IDENTITY OF THE ENDOSYMBIONT OF PERIDINIUM FOLIACEUM (PYRROPHYTA): ANALYSIS OF THE rbcLS OPERON1

Extant chromophytic algae have been suggested to have originated via the engulfment of a photo synthetic alga by a colorless protist. The dinoflagellate Peridinium foliaceum (Stein) Biecheler contains a reduced chlorophyll c–containing endosymbiont and, thus, represents an evolutionary intermediate stage in the establishment of chloroplasts. Although the exact phylogenetic relationship of the symbiont to extant algal species is unknown, it had been suggested that the P. foliaceum symbiont was either a diatom or a chrysophyte. Identification of the closest living relative of the P. foliaceum symbiont would provide a free-living model system with which the photosynthetic symbiont could be compared. Nucleotide sequence analysis of rbcL and rbcS (encoding the large and small subunits ofribulose-1,5-bisphosphate carboxylase/oxygenase) by the P. foliaceum symbiont was performed to provide insights into its identity. Cloned restriction fragments from a chloroplast DNA library were screened, and clones encoding the rbcLS operon were sequenced. Parsimony phylogenetic analysis was performed for each gene. Our data strongly suggest that the symbiont originated from a photosynthetic diatom.     

FREEZE-FRACTURE STUDY OF THE SINGLE MEMBRANE BETWEEN HOST CELL AND ENDOCYTOBIONT IN THE DINOFLAGELLATES GLENODINIUM FOLIACEUM AND PERIDINIUM BALTICUM1

The dinoflagellates Glenodinium foliaceum Stein and Peridinium balticum (Levander) Lemmermann harbor a chrysophytic endocytobiont which is bounded by only a single membrane. This unique membrane is of particular interest because it could correspond to an intermediate stage in the evolution of “complex” plastids found in many Plastids of this type are surrounded by three or membranes instead of the usual two. With freeze-fracture techniques, we show that the single membrane in P. balticum has a pronounced polarity with respect to the distribution of intramembrane particles (IMPs) on the two corresponding fracture faces. The inner face exhibited more IMPs than the outer. We suggest that this stdedness identifies the separating membrane as the plasma membrane of the endocytobiont. A symbiontophoric vacuole with a separate membrane apparently is lacking. In the endocytobiosis of G. foliaccum, the single membrane separating host and endocylobiont exhibits a symmetrical particle partition. Nevertheless, from the size distribution of the IMPs it appears likely that this membrane, too, corresponds to the plasma membrane of the symbiont.     

FURTHER EVIDENCE FOR A MEMBRANE-BOUND ENDOSYMBIONT WITHIN THE DINOFLAGELLATE PERIDINIUM FOLIACEUM1

The fine structure of the binucleate, fucoxanthin-containing dinoflagellate Peridinium foliaceum (Stein) Biechler was re-examined for evidence of an endosymbiout. The eucaryotic nucleus, chloroplasts and associated ribosome-dense cytoplasm were separated by a single invaginating membrane from the rest of the dinoflagellate cell. The triple membrane-enclosed eyespot, mesocaryotic nucleus, trichocysts and accumulation bodies resided in the dinoflagellate cytoplasm. These observations suggest that P. foliaceum contains a membrane-bound endosymbiont, similar to that already described for the closely related species. P. balticum (Levander) Lemmermann.     

NUTRIENT REQUIREMENTS OF THE DINOFLAGELLATE PERIDINIUM GATUNENSE1

Optimum nutrient conditions for growth and photosynthesis of Peridinium gatunense (Nygaard) (Peridinium cinctum fa. westii) were investigated using axenic clones in batch cultures. Selenium (Se) had previously been found to be an indispensable growth factor for P. gatunense. Optimal, suboptimal, and supraoptimal concentrations of HCO₃^?, N, Ca, Cl, Mg, P, K, S, Si, EDTA-Na, Fe, Mo, Zn, Mn, Co, Se, B, Br, I, and various trace element mixtures were determined by measuring biomass development, growth rates, ¹⁴C uptake, and/or oxygen production at various concentration gradients of these elements. The general characteristics of the best formulation, medium-L 16, relative to other media, are its high content of NaHCO₃ (1 meq · L^?1) and Mo (0.2 μM) but low concentrations of NO_3-N (150 μM), PO_4-P (10 μM), and Fe (0.4 μM), in addition to its content of Se. The total content of trace metals, except for Se, may be reduced to one-fourth of that in medium-L 16 without altering the major growth-promoting properties of the medium. Medium-L 16 deviated considerably from Lake Kinneret (Israel) water, being much lower in macroelements except for N and P. The pH (8.1–8.4) was in the same range, but the values of conductivity (140 μS · cm^?1), alkalinity (1 meq · L^?1) and NaCl (200 μM) were > 8, 2, and 30 times higher, respectively, in the lake water. Selenium deficiency may limit the growth of P. gatunense in this lake.     

CARBONIC ANHYDRASE ACTIVITY IN THE BLOOM-FORMING DINOFLAGELLATE PERIDINIUM GATUNENSE

The response of carbonic anhydrase (CA) activity in Peridinium gatunense Nygaard, the natural bloom-forming dinoflagellate in Lake Kinneret, to diel and seasonal variations in environmental conditions was characterized under controlled laboratory experiments. Simulated diel cycles demonstrated large changes in the ambient concentration of dissolved CO₂ and parallel changes in CA activity. The CA activity depended on the total concentrations of inorganic carbon (C₁) and in particular on the dissolved CO₂. Lowering the C₁ concentrations resulted in a large increase in CA activity within several hours. Light and photosynthesis were both required for the induction of CA activity. Under CO₂ -limited conditions, the dependence of the photosynthetic rate on CA (estimated from the ratio of photosynthetic rates in the presence or absence of CA inhibitors) was greater in P. gatunense than in other eukaryotic microalgae. This points to the ecological significance of CA in photosynthetic carbon uptake mechanisms of a large, dominant alga in a natural ecosystem .     

DISCRIMINATIVE POWER OF NUCLEAR rDNA SEQUENCES FOR THE DNA TAXONOMY OF THE DINOFLAGELLATE GENUS PERIDINIUM (DINOPHYCEAE)1

The genus Peridinium Ehrenb. comprises a group of highly diversified dinoflagellates. Their morphological taxonomy has been established over the last century. Here, we examined relationships within the genus Peridinium, including Peridinium bipes F. Stein sensu lato, based on a molecular phylogeny derived from nuclear rDNA sequences. Extensive rDNA analyses of nine selected Peridinium species showed that intraspecies genetic variation was considerably low, but interspecies genetic divergence was high (>1.5% dissimilarity in the nearly complete 18S sequence; >4.4% in the 28S rDNA D1/D2). The 18S and 28S rDNA Bayesian tree topologies showed that Peridinium species grouped according to their taxonomic positions and certain morphological characters (e.g., epithecal plate formula). Of these groups, the quinquecorne group (plate formula of 3′, 2a, 7″) diverged first, followed by the umbonatum group (4′, 2a, 7″) and polonicum group (4′, 1a, 7″). Peridinium species with a plate formula of 4′, 3a, 7″ diverged last. Thus, 18S and 28S rDNA D1/D2 sequences are informative about relationships among Peridinium species. Statistical analyses revealed that the 28S rDNA D1/D2 region had a significantly higher genetic divergence than the 18S rDNA region, suggesting that the former as DNA markers may be more suitable for sequence‐based delimitation of Peridinium. The rDNA sequences had sufficient discriminative power to separate P. bipes f. occultaum (Er. Lindem.) M. Lefèvre and P. bipes f. globosum Er. Lindem. into two distinct species, even though these taxa are morphologically only marginally discriminated by spines on antapical plates and the shape of red bodies during the generation of cysts. Our results suggest that 28S rDNA can be used for all Peridinium species to make species‐level taxonomic distinctions, allowing improved taxonomic classification of Peridinium.     

INTRASPECIFIC VARIATION IN THE DINOFLAGELLATE PERIDINIUM VOLZIP1

Clonal isolates of Peridinium volzii Lemmerman were analyzed morphologically and biochemically. Morphological observations at the light microscope level show the clones to be different varieties and forms of the same species. Biochemical analysis by enzyme electrophoresis and flow cytometric determination of nuclear DNA quantities indicates that these isolates are genetically heterogeneous without any clear correlation existing between morphological variation and biochemical variation. Isozyme analysis, however, indicates that strains from the same location are generally more related to each other than they are to isolates with other geographical origins. In general, our results suggest the presence of genetic redundancy and a multiclonal origin for individuals of the same species present in the same locale.     

A UNIQUE MITOTIC VARIATION IN THE MARINE DINOFLAGELLATE OXYRRHIS MARINA (PYRROPHYTA)1

Mitosis is described in the flagellate Oxyrrhis marina Dujardin and is compared in related genera. Dense plaques develop in the nuclear envelope at prophase and give rise to an intranuclear spindle. Some of the microtubules associate with the chromosomes while others extend across the nucleus. The basal bodies migrate toward the poles early in division and retain a position lateral to the nuclear poles throughout mitosis. Microtubules are not present between the nucleus and the basal bodies. The nucleolus is persistent and elongates throughout anaphase and telophase. Chromosomal separation is accomplished by sliding of non-chromosomal microtubules and by elongation of the nuclear envelope rather than by shortening of the spindle microtubules. The nuclear envelope begins to constrict in the center early in anaphase. Continued constriction of the envelope and elongation of the nucleus leads to the formation of a dumbbell-shaped nucleus by late telophase. Mitosis culminates by the constriction of the nucleus into two daughter nuclei. The taxonomic position of Oxyrrhis marina is discussed in light of these findings.     

GYMNODINIUM ACIDOTUM NYGAARD (PYRROPHYTA), A DINOFLAGELLATE WITH AN ENDOSYMBIOTIC CRYPTOMONAD1

Ultrastructural examination of the freshwater, blue-green dinoflagellate Gymnodinium acidotum Nygaard revealed the presence of an endosymbiotic cryptomonad. Features of the endosymbiont allying it with the Cryptophyceae include mitochondria with flattened cristae, paired thylakoids with electron-dense contents, and nucleomorphs, bodies unique to the Cryptophyceae. This report is the first conclusive documentation of a symbiosis involving these two groups.     

PHOTOINHIBITION OF MECHANICALLY STIMULABLE BIOLUMINESCENCE IN THE AUTOTROPHIC DINOFLAGELLATE CERATIUM FUSUS (PYRROPHYTA)1

Ceratium fusus (Ehrenb.) Dujardin was exposed to light of different wavelengths and photon flux densities (PFDs) to examine their effects on mechanically stimulable bioluminescence (MSL). Photoinhibition of MSL was proportional to the logarithm of PFD. Exposure to I μmol photons·m^?2s^?1 of broadband blue light (ca. 400–500 nm) produced near-complete photoinhibition (≥90% reduction in MSL) with a threshold at ca. 0.01 μmol photons·m^?2·s^?1. The threshold of photoinhibition was ca. an order of magnitude greater for both broadband green (ca. 500–580 nm) and red light (ca. 660–700 nm). Exposure to narrow spectral bands (ca. 10 nm half bandwidth) from 400 and 700 nm at a PFD of 0.1 μmol photons·m^?2·s^?1 produced a maximal response of photoinhibition in the blue wavelengths (peak ca. 490 nm). A photoinhibition response (≥ 10%) in the green (ca. 500–540 nm) and red wavelengths (ca. 680 nm) occurred only at higher PFDs (1 and 10 μmol photons·m^?2·s^?1). The spectral response is similar to that reported for Gonyaulax polyedra Stein and Pyrocystis lunula Schütt and unlike that of Alexandrium tamarense (Lebour) Balech et Tangen. The dinoflagellate's own bioluminescence is two orders of magnitude too low to result in self-photoinhibition. The quantitative relationships developed in the laboratory predict photoinhibition of bioluminescence in populations of C. fusus in the North Atlantic Ocean.     

IMMUNOLOCALIZATION OF NITRATE REDUCTASE IN THE MARINE DINOFLAGELLATE GONYAULAX POLYEDRA (PYRROPHYTA)1

A polyclonal antibody, raised against nitrate reductase (NR) purified from the photosynthetic dinoflagellate Gonyaulax polyedra Stein, was used as a probe in immunogold-labeling experiments on thin sections prepared from cells harvested both during day and night phases. Previous experiments have shown that both NR activity and the amount of immunoreactive NR in cell extracts is greater when day-phase cells are examined, and this property was exploited as an internal control for the cytochemical labeling. We observed that in day-phase cells, chloroplasts contained approximately three times more gold particles than night-phase cells (highly significant difference; P < 0.0001), whereas cytoplasmic labeling levels remained relatively level between day and night. We conclude from the diurnal difference in labeling that our antibody faithfully reflects the distribution of NR in Gonyaulax cells. Thus, as in to some other higher plants and green algae, Gonyaulax compartmentalizes active NR in its chloroplasts.     

FERTILIZATION AND ZYGOTE DEVELOPMENT IN THE BINUCLEATE DINOFLAGELLATE PERIDINIUM BALTICUM (PYRRHOPHYTA)

Peridinium balticum (Pyrrhophyta) exists as a symbiosis between a nonphotosynthetic dinoflagellate host and a chlorophyll c-containing alga. It is hypothesized that P. balticum is an evolutionary link between primitive nonphotosynthetic and advanced photosynthetic dinoflagellates. This study documents pre- and postfertilization events of sexual reproduction in this unusual dinoflagellate for the first time. Light microscopy and TEM observations showed that gametes resemble vegetative cells except in the organization of their chloroplasts. Fusion of gametes occurred in a specific orientation, i.e., apical to sulcal area. The presence of an intact membrane between fusing pairs prior to plasmogamy was suggestive of enzymatic digestion of plates during fertilization. Rupture of this membrane triggers plasmogamy and karyogamy of the host, followed by that of the algal symbiont. A discussion of the cellular processes involved in gamete formation, fertilization, and zygote development is presented. The results of this investigation demonstrate that a synchronous sexual reproduction cycle has evolved for the symbiont and its dinoflagellate host.     

PHOTOINHIBITION OF MECHANICALLY STIMULABLE BIOLUMINESCENCE IN THE HETEROTROPHIC DINOFLAGELLATE PROTOPERIDINIUM DEPRESSUM (PYRROPHYTA)1

Photoinhibition of mechanically stimulable bioluminescence (MSL) in the heterotrophic dinoflagellate Protoperidinium depressum Bailey was investigated using samples collected from the Massachusetts and southern Texas coasts. The times for both photoinhibition of MSL (ca. 10 min) and dark recovery from photoinhibition of MSL (ca. 45 min) in this species were similar to those reported for autotrophic dinoflagellates. The degree of photoinhibition of MSL was a linear function of the logarithm of photon flux density (PFD). The threshold PFDs for the photoinhibition of MSL were 0.02, 0.6, and 21 μmol photons · m^?2· s^?1 for broad-band blue, green, and red light, respectively. These PFDs are lower than those required for photoinhibition of MSL by the autotrophic dinoflagellates Pyrocystis lunula and Ceratium fusus. We speculate that photosynthetic pigments in autotrophic dinoflagellates shield the photoreceptor that causes photoinhibition of MSL, thus lowering the sensitivity of these dinoflagellates to light. When field-collected P. depressum were kept in the laboratory without growth for a week, photoinhibition of MSL's sensitivity to light increased progressively along with 1) a decrease in its bioluminescence capacity (BCAP), 2) a decrease in the ratio of MSL to BCAP (MSL/BCAP), and 3) a decrease in the orange pigmentation (probably carotenoid) of the dinoflagellate. The action spectrum for photoinhibition of MSL in P. depressum was characterized primarily with a broad peak in the blue extending into the green. We suggest that carotenoid was not a photoreceptor for the photoinhibition of MSL in P. depressum because the peak of the action spectrum was too broad and extended too far into the green part of the spectrum, and because the orange pigment present decreased as photoinhibition of MSL became more sensitive to light.     

PIGMENTS OF THE DINOFLAGELLATE PERIDINIUM BALTICUM AND ITS PHOTOSYNTHETIC ENDOSYMBIONT1

An examination of the pigments of the binucleate dinoflagellate Peridinium balticum (Levander) Lemmerman revealed the presence of chlorophylls a, c₁ and c₂ and the carotenoids: fucoxanthin (most abundant), diadinoxanthin, diatoxanthin, an unidentified fucoxanthin-like xanthophyll, β-carotene, γ-carotene and astaxanthin. A comparison of the pigments of P. balticum and P. foliaceum (Stein) Biecheler, also a binucleate dinoflagellate, demonstrated similar compositions. However P. balticum lacked the β-carotene precursors (e.g. phytoene) which accumulated outside the chloroplast in P. foliaceum. This study indicates that P. balticum and P. foliaceum are closely related; each species is a heterotrophic dinoflagellate with a photosynthetic endosymbiont taxonomically affiliated with the Chrysophyta (Chrysophyceae or Bacillariophyceae).     

RESPONSE OF CATALASE ACTIVITY TO ENVIRONMENTAL STRESS IN THE FRESHWATER DINOFLAGELLATE PERIDINIUM GATUNENSE1

Catalase activity increased in Peridinium gatunense (formerly P. cinctum fa. westii) cells during the decline of the seasonal spring bloom period in Lake Kinneret. This was correlated with the low ambient total CO₂ concentration. The relationship was confirmed in laboratory experiments where maximum catalase activity occurred under an atmosphere composed of 30% O₂ and 0.003% CO₂. Conversely, high CO₂ concentrations inhibited catalase activity. The rise in catalase activity was not directly due to increasing environmental pH, as in vitro and in vivo measurements showed a characteristic broad pH curve with a constant activity from pH 6–10 for catalase. Photoinhibition of catalase occurred above 250 μmol photons · m^?2· s^?1. However, at high photoinactivating irradiances, photoinhibition was ameliorated under high pO₂/pCO₂. Such conditions prevail in the Kinneret at the end of the spring. We propose that the enhancement of photorespiration (under high pO₂/pCO₂) induces a temporary burst in catalase activity despite the progressively photoinhibitory conditions of early summer.     

THE DINOFLAGELLATE TRANSVERSE FLAGELLUM: THREE-DIMENSIONAL RECONSTRUCTIONS FROM SERIAL SECTIONS1

Serial sections through in situ transverse flagella of the dinoflagellate Peridinium cinctum f. irregulatum (Lindem.) Lefévre are presented. Three-dimensional reconstructions based upon tangential and radial series show a helically coiled axoneme lying external to and distinct from an accessory strand. Hitherto undescribed vesicles within the expanded flagellar sheath are suggested to provide a decoupling effect between axoneme and strand. The flagellar axis bears two types of hair but anchoring threads between cingulum and flagellum have not been found. Functional and taxonomic implications of these observations are briefly discussed.     

SMALL-SUBUNIT RIBOSOMAL DNA SEQUENCE ANALYSES AND A RECONSTRUCTION OF THE INFERRED PHYLOGENY AMONG SYMBIOTIC DINOFLAGELLATES (PYRROPHYTA)1

The small-subunit ribosomal RNA genes (SSU rDNA) from the four symbiotic dinoflagellates, Symbiodinium corculorum Trench isolated from the bivalve mollusc Corculum cardissa (from Belau, Western Caroline Is.), S. meandrinae Trench, from the scleractinian coral Meandrina meandrites (from famaica, W.I.), Gloeodinium viscum Banaszak et al. from the hydrocoral Millepora dichotoma (from the Gulf of Aqaba), and Amphidinium belauense Trench from the acoel flatworm Haplodiscus sp. (from Belau) have been amplified by the polymerase chain reaction, cloned, and sequenced. Following alignment of these complete sequences to homologous sequences from six other dinoflagellates, eight api-complexans, six ciliates, six chromophytes and oomycetes, three ascomycetes, two rhodophytes, two chlorophytes, and two myxomycetes (with Physarum polycephalum as the outgroup), phylogenetic reconstruction was conducted using Fitch and Margoliash distance, DNA maximum likelihood, and Wagner parsimony methods, with bootstrap resampling. All methods generated trees with similar topologies. The inferred “across Kingdom” phylogeny reemphasizes previous reports that show that the dinoflagellates, the apicomplexans, and the ciliates share a common ancestry and that the dinoflagellates are distantly related to the chromophyte-oömycete lineage. The evidence supports the concept of a polyphyletic origin of dinoflagellate-invertebrate symbioses, as symbiotic dinoflagellates represent seven genera in at least four orders. The three symbiotic species, S. corculorum, S. meandrinae, and S. pilosum, consistent with their morphological and biochemical similarities, cluster most closely. Symbiodinium pulchrorum Trench, the symbiontfrom the Hawaiian sea anemone Aiptasia pulchella, is more distantly related to them. Gloeodinium viscum is not closely related to the Symbiodinium species. Amphidinium carterae (free-living) and A. belauense (symbiotic) also appear to be distantly related to Symbiodinium. Some symbionts (e.g. S. corculorum, S. pilosum) from distant geographic locations (the Indo-Pacific and Caribbean, respectively) were found to be very closely related, whereas S. pulchrorum and S. corculorum from the Pacific were found to be distantly related. Analyses of 10 additional symbiotic and nonsymbiotic dinoflagellates, using partial SSU rDNA sequences to generate a tentative dinoflagellate phylogeny, indicate that members of the genus Symbiodinium cluster with most of the other (free-living) dinoflagellates in the genus Gymnodinium. The genus Amphidinium, as represented by A. carterae and A. belauense, appear to be distantly related to the other members of the Gymnodiniaceae. This analysis, combined with morphological and biochemical data, indicates that the symbionts S. pulchrorum (from Aiptasia pulchella) and S. bermudense Trench (from Aiptasia tagetes) from the Indo-Pacific and Caribbean, respectively, are very closely related but are not identical.     

THE FLAGELLAR APPARATUS OF OXYRRHIS MARINA (PYRROPHYTA)1

The three-dimensional structure of the flagellar apparatus in the dinoflagellate Oxyrrhis marina has been reinvestigated and found to consist of several previously unknown components and component combinations that appear strikingly similar to those of some gymnodinoid taxa. The flagellar apparatus of this dinoflagellate is asymmetric and extremely complex consisting of a longitudinal and a transverse basal body that gives rise to eight structurally different components. The only posteriorly directed component is the large microtubular root that consists of 45–50 microtubules at its origin and is attached proximally to a perpendicularly oriented striated fibrous component. Arising from each basal body, two striated fibrous roots with different periodicities extend to the cell's left. A single stranded microtubular root with associated electron dense material emanates from the transverse basal body and also extends to the cell's left. A striated fibrous connective arises from the longitudinal basal body and extends toward the cell's right ventral surface and terminates near the sub-thecal microtubular system. A compound root consisting of microtubules and electron dense material also originates from the longitudinal basal body and extends ventrally into the anterior region of the tentacle. Structural similarities between the parallel striated fibrous roots of Oxyrrhis and Polykrikos are discussed as are flagellar apparatus similarities among other gymnodinoid dinoflagellates. A diagrammatic reconstruction of the Oxyrrhis flagellar apparatus is also presented.     

SEXUAL REPRODUCTION OF PERIDINIUM WILLEI (DINOPHYCEAE)1

Sexual reproduction was induced in the dinoflagellate Peridinium willei Huitfeld-Kass when exponentially growing cells were inoculated into nitrogen deficient medium. Small, naked vegetative cells produced by division of thecate cells acted as gametes. The zygote remained motile 13–14 days, during which time it enlarged and the theca formed became warty. Fourteen to 15 days following plasmogamy the zygote was nonmotile with the protoplast contracted. A large red oil droplet appeared and the wall thickened, becoming chitinized. Hypnozygotes with 4 nuclei were observed 7–8 wk following formation. Meiosis was inferred. The hypnozygote germinated, within 8 wk producing one post-zygotic cell retaining the red oil droplet. This cell divided within 24 h into 2 daughter cells each with a prominent red oil droplet. These daughter cells divided after 2 to 3 days into ordinary vegetative cells. Attempts to induce sexual reproduction by inoculation of cells into media deficient in a number of basic elements were unsuccessful.     

SEXUAL REPRODUCTION OF PERIDINIUM GATUNENSE (DINOPHYCEAE)1

Sexual reproduction was induced in the dinoflagellate Peridinium gatunense Nygaard when exponentially growing cells were inoculated into nitrogen deficient medium. Small thecate cells produced by division of vegetative cells then acted as gametes. Thecae of fusing gametes broke in the girdle region and were lost. Zygotes thus formed remained motile 3–5 days during which time they enlarged slightly with the newly formed theca becoming warty. Three to 5 days following plasmogamy the zygote became nonmotile, the protoplast contracted, and the cell wall thickened. Hypnozygotes with 4 nuclei were observed ca. 10–12 h following formation. Meiosis was inferred. Hypnozygotes germinated within 12 h of formation producing 2 vegetative cells which divided within a 24 h period. Attempts to induce sexual reproduction by inoculation of cells into media deficient in a number of basic elements other than N were unsuccessful.