analyzing the graphs in P2 can be observed a similarity in cell
284
population (Fig. 4B).
285 286 287
3.5. Analysis of the cell cycle phases
288 289
The phases of the cell cycle had, on average, 27.5% ± 2.0% cellular debris in P1, 68%
290
G0-G1 of the rest, where the cells were at the beginning of cell growth, 30.2% in S-phase
291
and 1.9% ± 0.5% in G2-M phase, indicating that the cells were in preparation for cell
292
division. Then samples were analyzed after the P2 in cell culture, and the following values
293
were obtained: 10% of cell debris and of the cells 92.4% were in the G0-G1 phase, 6.8%
294
in S-phase, and 0.6% in G2-M phase (Fig.4C).
295 296
3.6. Immunophenotyping by flow cytometry
297 298
Immunophenotyping was performed by flow cytometry of Aedes aegypti
299
embryonated eggs in P2 and P4, and from the digestive tube of fourth instar larvae, at P4
300
and P6, to determine cell markers and cell characterization. The markers expressed by the
301
cells of Aedes aegypti embryonated eggs in P2 were: vimentin, Caspase 3, Stro-1, Nestin,
302
Sox2, HSP70, GM130 e RAB5 (Fig. 5A). InP4 the following markers were expressed:
303
Oct 3/4, Vimentin, Caspase 3, Stro-1, Nestin, Sox-2, HSP70, GM130 e RAB5 (Fig. 5A).
304
In the digestive tract, the markers: Caspase 3, Sox2, HSP70, GM130 and RAB5 (Fig. 5B)
305
were differentially expressed. In P4 of cell culture from fourth instar larvae intestine the
306
markers expressed were: Caspase 3, Sox2, HSP70, GM130 and RAB5 (Fig. 5B).
307 308 309 310
4. Discussion
10 311
The embryonic development of Aedes aegypti generally occurs 62 hours after
312
fertilization. At this stage the embryo has all features present in the first instar larvae
313
(Vital et al., 2010). Even without any water contact at ideal temperatures (12 °C to 35
314
°C), the egg keeps viable on average for a year (Farnesi et al., 2009). In the present study,
315
the embryos were at the end of the incubation period (after 62 hours), and at this stage
316
were divided into head, thorax and abdomen, in which eight abdominal segments were
317
identified, as described by Vital et al. (2010). The present study corroborates with the
318
morphological characteristics of Aedes aegypti intestines described by Clark et al. (2005),
319
Bernick et al. (2007).
320
Cell culture with insects encountered major problems with contaminants. This
321
data adds to the information presented by Lynn (1996), once the author reports that the
322
small size of cells tends to hinder their handling, in addition to the environment where the
323
insects live be predisposed to cell contamination, due to dirt accumulation and preexisting
324
microorganisms. The intestine is the largest organ in insect larvae due to its high need of
325
feeding during the immature period of its development, therefore the larvae ingest large
326
amounts of contaminants along with food (Lehane and Billingdley, 1996). A recent study
327
showed that the medium intestine lumen of Aedes is highly colonized by bacteria
328
(Gusmão et al., 2010), which complicates cell culture of the digestive tract.
329
Even so, the success of cell culture using insects was demonstrated for several
330
established cell lines. The development of a new cell line, into a new species, requires
331
adaptations to existing protocols (Lynn, 2001). Many studies have been made with insect
332
cells. Drosophila cell lines are largely used as experimental models for various
333
organisms. The two most studied cell lines are stem cells of ovarian follicles and intestinal
334
stem cells. A recent study with Drosophila middle posterior intestine cells aimed to create
335
a useful model for maintenance of adult intestinal stem cells (Roth, 2001; Navascués et
336
al., 2012; Panthak et al., 2012). There are several cell lines being studied in Drosophila.
337
Bello et al. (2008) studied neural stem cells in studies of neurogenesis in the brain of
338
larvae and proliferation of neuroblasts. In adult insects the medium intestine is
339
maintained/renewed by pluripotent stem cells. The digestive tract of vertebrates and
340
invertebrates show extensive similarity in their cell development and genetic control
341
(Ohlstein and Spradling, 2006).
342
Using immunohistochemistry, a positive staining to marker of cellular
343
proliferative activity, PCNA, was observed. This demonstrates the proliferative potential
11 344
of embryonated eggs, as observed by Vital et al. (2010). For cell pluripotency markers,
345
the Oct 3/4 showed low labeling where as Sox2 expression was higher. When these
346
markers are expressed in cells whose objective includes a cell treatment it is required
347
great attention due to the potential of these cells on differentiating into some types of
348
tissues, thus they have great chance of becoming genetically unstable (Abreu et al., 2008;
349
Carrion et al., 2009; Yarak and Okamoto, 2010).
350
The Stro-1 and vimentin mesenchymal cell markers were identified. When
351
isolated, these cells are largely used in regenerative medicine, due to its high cellular
352
differentiation capacity (Nikolić et al., 2013). In vertebrates, they can differentiate into
353
three cell types: osteogenic, adipogenic and chondrogenic (Bydlowski et al., 2009), but
354
in invertebrates only adipogenic is seen.
355
The specific insect markers, GM130 and RAB5, were expressed by the embryos.
356
These data are consistent with results reported by Wang et al. (2014) in which GM130 is
357
strongly expressed in Drosophila eyes, and Zschätzsch et al. (2014) where RAB5 was
358
expressed in areas where primary neurons are found.
359
In cell culture of Aedes aegypti several different cell types were seen. In a study
360
of Aedes aegypti cell lines larvae (Sudeep et al., 2009) the authors described three cell
361
types: epithelial (92%), fibroblast (7%) and giant cells (1%).
362
The data regarding the phases of cell cycle in cultures demonstrated that cells
363
underwent their natural processes in interphase and cell division. The cell cycle is
364
required to produce cellular copies responsible for maintaining dead cells of a tissue, and
365
also for embryonic development, in which the individual is formed from a single cell
366
through the processes of cell multiplication and differentiation (Albert, 2002).
367
The pluripotency Oct 3/4 and Sox2 markers were expressed in cell cultures.
368
According to Zang and Cui (2014), these markers are expressed in embryonic cells. The
369
mesenchymal cell markers, represented by Stro-1 and Vimentin, showed positive staining
370
in egg cultures but were not expressed in intestine cultures. These data are an addition to
371
those reported by Loeb et al. (2003) in Heliothis virescens intestine cell culture. The
372
following cell types were found: monolayer epithelium, goblet cells, columnar epithelial
373
cells and a few secretory stem cells which are responsible for replacement of intestinal
374
cells in adults. The apoptosis marker, caspase3, was present in all cell culture passages,
375
and its expression decreased in the sixth passage culture of intestine cells. Death by
376
apoptosis serves to maintain homeostasis in multicellular organisms (Meça et al., 2010;
377
Bernick, et al., 2008), as well as in embryonic tissues, when these proteins undergo self-
12 378
degenerating regression (Hengartner, 2000). Thus, as embryo cells are differentiating
379
over time it is common to find some cells in apoptosis.
380
The two specific markers used for Aedes aegypti cells had positive staining. The
381
first, GM130, a marker that expresses protein belonging to the Retremer group complex
382
involved in retrograde transport of proteins from endosomes to the Trans-Golgi network
383
of the Golgi apparatus. The Retremer complex is required for recycling rhodopsin. This,
384
in turn, is a protein coupled receptor that acts as the light sensor of the insect compound
385
eyes photoreceptors. The Drosophila has on average 800 hexagonal units, and each one
386
has about eight sites expressing rhodopsin (Wang et al., 2014). The second specific
387
marker, which is also present in endosomes, showed strong labeling in culture..
388
Zschätzsch et al. (2014) showed that at the beginning of endosome recycling the factor
389
EGFR is present (Epidermal Growth Factor Receptor) which is responsible for location
390
of primary neurons in Drosophila. The endocytosis regulates spatial distribution of EGFR
391
signaling in several types of epithelial cells in vitro and also of neurons in vivo
392
(Zschätzsch et al., 2014).
393
Many studies involving insect cell lines are being carried out, especially to
394
mosquitoes of medical importance such as Aedes aegypti. New studies of insect cells
395
infected with arboviruses are being conducted to improve results in the fight against these
396
diseases (Bello et al., 2008; Lawrie et al., 2004; Sudeep et al., 2009). For the first time, a
397
cell culture protocol was established for embryonated egg cells and digestive tube of
398
fourth instar Aedes aegypti larvae without contaminants. This allows to characterize
399
undifferentiated cells with great potential for cell proliferation. The morphological
400
description of the obtained cell of Aedes aegypti may contribute to the development of
401
new biotechnologies with the potential for combating diseases transmitted by Aedes
402
aegypti.
403 404
Acknowledgements
405 406
The authors acknowledge FAPESP (Support Foundation of São Paulo Research), for
407
funding this research [grant number 2013/07649-2]; researchers: Dr. Connie McManus,
408
Dr. Juliana Guimarães, Dr. Margareth de Lara Capurro-Guimalhães, Ms. Danilo de
409
Oliveira Carvalho, Dra. Valeria Cristina Soares Pinheiro, Dr. Vera Margarete Scarpassa,
410
Ms. Gersonval Leandro Monte, Dr. Ronildo Baiatone Alencar, Dr. Ormezinda Celeste
411
Fernandes Christ, Ms. Silmara Mundim, Dr. Maria Marta Antoniazzi, Dr. Claudia Regina
13 412
Stricagnolo; the technicians Dr. Rose Eli Grassi Rici; Ronaldo Augustine; Claudio
413
Arroyo, and to all who directly or indirectly contributed to the realization of this research.
414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460
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Legends
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Fig.1: Morphological analysis of embryonated eggs and of larval gut of Aedes aegypti.
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In A, electron microscopy of an Aedes aegypti embryonated egg showing its flattened
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shape (O). In B, photomicrograph of Aedes aegypti embryonated eggs, we observe
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embryonic divisions: head (H), esophageal ganglia (*), thorax, abdomen and final portion
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of the embryo (seta). In C, thoracic and abdominal segments (S) and intestinal cells
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(arrowhead). In D, E and F dissection of the intestine of fourth instar of Aedes aegypti
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larvae. Images were obtained using a stereomicroscope (Leica Mod. S6E). In D, superior
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portion of the intestine with gastric cecum (Gc) and foregut (Fg). In E, midgut (Mg),
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hindgut (Hg) and Malpighian tubules (Mt). In F, overview of the digestive tract, showing
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the foregut (Fg), midgut (Mg), Malpighian tubules (Mt) and hindgut (Hp).
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Fig. 2: Immunohistochemistry of Aedes aegypti embryo at the end of embryonic period.
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In A, PCNA expression, nuclear marker for cell proliferation (arrow) in the thoracic
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region (TR), abdominal region (AR) and posterior region (PR) from larvae. In B, positive
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staining for Stro-1, endothelial marker expressed in the abdominal region. In C, there was
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poor marking to Oct3/4, nuclear marker the abdominal segments, in Aedes aegypti
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embryo (*). In D, strong labeling for Vimentin (arrow), cytoplasmic marker used to detect
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proteins in the head region (HR), thoracic region (TR), abdominal region (AR). In E, there
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was strong marking for Sox2 (arrow), nuclear marker in the head region (HR).In F, poor
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marking to Nestin (*), a cytoplasmic marker expressed in the region of the head (HR),
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thoracic region (TR) and abdomen region (AR), especially in the abdominal segments
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(*).
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Fig. 3: Immunohistochemistry of Aedes aegypti embryo at the end of embryonic period.
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In A, RAB5 expression, cytoplasmic marker (arrow) for endosomal cells in Aedes aegypti
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in the head region (HR), thoracic region (TR) and abdominal region (AR). In B, GM130
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expression (arrow), cytoplasmic marker responsible for the Golgi Complex proteins, in
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Aedes aegypti in the head region (HR), thoracic region (TR) and abdominal region (AR).
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In C, positive control of GM130 marker, (arrow), in Drosophila spp showing the
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compound eyes (CE) in the head. In D, positive control of RAB5 marker showing the
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compound eyes (CE) in the head (arrow), in Drosophila spp.
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Fig.4: Cell Culture of embryonated eggs and larval gut of Aedes aegypti and transmission
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electron microscopy of the progenitor cells from embryonated eggs of Aedes aegypti . In
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A embryonated eggs (arrow) in P2, magnification: 10x. In B, intestine cells (arrow) in
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P2, magnification: 20x. In C and D embryonated eggs in P2, the cells had an ovoid shape
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with nuclei (N) located in the periphery, dispersed and evident chromatin (Cr), and central
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nucleolus (Nc). The plasmatic membrane (Pm) delimited the cytoplasm (Ct), which
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showed several mitochondria (Mt), droplet vesicles (Dv), and lysosomes (Ly).
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Fig.5: Cell cycle analysis by flow cytometry of Aedes aegypti embryonated eggs. In A,
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the cell granularity analysis of culture in P1 (first passage). In B, the cell granularity
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analysis of culture in P2 (second passage). In C, analysis of cell cycle phases cell debris
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(sub-G1) G0/G1, S and G2M in cell culture from embryonated eggs in P1 and P2.
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Fig.6: Analysis of Aedes aegypti embryonated eggs and larval gut markers by flow
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citometry. In A, comparison of markers by flow cytometry of embryonated eggs from
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Aedes aegypti in first and second passages. In B, comparison of markers by flow
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cytometry of embryonated eggs from Aedes aegypti in fourth and sixth passages.
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