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C-type lectin-producing tissue, designated as CLPT, constitutes mouthparts of the flesh fly (Sarcophaga peregrina) and produces a C-type lectin, CLEM 36, which is found to be secreted into saliva. In the present study, CLPT was investigated by immunohistochemical method using a transmission electron microscope (TEM) and scanning electron microscope (SEM), in order to clarify the secretion pathway of CLEM 36. CLPT was found to consist of cells having a microvillus. Each CLPT cell was connected to a fine duct. The fine ducts were bundled together and connected to the food canal. CLEM 36 was densely detected in the microvilli of CLPT cells, and also detected in cytoplasm, and ducts of CLPT. CLPT was found not to be connected to the salivary glands. Since little expression of CLEM genes including CLEM 36 was detected in the thorax and abdomen including the salivary glands in our previous study, when taken together, our current findings suggest that CLPT is a novel secretory gland which is independent of salivary glands, and has its own role in the intake of foods. Discipline: Insect pest Additional key words: azan staining, toluidine blue staining, whole mount immunostaining *Corresponding author: e-mail mami3y@affrc.go.jp Received 16 July 2014; accepted 2 April 2015. 09_Kihara_p057-062.indd 57 2015/11/26 21:15:02 58 JARQ 50 (1) 2016 M. Yamamoto-Kihara et al. (Yamamoto-Kihara & Kotani 2004, Yamamoto-Kihara et al. 2011). CLEM 20 and 36 were characterized as being highly and continuously transcribed from the second day after eclosion only in one tissue, which was designated as C-type lectin-producing tissue (CLPT), located at the entrance to the food canal between the labellum and the haustellum. CLEM 20 and 36 are secreted into saliva, suggesting that they have physiological roles different from Sarcophaga lectin and granulocytin. We also found CLEM 36 protein in CLPT cells using immunostaining of sections (Yamamoto-Kihara et al. 2011). However, the existence of CLEM 36 in the cells would not be decisive evidence yet to suggest that CLPT is a secretory organ. In the present study, to obtain a clue for elucidating the roles of CLEM 20 and 36 in the adult flesh fly, we have morphologically investigated CLPT and performed comprehensive analysis for the existence of CLEM 36 in CLPT using whole mount and TEM-based immunostaining. We then identified it as a novel secretory gland producing C-type lectin in the flesh fly. Materials and methods 1. Fly samples Flesh flies (Sarcophaga peregrina) were maintained at 25°C and reared on 3% sucrose and fresh water. Pork liver was also provided for depositing larvae. Larvae were reared on pork liver at 20°C, and pupae were maintained at 20°C. For immunostaining and histological analyses excluding azan staining, flies were used 3 to 5 days after eclosion. For azan staining, tips of mouthparts were excised just before eclosion. 2. Azan staining Tissue samples were fixed at 4°C overnight by immersion in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB) (pH 7.4), and washed in 0.1 mol/L PB (pH 7.4). They were then dehydrated in a graded ethanol series and embedded via xylene intermedium into paraffin (Wako Pure Chemical, Osaka, Japan). Paraffin sections 4 μm thick were stained according to Mallory-Heidenhain (Bancroft & Gamble 2001), and observed by light microscope (DMLB; Leica Microsystems, Buffalo Grove, IL, USA). 3. Toluidine blue staining Tips of mouthparts were fixed as described above, and subjected to dehydration in a graded ethanol series and to infiltration via propylene oxide intermedium with EPON 812 resin (TAAB Laboratories Equipment Ltd., Berkshire, UK) (overnight in 50% EPON 812 in propylene oxide, 3 h in 75% EPON 812 in propylene oxide, 2 h in 100% EPON 812 and 24 h in fresh 100% EPON 812) at 4°C, followed by polymerization of the resin at 60°C overnight. Sections with a thickness of 1 μm were stained with toluidine blue, and inspected by light microscope (DMLB). 4. Whole mount immunostaining analysis Tips of mouthparts were fixed as described above and digested with Protease K according to the procedure described previously (Yamamoto-Kihara & Kotani 2004), followed by washing twice with phosphate buffer saline (PBS) containing glycine and with PBS containing 0.3% Triton X-100 (PBST). Blocking was performed in blocking buffer (PBST containing 3% BSA) for 2 h. Samples were incubated with the anti-CLEM 36 antiserum (1:800) (Yamamoto-Kihara & Kotani 2004) or the preimmune serum (1:800) in blocking buffer at 4°C for more than 16 h. CLEM 36 was recognized and detected using a VECTASTAIN Universal Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) and VectaStain VIP Substrate Kit for Peroxidase (Vector Laboratories), according to the manufacturer’s instructions. 5. Scanning electron microscopy Tips of mouthparts were excised, and the cuticles were removed from the tips. The samples thus obtained were then fixed with 4% paraformaldehyde in PBS at 4°C overnight, followed by washing with PBS. For SEM analysis, two types of specimens were prepared from the fixed samples; one was the outer matrix of the mouthpart tips treated with protease K at a concentration of 1 μg/ml for 10 min at 25°C; the other was the sagittally-dissected mouthpart tips without protease treatment. The protease K-treated samples were washed in PBS containing glycine and refixed with 4% paraformaldehyde in PBS for 2 h at 25°C, followed by washing with PBS. The resulting samples were dehydrated with ethanol series, and dried via isopentyl acetate by the critical point method using CO2 (Anderson, 1951). The dried samples were coated with gold, followed by examination with SEM (6301 F; JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV. 6. Transmission electron microscopy Tips of mouthparts were excised and frozen immediately in liquid nitrogen at high pressure using a Leica EM PACT high-pressure freezer (Leica Microsystems). Freeze substitution of the frozen specimens was conducted using an AFS system (Leica Microsystems) beginning at -90°C in 1% wt/vol osmium tetroxide in acetone, and ending in several changes of EPON 812 resin at 20°C through pure acetone at -50°C. The resin containing specimens was polymerized at 60°C for 24 h. Ultra-thin 09_Kihara_p057-062.indd 58 2015/11/26 21:15:02 59 C-Type Lectin-Producing Tissue (CLPT) of the Flesh Fly sections (60 nm) of the samples were obtained using a Leica Ultracut R (Leica Microsystems), and were stained with uranyl acetate and lead citrate. Samples were studied by TEM (JEM-1010, JEOL) at an accelerating voltage of 80 kV. 7. TEM-based immunostaining Tips of mouthparts were removed, cut in half, and fixed with 1% paraformaldehyde and 0.25% glutaraldehyde in 0.1 mol/L PB (pH 7.4) at 4°C overnight, followed by washing with 0.1 mol/L PB (pH 7.4). Samples were dehydrated in a graded ethanol series and infiltrated with Lowicryl K4M resin (Chemische Werke Lowi GmbH & Co., Waldkraiburg, Germany) overnight in 50% K4M in ethanol, 3 h in 75% K4M in ethanol, 2 h and 24 h in 100% K4M, at 4°C. After transferring the samples to gelatin capsules with fresh K4M resin, they were polymerized using ultraviolet light at 4°C for 65 h. Ultra-thin sections (60 nm) of the CLPT on nickel grids covered with 2% collodion solution in isoamyl acetate were treated with antibody against anti-CLEM 36 and then with 20 nm-gold labeled goat-rabbit IgG (H+L), according to the manufacturer’s instructions (BBInternational, Dundee, UK), and examined with TEM. Results Under SEM observation, a pair of CLPT was localized at the entrance to the food canal between the labellum and haustellum (Fig. 1iii), consistent with previous observations (Yamamoto-Kihara & Kotani 2004). Furthermore, SEM examination demonstrated that most CLPT cells were of a round shape with an approximate diameter of 25 μm (Fig. 1v). Each CLPT cell was connected to a fine duct, forming a botryoid shape (Figs. 1iv-vi). The fine duct showed a stripe structure on its surface, which was different from the cell membrane of the CLPT (Figs. 1vii-viii). The ducts were bundled and connected with the entrance to the food canal (Fig. 1iv). The Fig. 1. SEM images of flesh fly head region (i) Represents the head of the flesh fly; and (ii) is a schematic drawing of the proboscis shown in (i). (iii) (v) Images correspond to the respective areas in the drawing (ii). (vii) And (viii) present cells and ducts of CLPT, which are shown in (iii) (v). (vi) Represents a schematic drawing of CLPT seen in (iv) (v). The white arrow in (iv) indicates bundled ducts connecting to the food canal; white arrows in (vii), ducts; and the white arrow in (viii), protraction site of duct from a CLPT cell. Ro represents rostrum; Ha, haustellum; La, labellum; FC, food canal; M, muscle; and LN, labial nerve. 09_Kihara_p057-062.indd 59 2015/11/26 21:15:03 60 JARQ 50 (1) 2016 M. Yamamoto-Kihara et al. Fig. 2. Photographs of toluidine blue and azan staining of the tip of mouthparts (i) Schematic drawing of the proboscis. Red lines indicate the dissection positions for toluidine blue staining (ii), and azan staining (iii). CLPT is shown as a yellow ellipse in (ii). Black arrows indicate the cord linking CLPT with the pseudotrachea and interpseudotrachea. M indicates muscle; PT, pseudotrachea; and IPT, interpseudotrachea. Fig. 3. TEM micrographs of CLPT Axial (i) and longitudinal sections (ii) of CLPT are shown. N indicates nucleus; MV, microvillus; BC, body cavity; and D, ducts of CLPT. Fig. 4. Whole mount immunostaining using antibody against CLEM 36 (i) – (iii) Whole mount immunostaining using antibody against CLEM 36. CLEM 36 is shown by red staining. Yellow arrows indicate small red spots in cytoplasm, shown in (i) and (ii). Black arrows in (i) and (iii) indicate positions where CLEM 36 is densely localized in cytoplasm. Yellow ellipse in (iii) indicates CLEM 36 in the duct. (iv) Whole mount immunostaining using a preimmune serum. M shows muscle; FC, food cana

jarq/jarq. japan agricultural research quarterly

Identification of a Novel Secretory Gland Producing C-type Lectin in the Flesh Fly (&lt;i&gt;Sarcophaga peregrina&lt;/i&gt;)&lt;i&gt;, &lt;/i&gt;and its Characterization