To what extent are ephippia of Mexican Anomopoda (Crustacea, Cladocera) identifiable?

Diapausing embryos encased within cladoceran ephippia result from sexual reproduction and increase genetic diversity. They are also important means by which species bypass harsh environmental conditions and disperse in space and time. Once released, ephippia usually sink to the benthos and remain there until hatching. Using the Sars' method (incubating sediments to identify cladoceran hatchlings), ephippial egg bank biodiversity can be evaluated. Yet, even when samples are incubated under a variety of conditions, it is not possible to warrant that all have hatched. Few keys are available that facilitate the identification of cladocerans by using only ephippial morphology. Our goal was to analyze some cladoceran ephippia from Mexico, to develop a means to identify them using easily recognizable characteristics. Ephippia of 23 cladoceran species from waters in Aguascalientes (México) in 11 genera (Alona, Biapertura, Ceriodaphnia, Chydorus, Daphnia, Dunhevedia, Ilyocryptus, Macrothrix, Moina, Pleuroxus, and Simocephalus) were analyzed. In our analysis six morphological features were selected that permitted the identification of ephippia to species(-group) level. The results demonstrate that with a proper catalog of features, some ephippia can be identified.

Resilience of rotifers and cladocerans communities in four reservoirs with eutrophication pollution and lead concentrations in Aguascalientes, Mexico.

Pollution in aquatic ecosystems has been increasing drastically worldwide changing their water quality. Therefore, species must be adapted to these new scenarios. In Aguascalientes City, four representative urban reservoirs contain lead in the water column and extremely high concentrations of sediments. Therefore, an analysis was conducted to evaluate the resilience of zooplankton species to lead exposure in each reservoir using dormant and organisms. Results demonstrated a decrease range from 57.5 to 22.5% in overall diapausing egg hatching rate, while survivorship rate also decreased from 98 to 54% when organisms were exposed to the water of the four reservoirs and increasing lead concentrations. When Pb exposure increased, results showed a global negative effect on both hatching rate (decreasing from 58 to 30% at 0.09 mg L-1) and survivorship levels (decreasing from 100% to 0.07% at 0.09 mg L-1). We provide Species Sensitivity Distribution for both water reservoir dilutions and lead concentration to analyze diapausing eggs hatching and survivorship of offspring in the presence of same polluted conditions or lead of the autochthonous species found in reservoirs. Furthermore, specific analysis with two populations of the cladoceran Moina macrocopa showed clear dissimilar hatching patterns that suggested a different adaptive mechanism. Niagara population shows a hatching rate of approximately 25% in the first two days of reservoir water exposure, while UAA population drastically increased hatching rate to 75% on exposure at day seven. We provide the first record of bioaccumulation in ephippia of M. macrocopa.

Feeding behavior of early life stages of the zebrafish Danio rerio is altered by exposure to glyphosate.

Glyphosate levels and the transfer of glyphosate across trophic levels have rarely been studied in zooplankton. The food preferences of zebrafish during the first-feeding stage (which is critical for the survival of organisms), were analyzed because of the requirement for live food. Larval survival begins to be affected when glyphosate intake exceeds 0.3666 µg/larvae/day, in the case that only the food is contaminated; if the medium is also contaminated, the effects on survival start from 0.2456 µg/larvae/day. It was shown that glyphosate was more likely to be incorporated through the medium than through the food (zooplankton), which supports the results of previous studies that have ruled out the potential for biomagnification. The bioconcentration factor (BCF) of glyphosate was determined using an ELISA tests specific to measure glyphosate in the fish D. rerio, the rotifers Brachionus calyciflorus and Lecane papuana, and the cladoceran Ceriodaphnia dubia. The experimental design consisted in exposing seven zebrafish adults per replica (four replicates) in three treatments 1, 5, and 10 mg/L of glyphosate for 96 h to obtain bioconcentration factors in the gills, liver, and muscle. These concentrations were selected as potential glyphosate concentrations right after application as double highest reported concentration. Glyphosate levels in zooplankton can represent up to 6.26% of the total weight of rotifers (BFC = 60.35) and in zebrafish adult organs were less than 8 µg/mg of tissue (BCF values < 6). Although glyphosate does not biomagnify, our results suggest that glyphosate affected the dynamics between zooplankton and zebrafish larvae, diminishing survival and feeding rates, given that zooplankton species bioconcentrate glyphosate in large quantities. The BCF values found in this contribution are higher than expected. Glyphosate exposure affected energy metabolism and feeding behavior of zebrafish larvae, which presented high mortality rates at environmentally relevant concentrations.

A new species of Nitokra Boeck, 1865 (Copepoda, Harpacticoida, Ameiridae) from the Caribbean coast of Colombia.

Biological samples obtained from a coastal system of northern Colombia yielded male and female specimens of an undescribed harpacticoid copepod of the diverse ameirid genus Nitokra Boeck, 1865. The new species is a member of the genus group III. We describe the new species based on adult male and female individuals. Nitokrapuebloviejensis sp. nov., appears to be most closely related to N.vietnamensis Tran & Chang, 2012, but they can be separated by the following characters: 1) number of setal elements on second segment of mandibular palp, 2) P1ENP/EXP ratio, 3) relative lengths of P2, P3ENP/EXP, 4) number of elements on male P5EXP and ENP, and 5) segmentation of male antennule. In addition, N.puebloviejensis sp. nov. can be confused with two other congeners: N.taylori Gómez, Carrasco & Morales-Serna, 2012 from South Africa and Colombia and N.kastjanensis Kornev & Chertoprud, 2008 from the White Sea, but the new species can be distinguished from them by: 1) number of setae on the maxillule coxa, 2) P1ENP/EXP ratio, 3) P2,P3ENP/EXP ratio, 4) female and male P5 setophore, 5) setation pattern of female P5EXP and ENP, 6) structure of female P6, 7) ornamentation of female anal operculum, 8) number of setae on male P5EXP, and 9) the male antennule segmentation. Most importantly, the presence of a group of five short setae on the medial surface of the maxilliped syncoxa allows the new species to be readily distinguished from its congeners. Only two subspecies and one species of this genus have been hitherto recorded from Colombia. A key to the 23 known American species of Nitokra is provided.

A new species of Esola Edwards, 1891 (Crustacea, Copepoda, Harpacticoida, Laophontidae) from the Caribbean coast of Colombia.

A new species of the harpacticoid copepod genus Esola is described from specimens collected in Rodadero Beach, on Gaira Bay, on the Caribbean coast of Colombia. The species, E.wellsi sp. nov., is described, illustrated, and com-pared with its congeners. Esolawellsi sp. nov. differs from its known congeners in details of the armature of legs 1-4. It most closely resembles E.bulbifera (Norman, 1911) in the armature formula of P1-P5 but differs from the latter in several respects, including the female antennule segmentation (7-segmented in E.bulbifera but distinctly 6-segmented in E.wellsi sp. nov.) and in the shape and size of the male P3ENP2 apophysis, among other characters. This is the second species of the genus known from the Caribbean and the second record of Esola in the Northwestern Tropical Atlantic. The genus now contains eight species. A key to the known species of the genus is also included.

A new species of scavenger Cladocera Pseudochydorus Fryer, 1968 (Cladocera: Anomopoda: Chydoridae) from the Central Mexican Plateau.

A new species of the genus Pseudochydorus Fryer, 1968 is described from Central Mexico. P. margaritalfonsorum sp. nov. differs from the Old World species of the genus, P. globosus (Baird, 1843) and P. bopingi Sinev, Garibian Gu, 2016 in the morphology of thoracic limbs IIII. Analysis of existing literature data on distribution and morphology of Pseudochydorus in America suggest than P. margaritalfonsorum sp. nov. is an endemic of Central Mexican Plateau, and at least two more species of the genus are present in other regions of America.

Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization.

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30-35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve-hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.

A record of Disparalona hamata (Birge, 1879) (Cladocera: Chydoridae) in phytotelmata of Tillandsia aguascalentensis Gardner, 1984 (Poales: Bromeliaceae).

Bromeliads (Bromeliaceae) are an extremely diverse family of the angiosperms widely distributed in the tropical and subtropical regions of the Americas and West Africa. They often serve as phytotelmata, accumulating rainwater between leaves. Such water reservoirs can be inhabited by diverse organisms. But to date not much attention is paid to inventory of these organisms, with careful identification of each taxon. We found a microcrustacean Disparalona hamata (Birge, 1879) (Crustacea: Cladocera) in the bromeliad Tillandsia aguascalentensis Gardner, 1984 in Mexico. Investigated population included parthenogenetic females, gamogenetic females and males. Hereby the population apparently can pass the full life cycle under conditions of phytotelmata. Along with ecological observations, we provide additional taxonomic notes on the genus Disparalona Fryer, 1968 itself. Recently a series of morphological revisions was conducted for this genus. It was subdivided into two subgenera: Disparalona s.str. and Mixopleuroxus Hudec, 2010. But, in fact, the second taxon is a junior synonym of Leptorhynchus Daday, 1905. In this regard here we provide an updated list of taxonomic synonyms for the subgenus Leptorhynchus.

Brachionus paranguensis sp. nov. (Rotifera, Monogononta), a member of the L group of the Brachionus plicatilis complex.

The Brachionus plicatilis complex represents the most studied group of rotifers, although the systematics of the species complex has not been completely clarified. Many studies have been conducted trying to explore the diversity within the complex, leading to the recognition of three major morphotypes: large (L), small-medium (SM), and small (SS). Currently six species have been described and classified under these types and another nine taxa have been identified but not formally described. Within the L group, three species have been officially described [B. plicatilis s.s. (L1), B. manjavacas (L2), and B. asplanchnoidis (L3)], while a formal description of L4, unofficially known as B. 'Nevada', is still lacking. In the present study, a new species, Brachionus paranguensis sp. nov., is formally described and presented as a representative of the L4 clade. The species has been named after a high altitude saline crater lake from Central Mexico, where the specimens were collected. An integrated approach using DNA taxonomy through COI and ITS1 markers, morphology, and ecology was used to confirm the identity of the new species.

Morphological alterations in the freshwater rotifer Brachionus calyciflorus Pallas 1766 (Rotifera: Monogononta) caused by vinclozolin chronic exposure.

Vinclozolin (VZ) is a dicarboximide fungicide widely used on fruits, vegetables and wines, effective against fungi plagues. In this study we characterized the effects of VZ using a 4-day reproductive chronic assay with the freshwater rotifer Brachionus calyciflorus. The assay included observations of several features of asexual and sexual reproduction. Our results indicate that VZ: (a) increased asexual and sexual reproduction, (b) caused severe abnormality in females and (c) these abnormalities were inherited by sexual and asexual reproduction. At 1.2 mg/L three abnormal females were found out of 457 total females (0.66 %). This low percentage is consistent and reproducible according to further analysis, where we increased the number of replicates and total females exposed to 1.2 mg/L of VZ, and found 18 abnormal females out of 2868 total females (0.63 % abnormality). Interestingly, abnormal females found at 5.6 mg/L VZ exposure, were able to show mating behavior. Our results suggest that VZ behaves as a strong endocrine disruptor whose effects show the characteristic inverted-U-shape exposure concentration response curve regarding the intrinsic population increase and the percentage of abnormalities as endpoints.

Morphology and ultrastructure of the freshwater rotifer Brachionus bidentatus (Monogononta: Brachionidae) using scanning and transmission electron microscopy / Morfología y ultraestructura del rotífero de agua dulce Brachionus bidentatus (Monogononta: Brachionidae), utilizando microscopía electrónica de barrido y transmisión

The study of sexual reproductive behavior supported by ultrastructural evidence is important in rotifers to describe differences among potential cryptic species. In this research, the morphology of the rotifer Brachionus bidentatus is described at the ultrastructural level, using electronic microscopy, together with a brief description and discussion of its sexual reproductive behavior. The characteristics of the (a) male,(b) the female, (c) the sexual egg or cyst, (d) the partenogenic egg, (e) the no-fecundated sexual egg (male egg), and (f) the trophi, were described. Another part of this research is dedicated to the ultrastructure of the sex cells of the male rotifer B. bidentatus. Samples were obtained from La Punta pond in Cosio, Aguascalientes, Mexico (22°08’ N - 102°24’ W), and a culture was maintained in the laboratory. Fifty organisms, from different stages of the rotifer Brachionus bidentatus, were fixed in Formol at 4% and then prepared; besides, for the trophi, 25 female rotifer Brachionus bidentatus were prepared for observation in a JEOL 5900 LV scanning electronic microscope. In addition, for the observation of male sex cells, 500 males of Brachionus bidentatus were isolated, fixed and observed in a JEOL 1010 transmission microscope. Females of B. bidentatus in laboratory cultures had a lifespan of five days (mean±one SD=4.69±0.48; N=13), and produced 4.5+3.67 (N=6) parthenogenetic eggs during such lifespan. In the case of non-fertilized sexual eggs, they produced up to 18 eggs (mean±one SD=13±4.93; N=7). Sexual females produced a single cyst on average (mean±one SD=1±0; N=20). For the sexual cycle, the time of copulation between male and female ranged from 10 to 40 seconds (mean±one SD=17.33±10.55, N=7). The spermatozoa are composed of a celular body and a flagellum, the size of the body is of 300nm while the flagellum measures 1 700nm. The rods have a double membrane. Their mean length is almost 2.45µm±0.74, N=6; and their mean wide is 0.773µm±0.241, N=11. The evidence on the specific ultrastructural characteristics of the rotifer B. bidentatus is notorious, even more in the male and in the cyst cell. Regarding the ultrastructure of the spermatozoa and the rods, compared to other species they only differ in size, despite their structural resemblance. Our study of the ultraestructure of this species adds useful information that along with molecular data will help clarify the taxonomy of brachionid rotifers. Rev. Biol. Trop. 61 (4): 1737-1745. Epub 2013 December 01.

El estudio del comportamiento reproductivo sexual apoyado en evidencias ultraestructurales en rotíferos, es importante para describir diferencias entre especies potencialmente crípticas. En este trabajo se describe a nivel ultraestructural la morfología del rotífero Brachionus bidentatus, usando microscopía electrónica, junto con una breve descripción y discusión de su comportamiento sexual reproductivo. Se presentan las características del: (a) macho, (b) hembra, (c) huevo partenogenético, (d) huevo sexual no fecundado, (e) trofos. También se muestra un apartado sobre la ultra estructura de las células sexuales de rotíferos macho B. bidentatus. Para el estudio se utilizó una cepa proveniente del bordo La Punta (22°08’ N - 102°24’ W), ubicado en Cosió, estado de Aguascalientes, México y se cultivaron en el laboratorio. Para el procesamiento de las muestras se tomaron 50 organismos fijados en Formol al 4%, de los diferentes estadios del rotífero Brachionus bidentatus, mientras que para el trofos se tomaron 25 organismos hembra del rotífero Brachionis bidentatus y se prepararon para observarse en un microscopio electrónico de barrido JEOL 5900 LV, mientras que para las observaciones de las células sexuales del macho se aislaron 500 organismos machos del rotífero Brachionus bidentatus, se fijaron e incluyeron en resina epóxica (EPON) para su observación en un microscopio electrónico de transmisión JEOL 1010 operado a 80kv. Los análisis obtenidos de hembras de B. bidentatus en cultivos de laboratorio demuestran un ciclo de vida de cinco días (4.69±0.48; N=13), y una producción de 4.5±3.67 (N=6) huevos partenogenéticos. En el caso de los huevos no fertilizados, la hembra produce más de 18 huevos (13±4.93; N=7). Para los huevos sexuales solo se produce uno solo por hembra (1±0; N=20). En el ciclo sexual, los tiempos de copula entre el macho y la hembra están en el rango de 10 a 40 segundos (17.33±10.55, N=7). Los espermatozoides se componen de un cuerpo celular y un flagelo, el tamaño del cuerpo celular es de 300nm mientras que el flagelo mide 1 700nm. Los bastones presentan una doble membrana y su tamaño a lo largo va de 2.45µm±0.74; N=6 mientras que el ancho es de 0.773µm±0.241; N=11. La evidencia sobre las características específicas ultraestructurales del rotífero B. bidentatus son notorias, más aun en el macho y en el quiste. En cuanto a la ultraestructura de los espermatozoides y los bastones respecto a otras especies sólo difieren en su tamaño, a pesar de las similitudes estructurales las estructuras de los bastones se evidencian con mayor claridad y dan más evidencias sobre su funcionalidad. Nuestro estudio de la ultraesturctura de esta especie añade información útil que junto con un análisis molecular ayudarán a clarificar la taxonomía de rotíferos brachionidos.

A new species of Metacyclops Kiefer, 1927 (Copepoda, Cyclopidae, Cyclopinae) from the Chihuahuan desert, northern Mexico.

A new species of the freshwater cyclopoid copepod genus Metacyclops Kiefer, 1927 is described from a single pond in northern Mexico, within the binational area known as the Chihuahuan Desert. This species belongs to a group of Metacyclops species with a 3443 spine formula of swimming legs. It is morphologically similar to Metacyclops lusitanus Lindberg, 1961 but differs from this and other congeners by having a unique combination of characters, including a caudal rami length/width proportion of 3.5-3.8, a innermost terminal seta slightly longer than the outermost terminal seta, intercoxal sclerites of legs 1-4 naked, a strong apical spine of the second endopodal segment of leg 1 and one row of 6-8 small spinules at the insertion of this spine. The finding of this species represents also the first record of the genus in Mexico and the third in North America, where only two other species, Metacyclops gracilis (Lilljeborg, 1853)and Metacyclops cushae Reid, 1991 have been hitherto reported. This is also the first continental record of a species of Metacyclops from an arid environment in the Americas. This species appears to be endemic to the Chihuahuan Desert, thus emphasizing the high endemicity of this area.

Morphology and ultrastructure of the freshwater rotifer Brachionus bidentatus (Monogononta: Brachionidae) using scanning and transmission electron microscopy.

The study of sexual reproductive behavior supported by ultrastructural evidence is important in rotifers to describe differences among potential cryptic species. In this research, the morphology of the rotifer Brachionus bidentatus is described at the ultrastructural level, using electronic microscopy, together with a brief description and discussion of its sexual reproductive behavior. The characteristics of the (a) male, (b) the female, (c) the sexual egg or cyst, (d) the partenogenic egg, (e) the no-fecundated sexual egg (male egg), and (f) the trophi, were described. Another part of this research is dedicated to the ultrastructure of the sex cells of the male rotifer B. bidentatus. Samples were obtained from La Punta pond in Cosio, Aguascalientes, Mexico (22 degrees 08' N - 102 degrees 24' W), and a culture was maintained in the laboratory. Fifty organisms, from different stages of the rotifer Brachionus bidentatus, were fixed in Formol at 4% and then prepared; besides, for the trophi, 25 female rotifer Brachionus bidentatus were prepared for observation in a JEOL 5900 LV scanning electronic microscope. In addition, for the observation of male sex cells, 500 males of Brachionus bidentatus were isolated, fixed and observed in a JEOL 1010 transmission microscope. Females of B. bidentatus in laboratory cultures had a lifespan of five days (mean+one SD = 4.69 +/- 0.48; N=13), and produced 4.5 +/- 3.67 (N=6) parthenogenetic eggs during such lifespan. In the case of non-fertilized sexual eggs, they produced up to 18 eggs (mean+one SD = 13 +/- 4.93; N=7). Sexual females produced a single cyst on average (mean +/- one SD = I +/- 0; N=20). For the sexual cycle, the time of copulation between male and female ranged from 10 to 40 seconds (mean +/- one SD = 17.33 +/- 10.55, N=7). The spermatozoa are composed of a celular body and a flagellum, the size of the body is of 300 nm while the flagellum measures 1 700nm. The rods have a double membrane. Their mean length is almost 2.45 microm +/- 0.74, N=6; and their mean wide is 0.773 microm +/- 0.241, N=11. The evidence on the specific ultrastructural characteristics of the rotifer B. bidentatus is notorious, even more in the male and in the cyst cell. Regarding the ultrastructure of the spermatozoa and the rods, compared to other species they only differ in size, despite their structural resemblance. Our study of the ultraestructure of this species adds useful information that along with molecular data will help clarify the taxonomy of brachionid rotifers.

Bioconcentration and localization of lead in the freshwater rotifer Brachionus calyciflorus Pallas 1677 (Rotifera: Monogononta).

We studied how lead is bioconcentrated and distributed in the rotifer Brachionus calyciflorus using metal histochemistry to locate lead granules, Leadmium Green® analysis to establish the route of uptake, atomic absorption to determined the bioconcentration factor (BCF), and detected the presence of microelements in the cuticle by X-ray microanalysis with scanning electron microscopy. Our results indicate: (a) the digestive system is the main route of lead uptake in the rotifer B. calyciflorus, (b) after 24-h lead is deposited in granules in the mastax and vitellarium, (c) our energy-dispersive X-ray microanalysis indicates decalcification taking place in the cuticle of the rotifer after a 24-h lead exposure, and (d) we determined a BCF = 115 for lead after a 24 h exposure. However, the route of mobilization and storage of intracellular lead are still not fully understood in B. calyciflorus.

Illustrated record of the freshwater copepod Leptodiaptomus dodsoni (Calanoida, Diaptomidae) from central Mexico with comments on the distribution of the genus / Registro ilustrado del copépodo dulceacuícola Leptodiaptomus dodsoni (Calanoida, Diaptomidae) del centro de México con comentarios acerca de la distribución del género

The freshwater planktonic calanoid copepod Leptodiaptomus dodsoni Elías-Gutiérrez, Suárez-Morales & Romano, 1999, a rare species known only from western Mexico, is here recorded from the State of Aguascalientes, central Mexico. This species has a uniquely large, sinuous spine on segment 13 of the right male antennule, reaching the distal margin of segment 15. There are subtle differences between these two known populations; the morphological range of the species is complemented with data from the new specimens. This is the first record of L. dodsoni after its original description; it is probably restricted to a latitudinal fringe in central Mexico. Of the 22 nominal species of Leptodiaptomus currently recognized, up to 7 are known from the Neotropical region and some appear to be true endemics. Apparently, this primarily Nearctic genus radiated in North America and some species reached the Neotropical region where they diversified. A key for the identification of the species of the genus known from the Neotropical region is also provided.

El copépodo calanoide planctónico de aguas continentales Leptodiaptomus dodsoni Elías-Gutiérrez, Suárez-Morales & Romano, 1999, una rara especie conocida sólo en la zona occidental de México, se registra aquí para el estado de Aguascalientes, en el centro de México. Esta especie tiene una espina sinuosa distintivamente larga en el segmento 13 de la anténula derecha del macho, la espina alcanza el margen distal del segmento 15. Existen differencias sutiles entre estas dos poblaciones; se complementa el espectro morfológico de la epecie con datos de los nuevos especímenes. Este es el primer registro de L. dodsoni después de su descripción original; probablemente está restringida a una franja latitudinal del centro de Mexico. De las 22 especies nominales de Leptodiaptomus reconocidas, 7 se conocen en la zona Neotropical y algunas parecen ser verdaderos endémicos. Aparentemente, este género primariamente Neártico radió en América del Norte y estas especies alcanzaron la región Neotropical donde se diversificaron. Se presenta una clave para la identificación de las especies del género conocidas en la región Neotropical.

Ultrastructural analysis and identification of membrane proteins in the free-living amoeba Difflugia corona.

Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.