Eggs of the copepod Acartia tonsa Dana require hypoxic conditions to tolerate prolonged embryonic development arrest

Additional file 1. Raw data and calculations for all experiments as well as an overview of experiments in this study

FIGURE 1 in New species of Pisionidens (Sigalionidae, Annelida) from Akumal, México

FIGURE 1. Light microscopy images of Pisionidens ixazaluohae n. sp. (A) Whole specimen. (B) Anterior end, details of eyes. (C) Details of jaws. (D) Details of a midbody parapodia. Abbreviations: an, anterior; dc, dorsal cirri; ey, eye; gl, glands; jw, jaws; nac, neuroacicula; po, posterior; vc, ventral cirrus. Segments indicated by roman numerals

Pisionidens ixazaluohae Petersen, Gonzalez, Martínez & Worsaae, 2016, n. sp.

Pisionidens ixazaluohae n. sp. (Tables 1–2, Figs 1–2) Material examined. Half Moon Bay, Akumal, Quintana Roo, México, 15 specimens. Holotype: complete adult male (ZMUC-POL- 2435, stub-mounted for SEM), 13.3 mm long, 65 segments. Coarse sand, swash zone, 0.2 m depth, 20 ° 24.22 'N, 87 ° 18.28 'W. Collectors: B. C. Gonzalez & A. Martínez 19.06. 2014. Paratypes: 1 male (ZMUC-POL- 2436, stub-mounted for SEM), 4 females (1 stub-mounted for ZMUC-POL-2437, 3 preserved and stored in ethanol ZMUC-POL- 2439, ZMUC-POL- 2440), 4 incomplete specimens (ZMUC-POL- 2441), sex unknown (4.25–8.72 mm, 24–41 segments, stored in ethanol) and 4 specimens in bad condition (ZMUC-POL- 2442), sex unknown (7.88–17.75 mm, fixed in PFA, stored in PBS), same locality and sampling date as holotype. Diagnosis. Muscular and slender body, anteriorly and posteriorly tapering. One pair of anterior, basally fused antennae, two smooth lateral prostomial palps; one pair of ventral elongated cirri on segment 2, one pair of dorsal elongated cirri on segment 3. Pharynx eversible, with a terminal crown of 18 papillae and two pairs of hook-shaped jaws. Achaetous parapodia with distal glandular disc present from segment 8 onward. Males with one row of midventral pores from segment 11 to posterior end. One pair of club-shaped copulatory organs on segment 45. Description. All measurements from the holotype, parenthetical numbers from paratypes. Body slender, muscular, white to pale yellow (fixed specimens) (Fig. 1 A), with thick cuticle and smooth body surface. Length 13.3 mm (7.33–17.75 mm, n= 10), width including parapodia 0.45 mm (0.47–0.69 mm, n= 5), width excluding parapodia 0.34 mm (0.24–0.52 mm, n= 15), 65 segments (45–62, n= 5). Fused conical prostomium with segment 1, one pair of antennae fused at base (Fig. 2 A). One pair of palps, smooth, 0.28 mm long (0.22–0.34 mm, n= 7), emerging laterally from prostomium (Fig. 2 A). One pair of dorsal eyes in segment 2 (Fig. 1 B). Cerebral ganglion large, bilobed, extending from middle of segment 1 to segment 4. Mouth ventral with eversible pharynx, equipped with two pairs of hooked jaws (Fig. 1 C). Pharynx with a terminal crown of 18 digitiform papillae (Fig. 2 B), central papilla largest, decreasing in size laterally. Segments 1–3 lacking parapodia, segments 4-7 with rudimentary parapodia. Ventral cirri on segment 2 (buccal segment), long and tapering, similar in length to palps; dorsal cirri on segment 2 small, bottle-shaped (Fig. 2 A, C). Dorsal cirri on segment 3 (see Rouse and Pleijel 2001) longest among anterior appendages, approximately twice the length of palps (Fig. 2 A); ventral cirri on segment 3 small and bottle-shaped (Fig. 2 C). All anterior appendages smooth. From segment 4 posteriorly, dorsal and ventral cirri small with subdistal and distal cilia (Fig. 2 D). Rudimentary parapodia and cirri may be lacking in some specimens from segment 4 to segment 7 (Fig. 2 C). Parapodia initiating on segment 8, achaetous in mature specimens (unknown in juveniles). All parapodia cylindrical and slender, with subdistal tufts of compound cilia; distal disc with adhesive glands (Fig. 2 D). Notoacicula absent, neuroacicula straight (Fig. 1 D). Adult males with midventral line of segmentally repeated pores ca. 35 µm wide (n= 2) from segment 11 to last segment prior to pygidium, pore diameter smaller on copulatory segment (ca. 10–15 µm, n= 2) (Fig. 2 E); midventral pores lacking in females and juveniles. Paired lateral fields of ca. 35 epidermal pores (2-5 µm) present between parapodia. Pygidium with one pair of cirri, short and cylindrical (Fig. 2 F). Anus dorsal. One pair of male copulatory organs, on segment 45 in holotype (42, n= 1), with short dorsal cirri, parapodia and ventral cirri absent. Copulatory organ approximately same length as body width (0.40 mm, n= 1), club-shaped, curved latero-ventrally, with subdistal node-shaped process (Fig. 2 G) and small papillae distally; tip slightly curled, with small terminal process (Fig. 2 H), but lacking cuticular hooks. Females appear to differ from males externally only in the absence of copulatory organs and mid-ventral pores. Molecular information. Newly generated sequences for Pisionidens ixazaluohae n. sp. included 1800 bp of 18 S rDNA (Gb accession number KX 282503), 1039 bp of 28 S rDNA (Gb accession number KX 282504), 510 bp of 16 S rDNA (Gb accession number KX 282502), and 582 bp of COI (Gb accession number KX 282505). In previous molecular analyses (Gonzalez et al. 2015), Pisionidens ixazaluohae n. sp. was recovered next to Pisionidens sp. (Norlinder et al. 2012), forming the well-supported sister clade of all the species of Pisione included in the analyses. The COI gene fragment of Pisionidens ixazaluohae n. sp. presents 83.4 % similarity to the COI of Pisionidens sp. (Acc. Num JN 852943), being the only Pisionidens sequence available in GenBank. This similarity accounts for 94 mutations, three of them non-synonymous. Remarks. Pisionidens ixazaluohae n. sp. is characterized by having only one pair of copulatory organs, while the previously described species possess two to four pairs. P. ixazaluohae n. sp. differs from P. indica (Aiyar & Alikuhni 1940) and Pisionidens tchesunovi, Tzetlin 1987 by lacking true parapodia on segments with copulatory organs. This feature is similarly found in Pisionidens maturata, Yamanishi 1976. However, P. ixazaluohae n. sp. is distinguished from P. m at ur a t a and P. indica by having one continuous series of midventral pores (versus two separate series of ventral pores). A comprehensive comparison of taxonomic characters and distribution of all described species of the genus Pisionidens is given in Table 2. Holotype ZMUC-POL- 2435 Ađult Male 1 13.3 mm 64 45 0.28 mm 0.35 / 0.43 mm 0.45 / 0.34 mm Paratype 1 ZMUC-POL- 2436 Ađult Female 1 11.0 mm 54 0.26 mm 0.34 / 0.44 mm 0.51 / 0.37 mm Paratype 2 ZMUC-POL- 2437 Ađult Male 1 10.6 mm 61 42 0.22 mm 0.22 / 0.35 mm 0.49 / 0.30 mm Paratype 3 ZMUC-POL- 2438 Ađult Female 1 4.2 mm 24 * 0.31 mm 0.30 / 0.36 mm 0.47 / 0.29 mm Paratype 4 ZMUC-POL- 2439 Ađult Female 1 13.4 mm 62 0.32 mm 0.33 / 0.38 mm */ 0.34 mm Paratype 5 ZMUC-POL- 2440 ?? 2 8.2 mm 45 0.25 mm 0.19 / 0.35 mm */ 0.30 mm Ađult? 17.7 mm 52 * * 0.69 / 0.52 mm Paratype 6 ZMUC-POL- 2441 Ađult? 4 14.1 mm * * * */ 0.45 mm Ađult? 16.5 mm * 0.30 mm 0.31 / 0.45 mm */ 0.45 mm ?? 7.9 mm * * * */ 0.28 mm Ađult? 8.7 mm 41 *?/ 0.39 mm */ 0.35 mm Paratype 7 ZMUC-POL- 2442 Ađult? 4 6.2 mm * * * */ 0.38 mm Ađult? 13.1 mm * * * */ 0.26 mm ?? 7.3 mm * * * */ 0.24 mm Ađult? 6.2 mm * * * */ 0.44 mm Type locality. Half Moon Bay, Akumal, Quintana Roo, México. Habitat. Coarse sand in the swash zone of an exposed beach: 0–0.25 m depth. Several other annelids were found in the samples including Claudrilus cf. ovarium (Di Domenico et al. 2013), Saccocirrus sp., Polygordius sp., Microphthalmus sp., Macrochaeta sp., Pholoe sp., as well as high numbers of Ingolfiella sp. and otoplanid flatworms. Distribution. Caribbean Sea, Akumal, México. Etymology. Named after the Mayan goddess of water, Ixazaluoh.Published as part of Petersen, H. Cecilie B., Gonzalez, Brett C., Martínez, Alejandro & Worsaae, Katrine, 2016, New species of Pisionidens (Sigalionidae, Annelida) from Akumal, México, pp. 165-173 in Zootaxa 4136 (1) on pages 167-171, DOI: 10.11646/zootaxa.4136.1.8, http://zenodo.org/record/25549

Cancer Associated Aberrant Protein O-Glycosylation Can Modify Antigen Processing and Immune Response

<div><p>Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/− glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the <em>in vivo</em> response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.</p> </div

Peptide sequences for DC T cell hybridoma co-culture.

<p>1 and 2 (H2-Kb restricted fusion), 3 and 4 (I-Ab restricted fusion). Underlining indicate sites of glycosylation. Internal (I), terminal (T), N-Acetylgalactosamine (GalNAc).</p

Peptide glycosylation inhibits activation of antigen specific CD8+ T cell hybridoma.

<p>IL-2 production from OVA specific CD8+ T cell hybridoma (RF 33.70) co-cultured with bone marrow derived DCs (A) or CD11c+ DCs purified from mouse spleen (B). DCs were pulsed with two peptide variants with and without 2 and 4 glycan residues (GalNAc). Full length OVA was used as a positive control. Individually cultured T cells and DCs had an OD value equal to the background. C, D) Surface expression by flow cytometry of SIINFEKL in the H2kb peptide binding groove on DCs after pulsing with the two peptide variants (non-glycosylated peptide (thin purple dashed line), peptide with 2 GalNAcs (thick green line), or 4 GalNAcs (pink line)). E) Surface expression of SIINFEKL in the H2kb peptide binding groove on DCs without pulsing (blue line) and after pulsing with OVA control (purple line). Gray histograms represent cells stained only with secondary antibody.</p

<i>In vivo</i> response to degMUC1 in WT Balb/c and HLA-A2 transgenic mice.

<p>A) Serum reactivity to different mucin glycoforms from Balb/c mice immunized with degMUC1+/− GalNAc by ELISA. Data are representative of a minimum of 4 Balb/c mice immunized with each antigen. (B) T cell proliferation from mice Balb/c mice or (C) HLA-A2 mice immunized with GalNAc degMUC1 (white bars) and non-glycosylated degMUC1 (grey bars) for each peptide used for <i>in vitro</i> re-stimulation (100 ug/ml) as indicated on the x-axis. D) Lymphocytes from immunized HLA-A2 mice pulsed with the 9 mer degMUC1 peptide, ALGSTAPPV, with and without glycosylation. DegMUC1 specific CD8+ T cells were selected based on anti-CD8 Ab (x-axis) and anti-IFNγ Ab (y-axis) after 4 hrs of Golgi stop treatment. E) Spleen cells after 24 days of re-stimulation with the non-glycosylated ALGSTAPPV peptide. All T cell data is generated from spleen or lymph nodes from at least 4 mice, but in most cases 6 mice.</p

Increased density of peptide glycosylation increases uptake but inhibits activation of antigen specific CD8+ T cell hybridoma.

<p>A) DC uptake of uncoated fluorescent beads, MUC1-fluorescent beads, or GalNAc MUC1 fluorescent beads. DCs without beads were used as reference. B) The uptake of MUC2 fusion peptide +/−GalNAc was evaluated by flow cytometry after 48 hrs of peptide pulsing. Unstained DCs (purple filled) and DCs with no peptide load (green) were used as a background control. C) Evaluation of the surface expression by flow cytometry of SIINFEKL in the H2kb peptide binding groove. DCs were pulsed with the MUC2 fusion peptide (highest concentration from D) with GalNAc (green line) and without GalNAc (purple filled) 48 hrs before the staining. D) IL-2 production from SIINFEKL specific CD8+ T cell hybridoma (RF 33.70) co-cultured with bone marrow derived DCs pulsed <i>in vitro</i> for two days with MUC2 fusion peptide both with and without glycosylation in a concentration gradient. Full length OVA was used as a positive control. Representative data of at least two independent experiments is shown. E, F) Confocal imaging of DC internalized GalNAc MUC2 fusion (E, green) and MUC2 fusion (F, green) co-stained with endosomal marker EEA-1 (red) and lysosomal marker LAMP-2 (red).</p