Rutilus Rubilio Research Articles

Microplastics pollution in freshwater fishes in the South of Italy: Characterization, distribution, and correlation with environmental pollutants

In this study, we investigated the presence, abundance, and chemical nature of microplastics (MPs) in the freshwater fish gastrointestinal tract in the South of Italy, and evaluated the possible correlation between MPs and environmental pollutants. Fifty specimens belonging to five species (Scardinius erythrophthalmus, Barbus barbus, Rutilus rubilio, Leuciscus cephalus, Salmo trutta), from twenty sites were collected. MPs chemical feature was identified by means of Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) and Raman microscopy. MPs were represented by 34.86 % fragments, film, and foam (all together MPs) and 65.14 % by fibers (MFs). The mean number of MPs/MFs per fish ranged from 6.25 ± 4.35 in R. rubilio and 2.26 ± 1.94 in B. barbus. The highest number of MPs/MFs per g of GIT was found in R. rubilio (9.07 ± 9.66), and the lowest in S. erythrophthalmus (0.75 ± 0.53). The highest number of MPs/MFs per fish species was found in L. cephalus (16), and the lowest in S. erythrophthalmus (4). Black predominated in every type of plastic debris identified, followed by blue and white, respectively for MFs and MPs. Polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), and polypropylene (PP), were the main plastic polymers found. At fish sampling sites, comparing concentrations in soils of potentially toxic elements and persistent organic pollutants with the number of MPs/MFs in fish, a significant correlation was noted with polychlorinated biphenyls (PCBs) and, in particular, with PCB 105, PCB 118, PCB 156, PCB 157, and PCB 167. A strong correlation was also observed with all types of polycyclic aromatic hydrocarbon (PAHs) particularly with benzo(ghi)perylene, dibenz(a,h)anthracene, benzo(b)fluoranthene, benz(a)anthracene, benzo(a)pyrene, and pyrene. The results of this study would be useful to draft management and action plans, promote intervention plans aiming at removing threats to species and habitats, and address ways of renaturalization.

Status ofPseudorasbora parvain the Tiber River Basin (Umbria, central Italy) 20 years after its introduction

The aim of this research was to assess the distribution, abundance and growth of the non-native topmouth gudgeon Pseudorasbora parva that was recorded for the first time in the Tiber River Basin (central Italy) in 1994. The competitive interaction of P. parva with four native fish species was also investigated. The study area comprised 92 watercourses of the Umbrian section of the Tiber River Basin. Demographic and environmental data were collected during the period 1990−2014 in 171 sampling sites. The results of this study showed a wide distribution of P. parva in the study area, with records from 23.39% of all sampling sites (40 out of 171). This species inhabits the downstream reaches, where the presence of many non-native species and the poor environmental quality are associated with a decrease in native fish species. A total of 5570 specimens of P. parva were collected and five age classes (0+ to 4+ ) were identified. The equation for the total length-weight relationship of P. parva was W = 0.021T L 2.673±0.015 . For the chub Squalius squalus , the Tiber barbel, Barbus tyberinus, and the roach, Rutilus rubilio, the average values of the relative weight were significantly higher in the sites where P. parva was absent. The results of the present study suggest the need to undertake proper strategies for native biodiversity conservation.

Morphological variation in riverine cyprinids: a geometric morphometric contribution

This study aimed to contribute to understanding the role of changes in proportions related to variation in size in freshwater fish, particularly Rutilus rubilio, Squalius lucumonis and Telestes muticellus, from central Italy. The ontogenetic shape variation was described by a multivariate regression where the independent and dependent variables were size and a set of shape variables (the latter obtained from geometric morphometric analysis of 16 anatomical landmarks), respectively. Our analyses indicated: (1) proportional ontogenetic changes in several morphometric characters in all populations of the three species; (2) no geographical shape variation for each species; (3) interspecific differences. The main shape changes joining all the populations are the ocular area contraction, the tail peduncle enlargement, and the main changes involving the median body region. The shape variations showed similar modification patterns between R. rubilio and S. lucumonis (the silhouette passed from a streamlined to a more oval and higher shape for both species) and some differences between R. rubilio and S. lucumonis vs. T. muticellus (the latter approaching a more spindle-shaped body). The expression of these allometric trajectories may be associated with age-specific shifts in the ecological niche, optimizing some features such as foraging efficiency and movement.

Molecular Barcoding of an Atypical Cyprinid Population Assessed by Cytochrome b Gene Sequencing

A fish population of the carp family Cyprinidae with atypical phenotypic characteristics was observed in one of the main catchments of the Pollino National Park, a valuable, protected area in southern Italy. In this area, the Italian roach Rutilus rubilio (Bonaparte, 1837), a native endemic fish of Tyrrhenean regions, has been introduced in sympatric conditions with Squalius squalus (Bonaparte, 1837) and Telestes muticellus (Bonaparte, 1837). A molecular investigation was carried out to assess the genetic identity of the population with a view to conservation. Direct sequencing of a cytochrome b gene fragment was performed based on 30 individuals of cyprinid fish with atypical phenotype, in addition to 30 S. squalus, 10 T. muticellus, and 30 R. rubilio pure individuals collected in different Italian regions, which served as reference samples. Multiple sequence alignments demonstrated that 50% of atypical-cyprinid haplotypes were maternally inherited from either S. squalus or R. rubilio. No contribution by T. muticellus was determined. Our results indicate an intergeneric hybridization event between S. squalus and R. rubilio, as a consequence of trans-introduction activities of alien species.

Testing Species Delimitations in Four Italian Sympatric Leuciscine Fishes in the Tiber River: A Combined Morphological and Molecular Approach

Leuciscine fishes represent an important component of freshwater ichthyofauna endemic to northern Mediterranean areas. This lineage shows high intra-specific morphological variability and exhibits high levels of hybridization, two characteristics that contribute to systematic uncertainties, misclassification of taxa and, potentially, the mismanagement of biodiversity. This study focused on brook chub, Squalius lucumonis, an endemic taxon of Central Italy. The taxonomic status of this species has long been questioned, and a hybrid origin from sympatric leusciscines (S. squalus x Rutilus rubilio, or S. squalus x Telestes muticellus) has been hypothesised. A phenotypic (evaluating shape and meristic counts) and genetic (using mitochondrial and nuclear markers) investigation of these four taxa was conducted to test species delimitation in sympatric areas and to evaluate the taxonomic status of S. lucumonis. One hundred and forty-five individuals of all four taxa were collected within streams of the lowest portion of the Tiber River basin and analysed; this region encompasses a large portion of the S. lucumonis distribution. The different morphological and genetic approaches were individually examined, compared, and then combined in a quantitative model to both investigate the limits of each approach and to identify cases of misclassification. The results obtained confirm the cladogenetic non-hybrid origin of S. lucumonis, highlight the need for immediate conservation actions and emphasise the value of an integrated approach in the study of leuciscines evolution.

Molecular cytogenetic analysis of the Appenine endemic cyprinid fish Squalius lucumonis and three other Italian leuciscines using chromosome banding and FISH with rDNA probes

Karyotype and other chromosomal characteristics of the Appenine endemic cyprinid fish, Toscana stream chub Squalius lucumonis, were analysed using conventional banding and FISH with 45S and 5S rDNA probes. The diploid chromosome number (2n = 50) and karyotype characteristics including pericentromeric heterochromatic blocks and GC-rich CMA(3)-positive sites corresponding to both positive Ag-NORs and 45S rDNA loci on the short arms of a single medium-sized submetacentric chromosome pair were consistent with those found in most European leuciscine cyprinids. On other hand, 5S rDNA FISH in the Toscana stream chub and three other Italian leuciscines, S. squalus, Rutilus rubilio and Telestes muticellus, revealed a species-specific hybridization pattern, i.e. signals on four (S. lucumonis), three (S. squalus and R. rubilio) and two (T. muticellus) chromosome pairs. Whereas all the species shared the 5S rDNA loci on the largest subtelocentric chromosome pair, a "leuciscine" cytotaxonomic marker, S. lucumonis showed both classes of rDNA loci tandem aligned on the short arms of chromosome pair No. 12. The present findings suggest that the observed high variability of 5S rDNA loci provides a powerful tool for investigation of karyotype differentiation in karyologically conservative leuciscine fishes.

Assessing the impact of non-native freshwater fishes on native species using relative weight

The aim of the research was to test relative weight (W r ), a condition index which allows evaluation of fish well-being, as a tool to investigate the impact of the presence of non native species (NNS) on the condition of the key native species (NS) of the Tiber River basin (Italy): Barbus tyberinus Bonaparte, Leuciscus cephalus (Linnaeus), Leuciscus lucumonis Bianco, Rutilus rubilio (Bonaparte) and Telestes muticellus (Bonaparte). By means of Canonical Correlation Analysis, data from 130 sampling sites, distributed throughout Tiber River basin, were examined. W r of NS was related to densities of NNS and to environmental variables. Moreover, the correlation between W r of NS and density of NNS was investigated through linear regression analysis and covariance analysis. Preliminary results encourage the use of W r as a tool to assess the relationship between NS and ecological factors (such as the presence of NNS) and to explain the changes that occur along the longitudinal gradient of a river.

Infestation comparée deLigula intestinalis(Cestoda: Diphyllobothridae) chez deux poissons Cyprinidés :Rutilus rubilioetScardinius erythrophthalmusdans deux retenues de barrages en Tunisie

Monitoring quantitative parameters of parasitism by ligula intestinalis (Cestoda: Diphyllobothridae) was performed by examining 516 fish belonging to two introduced freshwater species: Rutilus rubilio (350 individuals) and Scardinius erythrophthalmus (166 individuals). These fish were collected in two dam reserves in Tunisia, Sidi Salem and Nebhana. The analysis of the aquatic bird's composition in these two reserves revealed the existence of piscivorous bird species that were previously reported as final host of Ligula. Monitoring the bird's composition highlighted higher relative abundance and frequency in Sidi Salem than in Nebhana dam reserve. The analyses of the prevalence, mean intensity and abundance of the parasite revealed the most important values in roach, Rutilus rubilio which seems to be the preferential second intermediate host of the parasite Ligula intestinalis in these environments. Comparative analysis of parasitism in both explored sites suggests that Ligula intestinalis presents two different strategies of infestation. In Sidi Salem reserve, which is larger than Nebhana and containing on important and diversified piscivorous species, the parasite infects a maximum of host individuals with low parasite mean intensity values. However, at Nebhana, which is a smaller reserve, the parasite infects fewer individuals than Sidi Salem but with higher mean intensity. The highest prevalence values were recorded in large size classes of roach species in Sidi Salem reserve.

Eurasian otter (Lutra lutra) prey selection in response to a variation of fish abundance

In 2001 and 2002 we studied Eurasian otter (Lutra lutra) fish selection in southern Italy by comparing otter diet (805 analysed spraints) with fish availability, estimated by electrofishing. The surveyed river was dominated by cyprinids, particularly by chub (Leuciscus cephalus), roach (Rutilus rubilio) and bleak (Alburnus sp.). Roach and chub were the most common prey recorded in spraints. Otters ate mainly small prey (total length < 90 mm). Chub were widely consumed according to abundance, whilst roach and bleak consumption was not proportionate to their availability. Otters selected roach both in 2001 and 2002 and responded to a decrease in roach availability (–27.3%) by increasing their frequency of consumption rather than switching to an alternative prey. Despite their high abundance, bleak seemed to be markedly avoided by otters. We hypothesized that differences in microhabitat selection may influence the susceptibility of cyprinids to otter predation. The possible effects on fish community were briefly discussed.

Feeding ecology of Leuciscus cephalus and Rutilus rubilio in southern Italy

The diet and feeding relationships of two cyprinids, the Italian orange‐fin roach Rutilus rubilio and the chub Leuciscus cephalus, were studied in southern Italy by the analysis of 189 gut contents. Fish species were sampled in conditions of sympatry/hybridisation in the River Sinni, and allopatry in the River Mercure‐Lao (roach) and in the River Raganello (chub). The diet of both fish species included both plant material and invertebrates. The broad spectrum of prey eaten showed that both fish species looked for invertebrates along the whole water column and particularly on the surface. For the River Sinni a high dietary overlap was found. The high contribution of plant material in the overall diet of roach in the presence of other cyprinids species, compared with the almost equal importance of plant material and animal food in allopatry, suggested that competition could be the cause of roach dietary shift towards suboptimal food sources. For the river Raganello, the high proportion of vegetation in the diet of chub, compared to the River Sinni, was explained as a consequence of habitat degradation caused by anthropogenic activities. Our results confirmed the generalist and opportunistic foraging behaviour of both fish species.

The presence of microcystins and other cyanobacterial bioactive peptides in aquatic fauna collected from Greek freshwaters

Toxic bloom-forming cyanobacteria can cause animal death and adversely affect human health. Blooms may contain microcystins (MCs), cyanobacterial heptapeptide hepatotoxins and other peptides such as anabaenopeptins and anabaenopeptilides. MCs have been shown to occur in various aquatic organisms including mussels, water snails, crustaceans and fish. Muscle and viscera samples from eight species of fish ( Acipenser gueldenstaedtii, Carassius auratus, Carassius gibelio, Cyprinus carpio, Perca fluviatilis, Rutilus rubilio, Silurus aristotelis and Silurus glanis), a frog ( Rana eperotica), a mussel ( Anodonta sp.) and a water snail ( Viviparus contectus) were analyzed by high-performance liquid chromatography (HPLC), protein phosphatase 1 (PP1) inhibition assay (PP1IA) and ELISA. MC(s) was detected in all fish, frog, mussel and water snail samples tested by PP1IA and ELISA, including the frog R. eperotica and the freshwater snail V. contectus, in which the occurrence of MCs was not previously known. MC concentration ranged from 20 to 1500 ng g −1 dw and from 25 to 5400 ng g −1 dw in muscle and visceral tissue of fishes and frogs, respectively. In mussel and water snail tissue MC concentration ranged from 1650 to 3495 ng g −1 dw. HPLC analysis revealed peaks having the same UV spectrum as anabaenopeptin- or anabaenopeptilide-like compounds, not previously known to occur in aquatic fauna tissue. The concentrations of the compounds detected ranged from 1.5 to 230 μg g −1 dw. Comparison of the PP1IA and ELISA showed that values obtained with PP1IA where higher than those obtained with ELISA. Anabaenopeptins and/or anabaenopeptilides occurring in faunal tissue may account for the higher PP1IA values as we found that PP1 activity was inhibited by the purified anabaenopeptins A (45–60% inhibition) and B (5–75% inhibition). Purified anabaenopeptilides 90A and 90B exhibited weaker PP1 inhibition activity (5–35 and 5–23% inhibition, respectively). This is the first report of MC occurrence in aquatic animals collected from freshwaters of southern Europe.

Anthropogenic changes in the freshwater fish fauna of Italy, with reference to the central region and Barbus graellsii, a newly established alien species of Iberian origin

In a survey of the west‐central Italian rivers Ombrone, Fiora, Albegna and Bruna, among established exotic species, the Iberian barbel Barbus graellsti was recorded in Italian fresh waters for the first time. Morphological identification was supported by comparison of cytochrome b sequences with those from related barbel species. Other exotics of particular note were Barbus barbus, Pseudorasbora parva and Leuciscus cephalus, together with the Padano‐Venetian Chondrostoma genei and Padogobius bonelli. Native species still present included Leuciscus lucumonis, Telestes muticellus, Rutilus rubilio and Padogobius nigricans, but were now more restricted to upper reaches and smaller watercourses. The deleterious effect of alien species on native forms is discussed with particular reference to probable competition between C. genei and L. lucumonis, and between the two gobies. The checklist of species introduced to the fresh waters of Italy is now updated to 34. For the Italian freshwater fish fauna in general, the conservation status of three native species (Acipenser naccari, Salmo marmoratus and Knipowitschia punctatissima) has been improved but five species are believed to be at risk (Salmo carpio, L. lucumonis, Scardinius scardafa, Gobio benacensis and P. nigricans) and four anadromous species (Petromyzon marinus, Lampetra fluviatilis, Acipenser sturio and Huso huso) no longer breed in Italian fresh waters. The processes of change in the composition of the Italian freshwater fish fauna as a whole may be summarized as successively ‘padanization’, ‘danubization’ and now ‘globalization’.

Egg to adult identification of 13 freshwater fishes from Italy: a biochemical-genetic key

The family Cyprinidae comprises most of the freshwater fauna of the North Mediterranean region and it is often numerically dominant. Taxonomy based on morphology is problematic, owing to the high intraspecific variation in phenotypic traits, their overlap between species and environmental plasticity of morphometric and meristic characters. A difficult diagnosis, particularly for young developmental stages, can increase resource mismanagement. Here we report on the occurrence of species-specific allozymic patterns, consistent from eggs to adults. These allowed the construction of a biochemical-genetic key for the identification of all stages of 13 freshwater fishes, mainly cyprinids native to the North Mediterranean region (Alburnus alburnus alborella, Barbus plebejus, Chondrostoma genei, Gobio gobio, Leuciscus cephalus, L. lucumonis, L. souffia, Rutilus rubilio, R. erythrophthalmus, Scardinius erythrophthalmus, Tinca tinca, Pseudorasbora parva and Padogobius martensii).

Comparative Analysis of Glucosephosphate Isomerase, Lactate Dehydrogenase and Malate Dehydrogenase Isozymes in 9 Cyprinid Species from Italy

The developmental and the tissue-specific expression of glucosephosphate isomerase (GPI), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) multilocus isozymes were analyzed in samples of Leuciscus cephalus and the adult patterns compared with those of 8 additional Italian cyprinid species: Alburnus alburnus alborella, Chondrostoma genei, L. lucumonis, L. souffia, Rutilus rubilio, R. erythrophthalmus, Scardinius erythrophthalmus and Tinca tinca, the taxonomic status of many of them being uncertain and highly debated. The spatial and temporal patterns of expression obtained generally agree with literature data. Main exceptions are the single expression of GPI-A* and MDH-A* loci of the liver in L. cephalus and the GPI pattern of the eye in all species examined. Since delayed appearence of the subunits coded by the GPI-B* locus and the very early ontogenetic expression of the sMDH-B* locus were found in L. cephalus, the onset of expression of orthologous loci can vary in related species. Genetic structure comparisons support a high genetic divergence of T. tinca from all other species.

Observations on the dynamics of Rutilus rubilio Cyprinidae in a man‐made hypertrophic basin (Arancio Lake, southwest Sicily)

Rutilus rubilio, recently introduced into Sicily, is now widespread in many man‐made basins and watercourses of the island. We investigated a population living in a man‐made hypertrophie basin of southwest Sicily (Lake Arancio) in 1995. Sampling was monthly by a trawl and trammel net. Morphometric and meristic features were taken, and sagittal otoliths were extracted to assess age. R. rubilio represented the dominant species in the fish community of the man‐made basin. The population was characterized by a slower isometric growth, as related to age, than the population of South Italy. Rutilus rubilio became naturalised successfully and has contributed in altering the composition of the fish community described in 1977 by Faranda.

Importance of water-level fluctuation on population dynamics of cladocerans in a hypertrophic reservoir (Lake Arancio, south-west Sicily, Italy)

During a period of three years (1990–1991 and 1993), we studied the population dynamics of planktonic cladocerans in a hypertrophic reservoir. Weekly sampling revealed that the five most common species followed a trend which reflects the peculiar hydrological characteristics of the reservoir and their key position in the pelagic food web. In particular, 1991 was characterized by a strong water inflow which probably interfered with the reproductive activities of the dominant fish population (Rutilus rubilio) and reduced the concentration of inedible planktonic algae allowing the development of small Chlorococcales. This event was associated with higher population densities of Daphnia hyalina compared to the other years of the survey. In spring 1991, an extended clear-water phase was observed and Secchi disk depth increased to 6 m, whereas in the other years it did not surpass 1 m. In addition, the D. hyalina population persisted throughout the summer in 1991, whereas it started to decline at the end of June in the other years. This development of D. hyalina probably influenced the population dynamics of the other cladoceran species in the reservoir, and in particular reduced the summer growth of Diaphanosoma lacustris and delayed the occurrence of Bosmina longirostris. Stomach analysis indicated that D. hyalina is the preferred food item of juvenile (less than two months old) R. rubilio. Overall, the hydrology of the reservoir was observed to interact with the trophic processes in the pelagic environment of the ecosystem in at least two different ways: via bottom-up processes, influencing phytoplankton dynamics, and via top-down processes, regulating the predation efficiency of the planktivores.

Organometallic complexes with biological molecules: V.In vivo cytotoxicity of diorganotin(IV)-amoxicillin derivatives in mitotic chromosomes ofrutilus rubilio (pisces, cyprinidae)

In order to test in vivo cytotoxicity of diorganotin(IV)-amoxicillin (amox) derivatives, mitotic chromosomes of Rutilus rubilio (Pisces, Cyprinidae) have been analyzed using two different chromosome-staining techniques. Results gathered after exposure of fish to the free amox.3H 2 O, R 2 SnClamox.2H 2 O, and R 2 Snamox 2 .2H 2 O (R = methyl, butyl and phenyl ; amox - = 6-[D(-)-β-amino-p-hydroxyphenylacetamido]penicillinate) suggest that methyl derivatives seem to exert a lower cytotoxicity than butyl and phenyl ones and that R 2 Snamox 2 .2H 2 O derivatives are more toxic than R 2 Snclamox.2H 2 O at both 10 -5 and 10 -7 mol dm -3 concentrations. The following structural lesions have been identified by comparative analysis of mitotic chromosomes from untreated specimens (controls) and specimens treated with diorganotin(IV)-amoxicillin derivatives : (1) differentially stained chromosome areas ; (2) granular deeply stained zones along the chromosomal body ; (3) arm breakages; and (4) side-arm bridges (pseudochiasmata).

Past and present state of the ichthyofauna of Lake Lugano

The evolution of the ichthyofauna of Lake Lugano (Lago di Lugano) during the past 120 years is characterized by the introduction of eight new fish species and by the possibly natural addition of the blenny (Blennius fluviatilis) in recent years. Introduced Salmonids such as charr (Salvelinus alpinus) and whitefish (Coregonus sp.) have established strong populations during the first half of this century, but have declined drastically thereafter. Likewise, the population of the native shad (Alosa fallax lacustris) has diminished significantly. A hydroacoustic survey of the pelagic fish stock of Lake Lugano in 1989 revealed the presence of a dense bleak (Alburnus albidus) population at 0 to 14 m depth. No other fish were found to be present in the pelagic zone. Oxygen and temperature distributions seem to confine the bleak to the uppermost layer in summer. In winter, very few fish were located by echosounding. Growth of bleak and roach (Rutilus rubilio) was difficult to interpret since data for comparison are lacking. Growth of perch (Perca fluviatilis) in the littoral zone was found to be slower than in other eutrophic lakes, for no obvious reason. Possible ways of restructuring the pelagic fish community are discussed.

Biometrical and biological features of Alburnus alburnus×Rutilus rubilio natural hybrids from Lake Mikri Prespa, northern Greece

Morphometric and meristic characteristics of a rare hybrid between the Adriatic roach. Rutilus rubilio, and the bleak, Alburnus alburnus, discovered in Lake Mikri Prespa, north‐western Greece are described. Axis 1 and 2 of a discriminant analysis using all morphometric and meristic features explains 99.5% of the total variability of the hybrid and both its parents. No back‐crossing is detected in the discriminant analysis: all hybrids found are F1 fish. Age and length of the hybrid and its parents were measured; the best‐fit growth model is the logistic curve. Growth of the hybrid is faster than that of the Adriatic roach and slightly faster than that of the bleak: this indicates heterosis. Problems in the systematics of genera of European cyprinids are discussed.

Rhabdospora thelohani Laguessé, 1895 (Apicomplexa): new host and geographic records with taxonomic considerations.

New fish species and geographic records for Rhabdospora thelohani Laguessé, 1895 (rodlet cells) are presented. Additionally, the ultrastructure of R. thelohani in Alburnoides bipunctatus ohridanus Karaman, Borostomias antarcticus (Lönnberg), Leuciscus cephalus albus Bonaparte and Rutilus rubilio (Bonaparte) is compared with that reported by other authors and with members of Subphylum Apicomplexa. The ultrastructure of R. thelohani was similar in all the fish species examined; however, the organism was not present in all members of any single species and had intertissue density variations. Rhabdospora thelohani is pyriform, averaging in size 7 X 12 micrometer, with a basal nucleus. The surface complex is composed of a layer (0.5 micrometer diameter) formed by microfilaments (9.3 nm) and an outer trilaminar membrane (9.3 nm). The cytoplasm contains structures identical to rhoptries, micronemes and subpellicular microtubules. Mitochondria, Golgi apparatus, and rough endoplasmic reticulum were not observed, althouth free ribosomes were present and arranged in a vesicular pattern. The observations suggest that the organism moves between cell of epithelial layers and is either released into a lumen intact or passively or actively discharges its contents into a lumen. Results from this study indicate that R. thelohani should be considered a member of Apicomplexa unless definitive evidence is presented to the contrary.