Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment
© The Author(s). 2016
Received: 23 March 2016
Accepted: 12 July 2016
Published: 25 July 2016
Leishmania parasites are transmitted by phlebotomine sand flies and a crucial step in their life-cycle is the binding to the sand fly midgut. Laboratory studies on sand fly competence to Leishmania parasites suggest that the sand flies fall into two groups: several species are termed “specific/restricted” vectors that support the development of one Leishmania species only, while the others belong to so-called “permissive” vectors susceptible to a wide range of Leishmania species. In a previous study we revealed a correlation between specificity vs permissivity of the vector and glycosylation of its midgut proteins. Lutzomyia longipalpis and other four permissive species tested possessed O-linked glycoproteins whereas none were detected in three specific vectors examined.
We used a combination of biochemical, molecular and parasitological approaches to characterize biochemical and biological properties of O-linked glycoprotein of Lu. longipalpis. Lectin blotting and mass spectrometry revealed that this molecule with an apparent molecular weight about 45–50 kDa corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis. We produced a recombinant glycoprotein rLuloG with molecular weight around 45 kDa. Anti-rLuloG antibodies localize the native glycoprotein on epithelial midgut surface of Lu. longipalpis. Although we could not prove involvement of LuloG in Leishmania attachment by blocking the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of Leishmania promastigotes.
We characterized a novel O-glycoprotein from sand fly Lutzomyia longipalpis. It has mucin-like properties and is localized on the luminal side of the midgut epithelium. Recombinant form of the protein binds to Leishmania parasites in vitro. We propose a role of this molecule in Leishmania attachment to sand fly midgut.
Leishmania protozoans, the causative agents of leishmaniases, are transmitted by bites of female phlebotomine sand flies. In the sand fly vector, Leishmania parasites must overcome various barriers to generate transmissible infections and ensure continuation of the life-cycle (reviewed by ). A crucial step in the vector phase of the Leishmania life-cycle is the binding of promastigotes to the sand fly midgut. Promastigotes insert their flagella into the microvillar border of the midgut epithelium and anchor themselves to the midgut surface [2, 3]. This attachment enables them to persist in the sand fly gut when the blood meal remnants are defecated.
A series of studies on the parasite-vector combination Leishmania major-Phlebotomus papatasi established an important paradigm; galactose residues on mono-galactosylated phosphodisaccharide repeats of the major surface glycoconjugate lipophosphoglycan (LPG) have been shown to bind to a galectin located on the surface of the sand fly midgut [4–7]. However, a more recent study reported partial involvement of the flagellar protein FLAG1/SMP1 in attachment of L. major to the midgut epithelium of P. papatasi , and earlier experiments with LPG-deficient mutants revealed that LPG is not required for the Leishmania attachment in many other sand fly species [9–11]. These studies indicate that alternative attachment molecules can be involved in midgut binding in addition to LPG-galectin.
Laboratory studies on vector competence to Leishmania parasites suggest that sand flies fall into two broad groups. Three species, i.e. P. papatasi, P. duboscqi and P. sergenti, are termed “specific” or “restricted” vectors that support the development of one Leishmania species only (L. major and L. tropica, respectively). Other sand fly species, importantly including vectors of the visceralising parasites L. donovani and L. infantum, belong to so-called “permissive” vectors, which are susceptible for a wide range of Leishmania spp. In laboratory conditions Leishmania parasites are capable of developing in any permissive vector, if given the opportunity (reviewed by [1, 12]).
In a study exploring the molecular basis of vector competence Myšková and colleagues  revealed a remarkable correlation between specificity vs permissivity of the vector and the glycosylation of its midgut proteins. The Helix pomatia agglutinin (HPA), a lectin specific for terminal N-acetyl-galactosamine (GalNAc) present on O-linked glycoconjugates, bound to midgut proteins from permissive but not from specific vectors. All five permissive species tested possessed HPA positive bands, whereas none were detected in the three specific vectors examined [9, 10, 13]. These findings suggested a hypothesis for the role of O-linked glycoconjugates in Leishmania binding, which was supported by two further observations. Fluorescent-labelled HPA (FITC-HPA) showed specific localisation of O-glycosylated epitopes in the microvillar border of sand fly midgut, the location required for Leishmania attachment, and the O-linked glycoconjugates recognised by FITC-HPA bound to the surface of Leishmania promastigotes .
In the present work we describe an O-linked glycoconjugate of the permissive vector Lu. longipalpis and show that it has mucin-like properties. Functional testing was performed using the recombinant form of the Lu. longipalpis glycoconjugate (rLuloG) and specific anti-rLuloG antibodies using in vivo and in vitro methods. For in vivo studies, experimental infections of Lu. longipalpis with L. infantum were performed as described previously for L. major and P. papatasi galectin : L. infantum parasites were mixed with blood reconstituted with specific anti- rLuloG serum to test for blocking of parasite development in the sand fly midgut. In vitro experiments were performed to investigate the binding of rLuloG to Leishmania parasites.
Sand fly colonies and parasites
Laboratory colonies of two sand fly species were used: Lutzomyia longipalpis (origin from Jacobina, Brazil) and Phlebotomus papatasi (origin from Turkey). Colonies were maintained in conditions described previously  and fed on 50 % sucrose. Most experiments were done with 4–7 day-old females, and the expression of LuloG was compared in females aged from 1 to 12 days.
The following parasite isolates were used: Leishmania infantum (MHOM/BR/76/M4192) and Leishmania major LV561 (MHOM/IL/67/LRC-L137). Parasites were maintained at 26 °C on Medium 199 supplemented with 20 % foetal bovine serum (Thermo Fisher Scientific, Waltham, USA). Prior to sand fly infections, Leishmania parasites were washed by centrifugation and resuspended in saline.
Detection of O-linked glycoconjugates in Lutzomyia longipalpis midguts
Female midguts were homogenized mechanically by repeated freezing and thawing, proteins were separated by SDS-PAGE (10 % gel, reducing conditions, 10 guts per lane), transferred to nitrocellulose and analyzed by lectin blotting. The nitrocellulose membrane was incubated with Tris-NaCl-Tween (20 mM Tris, 150 mM NaCl, pH 7.6) and blocked with 5 % (w/v) bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, USA; diluted in Tris-NaCl-Tween) overnight at 4 °C and then for 1 h at room temperature with biotinylated Helix pomatia agglutinin (HPA; 1 μg/ml; Sigma-Aldrich). After repeated washing the blots were incubated with streptavidin peroxidase (2.5 μg/ml; Sigma-Aldrich, St. Louis, USA; in Tris-NaCl-Tween) and the peroxidase reaction was developed in 4-chloro-1-naphthol solution (Sigma-Aldrich). Experiments were done with females of various ages and physiological states (e.g. females which did not feed on blood, females after blood-feeding) as indicated.
Characterization of the midgut glycoconjugate
Lysates were prepared from 20 midguts of Lu. longipalpis females. Triton X-114 was used for separation of hydrophilic and amphiphilic proteins as previously decribed by Bordier  with following modifications: the lysates were incubated with the 1 % Triton X-114 for 90 min on ice and the separation was done without sucrose cushion. Triton X-114 is a non-ionic detergent with a low cloud point (about 20–23 °C) enabling protein solubilization with phase-partitioning of hydrophilic from amphiphilic proteins. The aqueous and the detergent phase were then analyzed by SDS-PAGE and blotting with HPA (as decribed above). To study the presence of a glycophosphatidylinositol (GPI) anchor, lysates in Tris-NaCl were incubated for 1.5 h at 37 °C with protease inhibitors (Complete, Roche, Mannheim, Germany) and 1 unit phosphatidylinositol-specific phospholipase C (PI-PLC), a highly purified enzyme prepared from Bacillus cereus (Sigma-Aldrich). The sample was then fractionated with Triton X-114 and analyzed by SDS-PAGE followed by blotting with HPA.
To check the isoelectric point of the candidate glycoconjugate, a lysate of 50 midguts was analyzed by 2D electrophoresis (IEF/SDS-PAGE), followed by blotting with HPA incubation as described above.
To generate sufficient material for protein identification, HPA affinity chromatography was used to purify the O-linked glycoconjugate from midguts of Lu. longipalpis females. Lysates of 1400 midguts were incubated with 9 units of PI-PLC in Tris-NaCl and protease inhibitors (Complete, Roche) for 1.5 h at 37 °C and loaded onto a HPA-agarose column (Sigma-Aldrich). After washing, specifically bound molecules were eluted with 0.5 M GalNAc in Tris-NaCl and analyzed by SDS-PAGE (10 % gel, reducing conditions) or 2D electrophoresis (IEF/SDS-PAGE). The glycoconjugate band/spot was stained by Pro-Q-Emerald glycoprotein stain (Thermo Fisher Scientific), a technology available for detection of glycoconjugates in gels. The protein band/spot was excised from the gel, cut into small pieces and washed using 50 mM 4-ethylmorpholine acetate (pH 8.1) in 50 % acetonitrile (MeCN). The protein was reduced with 30 mM Tris(2-carboxyethyl)phosphine (TCEP) in 100 mM Tris-HCl (pH 8.0) at 65 °C for 30 min and alkylated by 30 mM iodacetamide in 100 mM Tris-HCl (pH 8.0) for 60 min in the dark. The gel pieces were then incubated overnight at 37 °C in a cleavage buffer containing 25 mM 4-ethylmorpholine acetate, 5 % MeCN and trypsin (100 ng; Promega, Woods, USA) or Asp-N protease (30 ng; Roche). The resulting peptides were extracted into 40 % MeCN/0.1 % TFA. One μl of the peptide mixture was deposited on a MALDI plate, allowed to air-dry at room temperature and overlaid with 0.4 μl of MALDI matrix (aqueous 50 % MeCN/0.1 % TFA solution of α-cyano-4-hydroxycinnamic acid; 5 mg/ml; Sigma-Aldrich). Mass spectra were measured on an Ultraflex III MALDI-TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with LIFT technology for MS/MS analysis. MS data were searched using an in-house MASCOT search engine against the NCBI nr database subset of eukaryotic proteins.
Expression and purification of rLuloG in H5 insect cells
RNA was isolated from 10 midguts of Lutzomyia longipalpis females using a High Pure RNA isolation kit (Roche) and reverse-transcribed using Superscript III reverse transcriptase (Thermo Fisher Scientific) following the manufacturer’s instructions. The sequence of the cDNA corresponding to transcript GenBank: EU124597 was amplified using Q5 High Fidelity DNA polymerase (BioLabs, Ipswich, USA) (forward primer: 5′-CTT AAA TGC TAC AAT TGC AAT TCC T-3′ and reverse primer: 5′-ACT ACT CTC AGT TGT AGT TGG A-3′) omitting the predicted signal peptide (SignalP, Lyngby, Denmark) and GPI anchoring signal (PredGPI, gpcr2.biocomp.unibo.it). The resulting amplicon was cloned into pFastBac HBM TOPO vector and expressed in High Five Trichoplusia ni cells using the Bac-to-Bac HBM TOPO secreted expression system (Thermo Fisher Scientific) following the manufacturer’s instructions. Infected High Five cells were scraped from Petri dishes and transfered to a microcentrifuge tube. The solution was centrifuged first at 1500 rpm for 7 min and the supernatant was concentrated using 30 kDa filters Amicon Ultra-15 (Millipore, Carrigtwohill, Ireland), according to the manufacturer’s instructions. The resulting sample was incubated with HPA agarose (Sigma-Aldrich) for 2 h. The recombinant protein named rLuloG was specifically eluted with 0.5 M GalNAc in Tris-NaCl and washed several times in Tris-NaCl on 30 kDa filters. The purity and molecular weight was confirmed by SDS-PAGE (10 % gel, reducing conditions) followed by Coomassie blue staining and blotting with HPA (as described above). The band was excised from the gel, digested by trypsin and analyzed by MALDI-TOF mass spectrometry as described above.
Rabbit anti-rLuloG antibodies: western blotting and indirect immunofluorescence with Lu. longipalpis midguts
Polyclonal anti-rLuloG antibodies were commercially prepared by repeated immunization of rabbit with 2 mg of purified rLuloG (Eurogentec, Seraing, Belgium). Antibody binding to rLuloG and to native protein from Lu. longipalpis midguts (10 guts per lane) was analyzed by SDS-PAGE (10 % gel, reducing conditions) followed by western blotting. The nitrocellulose membrane was incubated with Tris-NaCl-Tween (20 mM Tris, 150 mM NaCl, pH 7.6) and blocked with 6 % milk diluted in PBS/Tween overnight at 4 °C. The membrane washed with PBS/Tween was incubated first for 1 h with anti-rLuloG serum (1: 15,000 in 3 % BSA in PBS/Tween), repeatedly washed and then incubated with peroxidase-conjugated anti rabbit IgG SwAR/Px (Sevapharma a.s., Prague, Czech Republic), 1:1000 in 3 % BSA in PBS/Tween, as the secondary antibodies. After repeated washing the colour reaction was developed in 4-chloro-1-naphthol solution. Preimmune rabbit sera or midguts of the specific vector P. papatasi were used as negative controls. Incubation of separated rLuloG with HPA and inhibiton of this reaction with specific GalNAc were also tested.
Anti-rLuloG antibodies were also used to localize LuloG in sand fly midguts. To mimic the natural situation during Leishmania infections, midguts were dissected from Lu. longipalpis females which had defecated blood meal remnants (4–6 days post-blood meal). Midguts were longitudinally opened, fixed in 2 % paraformaldehyde for 20 min, washed twice in Grace’s Insect Medium (GIM, Sigma-Aldrich) and incubated with preimunne serum or anti-rLuloG antibodies (1:300 in 1 % BSA in GIM) for 1 h in RT. Guts were then washed four times in GIM and incubated with FITC anti-rabbit IgG (1: 50 in 1 % BSA in GIM) for 1 h. After washing in GIM, guts were placed in 2 μl of cooled CyGel (Biostatus Limited, Leicestershire, UK) on a microscope slide, carefully flattened, mounted in Prolong Gold antifade reagent (Thermo Fisher Scientific) and examined under a fluorescent microscope Olympus BX51.
Binding of rLuloG to Leishmania promastigotes
A 3-day culture of L. major LV561 was washed by centrifugation with PBS, fixed with 2 % formaldehyde for 30 min, washed again, diluted to a final density of 107 promastigotes/ml, spotted onto a slide and air-dried. Fixed parasite cells were rehydrated in PBS for 5 min, blocked with 0.1 % BSA in PBS for 5 min and incubated with rLuloG for 1 h. Slides incubated with preimunne serum or 0.1 % BSA served as negative controls. After washing (PBS for 15 min) the cells were incubated first with anti-rLuloG serum (1:500 in 0.1 % BSA in PBS) for 1 h, washed and then incubated with ALEXA fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific; 1:500 in 0.1 % BSA in PBS) in the dark. Slides were dried and mounted in Prolong Gold antifade mounting medium (Thermo Fisher Scientific) and examined under the fluorescent microscope Olympus BX51.
Leishmania development in Lu. longipalpis: effect of anti-rLuloG serum
Sand fly females were infected by feeding through a chick skin membrane with L. infantum promastigotes from 3-day-old cultures (cell density of 5 × 105/ml) in heat-inactivated rabbit blood (Bioveta, Ivanovice na Hané, Czech Republic) reconstituted either with anti-rLuloG serum or control preimmune serum. Blood-engorged females were separated and maintained at 26 °C. On day 2 post-blood meal (PBM) (i.e. before defecation) or day 4 and 7 PBM (i.e. after defecation of blood meal remnants) the females were dissected for microscopical examination.
Parasite density in dissected midguts was graded according to criteria reported previously as light (< 100 parasites/gut), moderate (100–1000 parasites/gut) or heavy (> 1000 parasites/gut) . Experiments were repeated three times. Infection rates (number of infected vs uninfected females) and intensities of infection (heavy, moderate, light, zero) were compared between groups using Chi-square test and S-PLUS 2000 programme.
Detection and characterization of midgut glycoconjugate
rLuloG, anti-rLuloG and their binding to Leishmania promastigotes and sand fly midguts
Development of L. infantum in Lu. longipalpis midgut fed with anti-rLuloG antibodies
Data reported by Myšková and colleagues  suggested that a 45 kDa O-linked glycoprotein on the Lu. longipalpis midgut epithelium may function as a parasite ligand. Here we used a combination of biochemical, molecular and parasitological approaches to characterize the biological properties of this glycoconjugate. We provide evidence that the molecule with an apparent molecular weight 45 kDa on SDS-PAGE corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis . The difference between the size of the native protein and predicted protein from cDNA library is assumed to be caused by a high degree of glycosylation. This is in agreement with the numerous predicted glycosylated serine and threonine residues found in the C- terminal part of the protein sequence, and also corresponds with the smeared appearance of the 45 kDa band on the gels or blots.
O-glycosylation prediction on the entire set of putative proteins identified in the midgut transcriptome of Lutzomyia longipalpis  did indeed find LuloG as the most heavily O-glycosylated protein. Beside LuloG, one more putative protein with extensive glycosylation was identified: ABV60335. It is a putatively secreted protein of 16.6 kDa with no other apparent conserved domains. No protein with a similar degree of O-glycosylation (a cluster of more than 40 putatively glycosylated Ser/Thr residues) was identified in the P. papatasi midgut transcriptome data .
An essential prerequisite for this GalNAc displaying molecule of permissive sand flies to function as a midgut receptor for Leishmania is its abundance and localization on midgut epithelial cells. Figure 7 shows that antibodies against the recombinant LuloG protein recognized round/spherical cells of dissected Lu. longipalpis midguts, typical of the luminal surface of epithelial midgut cells . On the other hand, preimunne serum recognized mostly musculature, consisting of circular and longitudinal fibres, which is located on the external side of sand fly midgut .
Although we could not prove involvement of LuloG in Leishmania attachment by blocking of the native protein on the epithelial surface with anti-rLuloG during sand fly infections, we demonstrated a strong binding ability of rLuloG to whole Leishmania body by in vitro indirect imunofluorescence experiments. This suggests that the receptor for the sand fly glycoconjugate is localized on the entire surface of promastigotes, including the body and flagellum and is accessible to the sand fly midgut ligand. Our data correspond with the observations of Myšková and colleagues , where midgut lysates of the permissive sand fly P. halepensis bound to Leishmania promastigote surfaces, and this interaction was visualized by FITC labelled HPA, the lectin specific for GalNAc.
We can speculate about the reason for the in vivo blocking attachment results. First, anti-rLuloG antibodies could be proteolytically degraded during blood digestion in the sand fly midgut, which is commonly observed in oral immunotherapy used for mammals. Proteolytic enzymes involved in the degradation of orally administered immunoglobulins in humans include pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase . In P. papatasi and Lu. longipalpis midguts, trypsin-like proteases were described as the most abundant. Secondly, the effectivity of anti-rLuloG antibodies in Leishmania attachment could be limited due to the recombinant protein rLuloG being expressed in soluble form without a GPI anchor, which was used for antibody production. Important roles for GPI anchors in binding of antibodies was demonstrated by numerous studies where removal of the GPI lipid moiety can influence ligand binding properties, most likely due to conformational changes in the protein. Studies showed that antibodies raised against the soluble form of proteins sometimes do not react well with the membrane-bound protein on the living organism, reviewed in . Seeing this unsuccesful inhibiton of Leishmania attachment in vivo, we performed ex vivo inhibition of Leishmania infantum binding to Lutzomyia longipalpis midgut by binding experiments. Details of the method are given by Wilson and coworkers . Although we did not find significant reduction of attached parasites in the presence of anti rLuloG, again a tendency to lower parasite numbers was observed (data not shown).
Nevertheless, a key question is what kind of parasite molecule could interact with this glycoconjugate. We may speculate that the parasite receptor for the 45 kDa glycoconjugate is a lectin-like molecule with predicted specificity for GalNAc, mimicking the activity of HPA lectin. Lectin activities and heparin-binding activities were demonstrated in Leishmania promastigotes by various authors [24–28]. More recent studies suggest that 65 kDa and 55 kDa heparin-binding proteins from the surface of L. braziliensis promastigotes can be involved in parasite adhesion to Lu. longipalpis cells through heparan sulphate bridges [29–31]. These heparin-binding proteins were reported to possess metallo-proteinase like activity . As they were not characterized by mass spectrometry, one cannot exclude the possibility that they are in reality identical with some isoforms of the major surface protease, leishmanolysin/gp 63 (reviewed in ).
We predict that the parasite receptor would be expressed at a reasonably high level, to ensure that specific binding to the midgut can occur despite the presence of other high copy surface molecules such as LPG. Reviewing current knowledge of the promastigote surface there is a variety of candidate molecules. These include leishmanolysin/gp63, PSA/gp46, mPPG, laminin-binding proteins or the already mentioned heparin-binding proteins, and maybe other unidentified proteins. A potential role for gp63 in parasite attachment has been previously proposed [33, 34]. These studies showed that the monoxenous trypanosomatids Herpetomonas samuelpessoai and Leptomonas species produce a metallopeptidase that has similar properties to Leishmania gp63 and contributes to parasite adhesion to Aedes aegypti guts or the Aedes albopictus cell line C6/36. These results are consistent with those obtained with Leishmania species; using an ex vivo by binding assay Jecna and colleagues  found that the gp63 of L. amazonensis gp63 down-regulated transfectants is functionally important for binding of its promastigotes to the Lu. longipalpis midgut.
In conclusion, while the L. major-P. papatasi work has established an important paradigm, recent evidence indicates that another mechanism of attachment could exist in permissive vectors. The present study has revealed interesting and novel properties of the “19 kDa midgut protein” from Lu. longipalpis midguts that can be briefly summarized as follows: (i) it is a mucin-like glycoconjugate with apparent weight of 45 kDa; (ii) it is located on the luminal surface of the midgut; and (iii) recombinant form rLuloG is able to bind to the surface of Leishmania cells. Nevertheless, additional studies will be necessary to prove the biological role of the “19 kDa midgut protein”, especially its role as a putative midgut ligand in permissive vectors.
In this study we characterized a glycoprotein from Lutzomyia longipalpis midgut which has been previously proposed as a molecule participating in Leishmania binding to the epithelium of the permissive sand flies. We identified that this glycoprotein corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis and named it LuloG. We demonstrate that this mucin-like molecule is GPI-anchored in the membrane of enterocytes and localized it on the luminal side of the midgut. These findings prove accessibility of the mucin for the parasites during blood meal digestion. In line with the putative role of mucin in parasite attachment, we indeed showed binding of a recombinant form of the glycoprotein to the surface of Leishmania promastigotes including flagella in vitro. However, antibodies raised against recombinat LuloG were not able to block Leishmania infection in Lu. longipalpis in vivo. In conclusion, our data support strongly the view that LuloG participates in Leishmania attachment to the Lu. longipalpis midgut and thus is important for parasite transmission.
We are grateful to Dr. Jitka Forstová and Dr. Martin Fraiberk and his colleagues who helped us to produce rLuloG in insect cells.
The work was supported by Grant 078937 from The Wellcome Trust. The authors gratefully acknowledge the support by the project LO1509 of the Ministry of Education, Youth and Sports of the Czech Republic and by Operational Program Prague – Competitiveness project (CZ.2.16/3.1.00/24023) supported by EU.
Availability of data and material
Data supporting the conclusions of this article are included within the article.
Substantial contribution to conception and design: PV, PB; acquisition of data: AD, LP, JM, PH; interpretation of data: JM, AD, LP, PH, PV; drafting the article: JM, PV, LP; revising the article for important intellectual content: PV, PB, JM. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Animals were maintained and handled in the animal facility of Charles University in Prague in accordance with institutional guidelines and Czech legislation (Act Number 246/1992 and 359/2012 coll. on Protection of Animals against Cruelty in present statutes at large), which complies with all relevant uropean Union and international guidelines for experimental animals. The experiments (including sand fly feeding) were approved by the Committee on the Ethics of Animal Experiments of the Charles University in Prague (Permit Number 24/773/08–10001) and were performed under the Certificate of Competency (Registration Number CZU945/05 ext. CZ02573) and the Permission Number 31114/2013–MSMT–13 ext. 24115/2014–MZE–17214 of the Ministry of the Environment of the Czech Republic.
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