Fine Structure and Molecular Phylogeny of Parametopidium circumlabens (Ciliophora: Armophorea), Endocommensal of Sea Urchins

Metopid armophoreans are ciliates commonly found in anaerobic environments worldwide; however, very little is known of their fine structure. In this study, the metopid Parametopidium circumlabens (Biggar and Wenrich 1932) Aescht, 1980, a common endocommensal of sea urchins, is investigated for the first time with emphasis on transmission electron microscopy, revealing several previously unknown elements of its morphology. Somatic dikinetids of P. circumlabens have a typical ribbon of transverse microtubules, an isolated microtubule near triplets 4 and 5 of the anterior kinetosome, plus two other microtubules between anterior and posterior kinetosomes, a short kinetodesmal striated fiber and long postciliary microtubules. In the dikinetids of the perizonal stripe, the kinetodesmal fiber is very pronounced, and there is a conspicuous microfibrillar network system associated with the kinetosomes. A new structure, shaped as a dense, roughly cylindrical mass surrounded by microtubules, is found associated with the posterior kinetosome of perizonal dikinetids. The paroral membrane is diplostichomonad and the adoral membranelles are of the “paramembranelle” type. Bayesian inference and maximum‐likelihood analysis of the 18S‐rDNA gene unambiguously placed P. circumlabens as sister group of the cluster formed by ((Atopospira galeata, Atopospira violacea) Metopus laminarius) + Clevelandellida, corroborating its classification within the Metopida.     

Kinetosome Production in Condylostoma Occurs During Cell Division*

Kinetosome counts in different stages of the cell cycle of Condylostoma indicate that somatic kinetosome production occurs during cell division. The kineties apparently increase in length during interphase by spacing-out of preexisting kinetosomes. This spacing-out may be passive and caused by expansion of the cortical membrane to which the kinetosomes are attached.     

Fine structure of the dorsal bristle complex and pellicle of Euplotes

The fine structure of the dorsal bristle complex and pellicle of non-developing Euplotes eurystomus is described in detail by scanning and transmission electron microscopy. The bristle-pit unit is a highly differentiated complex of organelles. The bristle complex is composed of a pair of kinetosomes (basal bodies) joined by a connective. The anterior kinetosome bears the bristle cilium, which contains a polarized network of particles (“lasiosomes”). The posterior kinetosome bears a very short, knob-like “condylocilium,” and has an associated striated fiber. Accessory ribbons of microtubules are also associated with the kinetosome couplets. Parasomal sacs, a septum connecting the bristle cilium to the anterior wall of the pit, core granules of the kinetosomes, and large membranous ampules are described. The organization of the bristle complex bears many similarities to the somatic ciliature of other ciliates. The pellicle of Euplotes is composed of a continucus outer cell membrane subtended by membranous alveoli, which contain a “fibrous mat.” Two sheets of subpellicular microtubules (longitudinal and transverse) are located just beneath the alveoli. The “epiplasm” seen in some other ciliates is apparently absent in Euplotes. The texture of the cell surface is a pattern of folds or rugae composed of the outer cell membrane and the upper membrane of the alveolus. The pattern of rugae probably defines the “silverline-system” of light microscopy.     

Etude Ultrastructurale du CiliéKuklikophrya dragescoi gen. n., sp. n.*

SYNOPSIS The cortical infraciliature of Kuklikophrya dragescoi gen. n., sp. n. is composed of double kinetosomes. Each kinetosome has transverse fibers. The anterior transverse fibers are associated with a sheet of dense material and the posterior transverse fibers are directed toward the posterior part of the body. The posterior kinetosome of a pair has only a short protuberance in the position of the kinetosomal fiber. The cortex has a well developed alveolar layer and a thick ecto-endoplasmic boundary. A distinctive characteristic of the buccal ciliature is the circumoral ciliature whose infraciliature is made up of pairs of cilia-bearing kinetosomes. The antero-posterior polarity of the paroral segment is in inverse relationship to that of the remaining ciliature of the organism. The adoral and preoral ciliary organelles consist of 2 rows of kinetosomes, each of which bears postciliary fibers. A frame of nematodesmata surrounds the cytopharynx which is supported by microtubular bands which impart to it a very specific laminated appearance. The “phagoplasm” is formed by “vermicelli”-like vesicles. The micronucleus is found in the perinuclear area of the macronucleus.     

The Mastigont System of Trichomonas gallinae (Rivolta) as Revealed by Electron Microscopy

SYNOPSIS. The fine structure of Trichomonas gallinae has been examined by electron microscopy and correlated with previous light microscope observations. A composite diagram of the flagellate, derived from both types of examination, is presented. Details of relationships of various mastigont organelles are documented by electron micrographs. The extent of the pelta and its connection to the capitulum of the axostyle have been determined. Four types of kinetosome rootlets have been described. One consists of superficial “filaments” radiating from each of the 9 triplet microtubules of kinetosomes #1, #2 and #3. A 2nd type of rootlet structure is represented by single comma-shaped filaments emerging clockwise from kinetosomes #1 and #3. The filament from kinetosome #1 has a periodic structure similar to that of the marginal lamella with which it is believed to connect. A 3rd type of rootlet emerges from kinetosome #2 as a sheet of about 9 filaments which traverse a sigmoid course and terminate on the inner surface of the microtubules of the pelta near the peltar-axostylar junction. The 4th set of structures consists of the costa and parabasal filaments. These structures have major periodicities of similar dimension but have readily differentiable repeating units. The costa appears to originate at the kinetosome of the recurrent flagellum, but its origin is also contiguous with that of parabasal filament 2 which has some continuity with kinetosomes #2 and #3. Parabasal filament 1, on the other hand, arises solely from or near kinetosome #2. Occasional observations of a costa and a parabasal filament in juxtaposition over a great part of their length has led to the suggestion that the parabasal filament may play a role in the development of the costa. Periodic and filamentous structures have been observed in paraxostylar and paracostal granules and in nearby cytoplasm. Their possible role in providing substance for the developing axostyle and the costa is discussed. The results are discussed in the light of available information pertaining to structure of various trichomonad species as revealed by light and electron microscopy.     

Zoosporulation in Labyrinthula sp.; an Electron Microscope Study1

SYNOPSIS. Zoosporulation in Labyrinthula sp. in monoxenic culture was initiated by aggregation of spindle cells into reticulate sori. The spindle cells then changed into rounded or oval cells and formed, de novo, 2 pairs of centrioles at opposite sides of each nucleus. A pair of granular aggregates (protocentrioles) ～ 240 mμ in diameter served as precursor bodies during centriole formation. Spindle microtubules around the prophase nucleus connected the pairs of centrioles but were not found in the nucleoplasm until nuclear envelope fragmentation occurred. Prophase nuclei of uninucleated sporangia contained synaptinemal complexes; therefore, meiosis is presumed to occur. The envelope fragments moved toward the centrioles and regrouped to form the nuclear membranes of the daughter cells. Alternating nuclear and cytoplasmic divisions subdivided the preparation into 8 cells which differentiated into laterally biflagellated zoospores. Flagellar development involved growth of the kinetosome microtubules into a bud which formed over the kinetosome tangential to the cell surface. Kinetosomes were derived directly from centrioles with little differentiation other than addition of an electron-dense core to the lumen of the centriole. Zoospore ultrastructure included a stigma comprised of a row of electron-dense granules located slightly under the plasmalemma and posterior to the pair of kinetosomes. A single row of 17–21 microtubules lay parallel to the stigma granules, one or more being connected to the anterior kinetosome. A striated fiber apparatus similar to that found in some phytoflagellates connected the midregions of the kinetosomes. Fibers 1.0–1.2 μ long were attached to the plasmalemma around the base of the anterior flagellum. Zoospores settled on the substrate and differentiated directly into spindle cells. Since synaptinemal complexes were observed the planonts are probably haploid zoospores and probably not gametes since planogametic copulation was not observed.     

The Structure and Development of the Dorsal Bristle Complex of Oxytricha fallax and Stylonychia pustulata*

SYNOPSIS. Oxytricha fallax and Stylonychia pustulata possess 6 rows of dorsal bristle units. Each dorsal bristle unit consists of a pair of kinetosomes; the anterior kinetosome has a cilium and the posterior kinetosome a ciliary stub. The kinetosome pair, located at the bottom of a cortical pit surrounding the cilium and ciliary stub, is surrounded by an asymmetrical fibrillar mass. Future rows 1-4 are formed from 2 sets of primordia originating within mature dorsal rows 1-3. Rows 5 and 6 originate from the anterior regions of both right marginal cirral primordia. Old dorsal bristle units utilized in formation of primordia are presumably maintained in the new rows of the proter and opisthe; those outside the primordia are resorbed. The morphogenetic pattern of the Oxytrichidae is similar to those of the Urostylidae and Holostichidae, but quite different from that of the Euplotidae.     

The Ultrastructure of Specimens of Paramecium multimicronucleatum Impregnated with Silver by the Fernández-Galiano Method1

Transmission and scanning electron microscopy of specimens of Paramecium multimicronucleatum treated with the Rio-Hortega silver-impregnation method as modified by Fernández-Galiano demonstrate that considerable deposition of silver occurs around the kinetosomes, especially at the level of the basal plate and also at the proximal end of the kinetosome. In addition, silver is heavily deposited within the kinetodesmal fibers, in the fibrous matrix that surrounds the postciliary and transverse microtubules, in the connective structures observed between the two kinetosomes of a pair and between the kinetodesmal fiber and the anterior kinetosome, and in the trichocysts. Differences and similarities in sites of deposit when other methods of silver impregnation are employed are discussed and the particular value of the present technique in studies of ciliate systematics and phytogeny is stressed.     

Centriole maturation in the amoebae ofPhysarum polycephalum

Summary The precise geometry of pro-centriole formation has been studied inPhysarum polycephalum amoebae. The spatial references used were the posterior and the anterior kinetosomes which are unequivocally defined by the presence of the posterior para-kinetosomal structure, the microtubular array 4 and the microtubular arrays 1, 2, and 3. The observations made suggest that pro-centrioles follow a maturation process. A pro-centriole formed during the n^th cell cycle becomes the posterior kinetosome during the (n + 1)^th cell cycle and the anterior one during all the following cell cycles. Pro-centriole formation occurs late in the cell cycle. This observation disagrees with a role of pro-centriole formation in the regulation of S phase in contrast to what has been suggested in other eucaryotic cells.     

The Behavior and Fine Structure of the Dorsal Bristles of Euplotes minuta,E. aediculatus,and Stylonychia mytilus (Ciliata,Hypotrichida)1

ABSTRACT. Studies of the bristle (dorsal) cilia of Euplotes minuta. E. aediculatus, and Stylonychia mytilus by light and electron microscopy indicate that these cilia do not beat metachronously in any of the species. The bristle cilia in Stylonychia may beat actively, but those in Euplotes stand erect or are bent in different directions with the flow of water. The duration and degree of bending appear correlated with the duration and velocity of the water current. The fine structure of the bristle complex is similar in both Euplotes species and like other reports of Euplotes in the literature. The complex consists of paired kinetosomes, the anterior bearing a short cilium containing four to six rows of fibrous balls (lasiosomes) oriented along the anterior surface of the axoneme, the posterior lacking a cilium but with a small cap. Microtubular ribbons are associated with the paired kinetosomes, and a collar with a pronounced alveolar ring underneath the pellicular membrane tightly surrounds the cilium at the opening of the bristle pit. The bristle complex in S. mytilus differs from that of Euplotes and other hypotrichs in that it has a single kinetosome in interphase cells and, attached to the kinetosome, a prominent fibrous structure (parakinetosomal body). Microtubules are attached to the parakinetosomal body. As in Euplotes, the bristle unit is surrounded by mucocyst-like organelles (ampules). Observations of behavior and fine structure suggest that the dorsal bristles may be sensory, perhaps responding to stimuli from water currents, although other functions are possible, too.     

Trimyema compressum Lackey, 1925: Morphology,Morphogenesis and Systematic Implications1

ABSTRACT Trimyema compressum is a species included in the family Trimyemidae with the single genus Trimyema. This species has 50–60 somatic kinetics and three rows of kinetosomes surrounding the oral cavity. Two isolated groups of kinetosomes can also be observed on the right side of the oral region. The morphogenesis of bipartition is telokinetal; all the new infraciliary structures of the opisthe come from the longitudinal and postero-anterior proliferation of the last kinetosome of all the somatic kinetics. In the proter there is a reorganization of the oral infraciliature. As a result of our observations, we suggest that the systematic position of the genus Trimyema be changed from the subclass Vestibulifera to the subclass Gymnostomata. We also consider that this genus must be included in the suborder Trimyemina Jankowski, 1980.     

Morphology and Infraciliature of a Marine Scuticociliate with a Polymorphic Life Cycle: Urocryptum tortum n. gen., n. comb.

A new genus and species combination are proposed for Urocryptum tortum n. gen., n. comb., a scuticociliate with a polymorphic life cycle. This marine ciliate was isolated from a sample taken at Gokasho Bay in Mie Prefecture (Japan). Specimens from different phases of the growth cycle were examined in vivo and with two silver staining techniques. Three life-history stages were observed: an exponential growth phase stage (trophont), a stationary phase stage (tomite), and finally a resting stage (cyst). The exponential growth form is laterally flattened and ovoid; it has 20-24 somatic kineties (SK) and a typical complement of scuticociliate oral structures. Polykinetid 1 (Pk1) has two longitudinal files of 6 kinetosomes (Ks); sometimes one or two additional kinetosomes are located anteriorly. Polykinetid 2 (Pk2) has two files of 6 or 7 Ks; a third file of three widely spaced kinetosomes is located on its right side, as well as a small curved row of 5 Ks positioned on the right hand side of the posterior end of Pk2. Polykinetid 3 (Pk3) has 3 rows of 4, 5, and 7 Ks's perpendicular to the haplokinety (Hk) or paroral membrane. The stationary growth phase cell is spindle-shaped and has a similar number of SK that are much more closely spaced than in exponentially growing specimens. Oral infraciliary structures are reduced in size, having fewer kinetosomes and being positioned more anteriorly in the cell. Pk1 is composed of two files of 5-Ks, Pk2 has only two files of 6-7 Ks, and Pk3 has two to three rows of 3-4 Ks. The Hk is displaced anteriorly and becomes straight, losing the shape typical of exponential growth phase cells. Cysts are pyriform with a wide cyst wall; no infraciliary structures were visible.     

The Resorption of Cilia in Cyathodinium piriforme*

SYNOPSIS. The fine structure of the cilium, kinetosome, kinetodesmal fiber, and basal microtubules has been described in Cyathodinium piriforme. The ciliary axoneme is encased in an electron-dense jacket termed the axonemal jacket. This jacket surrounds the axoneme and is found midway between the axoneme and the ciliary membrane when viewed in cross section. Before division or reorganization the cilia are withdrawn into the cell. Intact cilia surrounded by their jackets are found in the cytoplasm during the early phases of retraction. Degradation of the axonemal microtubules precedes the dissolution of the axonemal jacket. Profiles of the jackets are observed after the microtubules have been resorbed. The cilia appear to detach from the kinetosomes. Barren kinetosomes are seen below the cell surface frequently with kinetodesmal fibers still attached. Whether all or some of these barren kinetosomes contribute to the formation of the new ciliary anlage cannot be ascertained.     

Structure of Monocercomonas sp. as Revealed by Electron Microscopy*

SYNOPSIS. Monocercomonas shares many fine-structural features with all other trichomonads. These include the basic arrangement of the kinetosomes as well as of the recurrent and 3 anterior flagella. The pelta-axostyle complex and the parabasal apparatus, i.e. the Golgi complex and the periodic filaments, also conform to the trichomonad pattern. Of interest with regard to the crucial evolutionary position of Monocercomonas, considered to represent the most primitive trichomonad type, is the fact that it has some structures in common with other Monocercomonadidae and Trichomonadinae and others in common with Devescovinidae and Tritrichomonadinae. Among the former organelles are the marginal lamella and the costal base, and among the latter, the comb-like organelle situated between the infrakinetosomal body and parabasal filament 2 as well as the infrakinetosomal body. No traces of either costa or undulating membrane have been noted, but a complex structure homologous to the marginal lamella of Hypotrichomonas and Trichomonadinae is found underlying the short anteriormost portion of the recurrent flagellum that is attached to the body surface. Observations of sections of selected division stages indicate the potential of parental kinetosomes #1 and #3 to become daughter kinetosome #2.     

The Ultrastructure of Chaenea teres and an Analysis of the Phylogeny of the Haptorid Ciliates

Chaenea teres has typical haptorid ultrastructure. The somatic monokinetid has two transverse microtubular ribbons, an overlapping postciliary microtubular ribbon, and a laterally directed kinetodesmal fiber. The evered cytopharynx forms a dome at the apical end of the cell. The base of the dome is surrounded by oral dikinetids. The left, anterior kinetosome of the oral pair is not ciliated and has a transverse microtubular ribbon, a nematodesmata and a single postciliary microtubule. The right, posterior kinetosome is ciliated and has only postciliary microtubules. The kinetosomes at the anterior ends of the somatic kinetics are close together and their transverse microtubules and nematodesmata contribute to the support of the cytopharynx. The transverse microtubules of these oralized somatic kinetosomes, together with those from the oral dikinetids, line the cytopharynx. Accessory or bulge microtubules arise perpendicular to the transverse microtubules. A dorsal brush of three kineties of clavate cilia is found on the cell surface just posterior to the oral region. Mucocysts and a single type of toxicyst are present. The toxicysts are confined to the oral region. There are multiple ovoid macronuclei that stain weakly. Micronuclei were not observed. Cladistic analysis indicates the Chaenea may be most closely related to Fuscheria and Acropisthium. The cladistic analysis also suggests that existing taxonomies of the subclass Haptoria need to be revised. We propose some modifications to Foissner & Foissner's classification that include transferring Helicoprorodon, Actinobolina, the buetschiliids, and the balantidiids to the order Haptorida and recognizing the close relationship between pleurostomes and spathidiids.     

Transmission Electron Microscopical Observations on Stomatogenesis during Metamorphosis of Eufolliculina uhligi (Ciliophora: Heterotrichida)1

Stomatogenesis during metamorphosis of the marine loricate ciliate, Eufolliculina uhligi, was observed by transmission electron microscopy. Kinetosome proliferation in the stomatogenic territory leads to the formation of an anarchic field. This separates into the left adoral and the right paroral primordia. Both primordia consist of pairs of kinetosomes. One kinetosome of a pair is associated with one transverse and two postciliary microtubules; the other has one transverse microtubule. The postciliary microtubules of the adoral kinetosomes become divergent; those of the paroral kinetosomes become convergent. The adoral kinetosomes arrange in promembranelles. Then a third row of kinetosomes is produced anteriorly to each promembranelle. This third row is short at the peristome but longer in the buccal area. The paroral kinetosomes form a stichodyad. The buccal part of the paroral primordium is resorbed during formation of the buccal cavity. Stomatogenesis ends with the development of a functioning cytostome. During this process, the postciliary microtubules of the buccal adoral membranelles elongate and become associated with cytopharyngeal vesicles. Fusion of these vesicles with the cytostome has been observed some time after the completion of the oral structures.     

Fine Structure of the Mastigont System in Tritrichomonas muris (Grassi)*

SYNOPSIS. Tritrichomonas muris shares many fine-structural details with the previously described members of the family Trichomonadidae, and especially with the organisms belonging to the subfamily Tritrichomonadinae. Among the features which T. muris has in common with all Trichomonadidae and in all probability with other Trichomonadida are the arrangement and structure of: the kinetosomes; many rootlet filaments, including the sigmoid filaments of kinetosome #2; the parabasal apparatus; and the pelta-axostyle complex. The structures which T. muris-type flagellates share with other Tritrichomonadinae, and especially with Tritrichomonas augusta-type species (including T. foetus), but not with Trichomonadinae that have been studied to date, are: the Type A costa; a comb-like structure, which appears to have replaced the costal base of Trichomonadinae and of Hypotrichomonas; the suprakinetosomal body, rudimentary in T. muris; and the infrakinetosomal body. The undulating membrane, like that of T. augusta-type organisms, consists of a proximal and a distal part. The proximal part, which contains the proximal marginal lamella, is less developed in T. muris than in T. augusta-type flagellates, being represented by a relatively low ridge for the entire length of the organism. The distal part of the membrane in T. muris, on the other hand, is more highly developed; it is a heavy cord, with a distal marginal lamella which consists of a large triangular organelle having a highly ordered structure and 2 less well defined cords distal to this organelle. The tubules of the recurrent flagellum occupy the area distal to the cords. The sigmoid filaments of kinetosome #2, unlike those of other Trichomonadidae examined to date, extend posteriorly to the peltar-axostylar junction; they seem to terminate within the cytoplasm near, but not connected to the axostyle. In addition to the Type A costa, there is a small rootlet filament, which appears to be homologous to the rudimentary costa noted in Hypotrichomonas. Some of the paraxostylar and paracostal granules consist of an outer, relatively dense layer and an inner “core” of moderate density; between the 2 there is a lucent ring. The discussion deals in some detail with the possible nature of the paraxostylar and paracostal granules in trichomonads. The taxonomic status of Tritrichomonas cricetus (Wantland) [Tritrichomonas criceti (Wantland) emend. Levine] and Trichomonas criceti Ray & Sen Gupta is discussed in an appendix; it is concluded that both of these names are synonyms of T. muris (Grassi).     

Ultrastructure of the cytoplasm of the lower holotrichous infusorian Tracheloraphis prenanti]

The ciliature of T. prenanti Dragesco 1960 (forma oligocineta Raikov et Kovaleva, 1968) consists of 14-18 ventral and lateral longitudinal kineties with paired kinetosomes, carrying either two cilia or one cilium per kinetosome pair (in the latter case, the nonciliated kinetosome is always the posterior one). The ectoplasmic fibrillar system belongs to the postciliary type. A pair of kinetosomes shares a common basal plate. The anterior kinetosome gives rise to a short ribbon of transverse microtubules, the posterior one, to a poorly developed kinetodesmal filament and to a strong ribbon of postciliary microtubules. The latter proceeds backwards along 8 to 12 kinetosome pairs, being incorporated into a laminated postciliodesma which accompanies each kinety on its right side. Rows of Golgi elements, sending secretory vesicles and channels towards the body surface, exist beneath the kinetosome bases. Each kinety is accompanied on its left by a microfibrillar myoneme, surrounded by perimyary vesicles and underlain by a row of mitochondria. The median part of the dorsal surface is nonciliated; the cytoplasm here is rich of membrane systems, contains peripheral, electron-dense, extrusible inclusions and sometimes also bacteria. The electron-dense inclusions develop in the endoplasm, in close contact with mitochondria. The endoplasm contains also large microfibrillar spheres of unknown nature.     

A Reexamination of the Ultrastructure of Didinium nasutum and a Reanalysis of the Phylogeny of the Haptorid Ciliates

ABSTRACT. The cilia of Didinium nasutum are restricted to two girdles encircling the cell. Each row of cilia in both girdles is made up of two to three anterior pairs of kinetosomes followed by several single kinetosomes. Each single kinetosome has two sets of transverse microtubules, an overlapping postciliary microtubular ribbon, and a laterally directed kinetodesmal fiber. The pairs of kinetosomes are homologous to the oral dikinetids of other haptorians: the nonciliated kinetosome of the pair has a transverse microtubular ribbon that extends to line the membrane of the proboscis, a single short postciliary microtubule, and a nematodesma; the ciliated kinetosome has a ribbon of postciliary microtubules and two sets of transverse microtubules. The presence of these characters in Didinium invalidates Leipe & Hausmann's conclusion that the Didiniidae should be removed from the subclass that contains the other haptorians (Leipe, D. D. & Hausumann, K. 1989. Somatic infraciliature of the haptorid ciliate Homalozoon vermiculare (Kinetofragminophora, Gymnostomata) Ditransversalia n. subcl. and phylogenetic implications. J. Protozool. , 36 :280–289). In light of this, the justification for a subclass Ditransversalia is challenged and shown to be unnecessary.     

High-level mucosal and systemic immune responses induced by oral administration with <Emphasis Type="Italic">Lactobacillus</Emphasis>-expressed porcine epidemic diarrhea virus (PEDV) S1 region combined with <Emphasis Type="Italic">Lactobacillus</Emphasis>-expressed N protein

To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus (PEDV) infection, the DNA fragments encoding spike protein immunodominant region S1 and nucleocapsid N of PEDV were inserted into pPG1 (surface-displayed) or pPG2 (secretory) plasmids followed by electrotransformation into Lactobacillus casei (Lc) to yield four recombinant strains: PG1-S1, PG2-S1, PG1-N, and PG2-N. After intragastric administration, it was observed that live Lc-expressing S1 protein combined with Lc-expressing N protein could elicit much more potent mucosal and systemic immune responses than the former alone (P < 0.001), however slightly inferior to the latter alone (P > 0.05). Furthermore, the surface-displayed mixture (PG1-S1+ PG1-N) revealed stronger immunogenicity than the secretory mixture (PG2-S1+ PG2-N) as well as PEDV-neutralizing potency in vitro (P < 0.001). On 49th day after the last immunization, splenocytes were prepared from mice immunized with surface-displayed mixture, secretory mixture and negative control to be stimulated by purified N and S protein, respectively. The results of ELISA analysis showed that N protein was capable of inducing a higher level of IL-4 (P < 0.001) and IFN-γ (P < 0.001) than S1 protein in the immunized mice. Taken together, Lc-expressed N protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by Lc-expressing S1 region.