Causative species of diarrhetic shellfish poisoning (DSP) in Norway

This study was performed at Vikane in the Sognefjord, Norway, from September 1987 to October 1988 on the blue musselMytilus edulis collected monthly at three different depths (3 to 6, 6 to 8 and 8 to 12 m). Cell numbers of three species ofDinophysis from mussel digestive glands and in seawater were counted for each specimen. Multivariable statistical methods were used to detect annual cycles of phytoplankton abundance in the fjord and to examine the contribution of each species to toxification of mussels. Cluster analysis and principal component analysis indicated the following results: (1) an annual cycle and an inter-specific association of threeDinophysis species populations were pointed out.D. acuta andD. norvegica were well associated over the entire period of this study, and exhibited an autumnal peak.D. acuminata was dissociated from these species and reached its maximum abundance between spring and early summer. (2) The threeDinophysis species induced toxicity in the blue mussels in different manners.D. acuta andD. norvegica were responsible for high autumnal toxicity, andD. acuminata for the spring peak. (3) A long persistence of diarrhetic shellfish poisoning (DSP) in blue mussels (from April to February), and a depth gradient of toxicity were observed — the toxicity value in the upper layer being double those of other depths.     

The effects of the diarrhetic shellfish poisoning toxins,okadaic acid and dinophysistoxin-1, on the growth of microalgae

The diarrhetic shellfish poisoning toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are potent phosphatase inhibitors produced by certain species of marine dinoflagellates. OA can cause hyperphosphorylation of a broad range of animal and higherpalnt proteins, but little is known regarding the effects of the DSP toxins on marine organisms or their biological function. A variety of microalgae, including a clone ofProrocentrum lima known to produce both OA and DTX-1, were incubated with solutions of OA and in one case DTX-1 or a combination of OA and DTX-1. OA inhibited the growth of all non-DSP-producing test species at micromolar concentrations, butP. lima was not affected even at much higher levels. This differential activity of OA suggests that the DSP toxins may play an allelopathic role and raises questions regarding the strategies adopted by DSP-producing dinoflagellates such asP. lima to avoid autotoxicity. The effects of DTX-1 on microalgal growth were found to be equivalentt to those of OA, and the effects of both toxins in combination were simply additive.     

Tidal height,rather than habitat selection for conspecifics,controls settlement in mussels

Settlement is a major determinant of intertidal populations. However, the energy costs of lost larvae are very high. Accordingly, arrival and attachment on suitable substrata are essential requirements for species’ survival. On the intertidal, the presence of cues left by adult or juvenile conspecifics could be vital for the successful establishment of larvae arriving on the shore. Two mussel species, the indigenous Perna perna and the invasive Mytilus galloprovincialis, co-occur on the lower eulittoral zone on the south coast of South Africa. P. perna dominates the low and M. galloprovincialis the high mussel zones, with co-existence in the mid mussel zone. This study tested the hypothesis of settlement selectivity for conspecifics in these two mussel species, to understand whether the final adult distribution of mussels on the shores is determined by active behavioural and chemical mechanisms. Preferential selection by larvae for conspecifics was tested in the field during the peak settlement period in 2004 in natural mussel beds across zones and through manipulative experiments in the mid-zone where the species co-exist. On natural beds, settlement was determined by counts of settlers attached over 48 h onto artificial collectors. Collectors were placed on beds of P. perna and M. galloprovincialis present at both high- and low-adult densities, as well as in mixed beds. On such natural beds, settlers of both species consistently favored low-zone P. perna beds. Settlement patterns over 24 h onto experimentally created mussel patches consisting of P. perna, M. galloprovincialis or the two species combined beds, set in the mixed zone, did not conform with the results of the natural beds study: settlers of both species settled with no discrimination among different patches. The results indicate that mussels, which are sedentary, lack attraction to conspecifics at settlement. This highlights the importance of tidal height in setting settlement rates, and of post-settlement events in shaping populations of these broadcast spawners.     

Paralytic shellfish poisoning due to Pyrodinium bahamense var. compressa in Mati,Davao Oriental,Philippines

On 26 August 1983, a single case of paralytic shellfish-poisoning (PSP) was reported in Davao City, Philippines. The poisoning was traced to ingestion of the green mussel Perna viridis Linnaeus, gathered from Balete Bay, Mati, Davao Oriental. Phytoplankton and zooplankton analyses on 12 October 1983 (47 d later), revealed the presence of the dinoflagellate Pyrodinium bahamense var. compressa, a cause of a series of red tides in the early and middle 1970's in Papua New Guinea, Sabah, and Brunei, and more recently, in Palau, and Western Samar and Leyte, Philippines. The dinoflagellate was not dominant; in fact the enumeration showed greater numbers of Ceratium sp., another dinoflagellate. Quantification of the neurotoxin by the standard mouse assay revealed a very high potency. Mussels collected from a new raft (transplanted in May 1983) had a toxicity of 7 960 mouse units (MU) per 100 g^-1 meat. Those from an old raft (transplanted in May 1982) had a toxicity of 9 620 MU per 100 g^-1 meat.SEAFDEC Contribution No. 145     

Assessment of natural selection in a hybrid population of mussels: evaluation of exogenous vs endogenous selection models

We examined natural selection within a population of marine mussels, sampled in southwestern England in June 1991, containing a high frequency of hybrids between Mytilus edulis L. and M. galloprovincialis Lmk. This system is particularly tractable for the assessment of natural selection because hybridization is common and individual mussels can be aged, allowing changes in the frequency of hybrid genotypes among age classes to be determined. We show that strong viability selection occurs among hybrid genotypes which results in the virtual elimination of M. edulis–like genotypes from the population over a period of 3 years. Recombinant hybrid genotypes are intermediate in fitness, with M. edulis–like genotypes having a lower survival rate and M. galloprovincialis–like genotypes having a higher survival rate than genotypes of mixed ancestry. Since intermediate fitness for hybrid genotypes is inconsistent with endogenous selection models we conclude that the structure and position of this hybrid zone is probably generated by exogenous selection. This pattern of selection is a recurring feature of this hybrid population and likely occurs elsewhere in the hybrid zone. Selection against M. edulis–like genotypes appears to be offset by extensive immigration of larvae dispersed from pure populations of M. edulis. Received: 14 July 1997 / Accepted: 24 February 1998     

Evolution of habitat use by deep-sea mussels

Previous phylogenetic studies proposed that symbiont-bearing mussels of the subfamily Bathymodiolinae (Bivalvia: Mytilidae) invaded progressively deeper marine environments and evolved from lineages that decomposed wood and bone to specialized lineages that invaded cold-water hydrocarbon seeps and finally deep-sea hydrothermal vents. To assess the validity of the hypotheses, we examined two nuclear (18S and 28S rRNA) and two mitochondrial genes (COI and ND4) from a broad array of bathymodiolin species that included several recently discovered species from shallow hydrothermal seamounts. Bayesian phylogenetic analysis and maximum-likelihood estimates of ancestral character states revealed that vent species evolved multiple times, and that reversals in vent and seep habitat use occurred within the sampled taxa. Previous hypotheses regarding evolution from wood/bone-to-seeps/vents are supported in that mid-ocean hydrothermal vent species may represent a monophyletic group with one noticeable reversal. Earlier hypotheses about progressive evolution from shallow-to-deep habitats appear to hold with a few instances of habitat reversals.     

Incorporation of organic aggregates by marine mussels

Two marine mussels, Geukensia demissa (Dillwyn) and Mytilus edulis (L.) collected in 1990 in Old Silver Beach, Falmouth, Massachusetts, incorporated nitrogen when fed ¹⁵N-labelled organic aggregates produced from dissolved organic nitrogen released by the brown sea-weed Fucus vesiculosis. Uptake of ¹⁵N on the aggregate diet was linear over the course of 24 h, and unincorporated ¹⁵N was eliminated from the gut after 48 h. Both species of mussels incorporated approximately five times more N when they were fed organic aggregates than when they were fed either ¹⁵N-labelled dissolved organic material (DOM) or particulate detritus, both of which were also derived from the seaweed. Nitrogen uptake was greatest in controls fed the diatom Thalassiosira weissflogii; mussels fed phytoplankton incorporated seven times more nitrogen than those fed aggregates. However, aggregates could supply an estimated 7 to 14% of the N requirements for both mussels, whereas DOM or particulate detritus could only supply 1 to 3%. These data provide evidence that a food web pathway exists from primary producer to released dissolved organic nitrogen to microbial organic aggregate to metazoan consumer, and, further, that it can be more important in a detrital food web than either particulate detritus or DOM.     

Presence of Alexandrium catenella and paralytic shellfish toxins in finfish, shellfish and rock crabs in Monterey Bay, California, USA

The central California coast is a highly productive, biodiverse region that is frequently affected by the toxin-producing dinoflagellate Alexandrium catenella. Despite the consistent presence of A. catenella along our coast, very little is known about the movement of its toxins through local marine food webs. In the present study, we investigated 13 species of commercial finfish and rock crabs harvested in Monterey Bay, California for the presence of paralytic shellfish toxins (PSTs) and compared them to the presence of A. catenella and PSTs in sentinel shellfish over a 3-year period. Between 2003 and 2005, A. catenella was noted in 55% of surface water samples (n = 307) and reached a maximum concentration of 17,387 cells L⁻¹ at our nearshore site in Monterey Bay. Peak cell densities occurred in the month of July and were associated with elevated shellfish toxicity in the summers of 2004 and 2005. When A. catenella was present, particulate PSTs were detected 71% of the time and reached a maximum concentration of 962 ng STXeq L⁻¹. Of the 13 species tested, we frequently detected PSTs in Pacific sardines (Sardinops sagax; maximum 250 μg STXeq 100 g⁻¹), northern anchovies (Engraulis mordax; maximum 23.2 μg STXeq 100 g⁻¹), brown rock crabs (Cancer antennarius; maximum 49.3 μg STXeq 100 g⁻¹) and red rock crabs (C. productus; 23.8 μg STXeq 100 g⁻¹). PSTs were also present in one sample of Pacific herring (Clupea pallas; 13.3 μg STXeq 100 g⁻¹) and one sample of English sole (Pleuronectes vetulus; 4.5 μg STXeq 100 g⁻¹), and not detected in seven other species of flatfish tested. The presence of PSTs in several of these organisms reveals that toxins produced by A. catenella are more prevalent in California food webs than previously thought and also indicates potential routes of toxin transfer to higher trophic levels. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrocarbon pollution of edible shellfish by an oil spill

A spill of 650,000 to 700,000 l of No. 2 fuel oil has contaminated the coastal areas of Buzzards Bay, Massachusetts (USA). Gas chromatography demonstrates the presence of this oil in the sediments of the affected area. Two months after the accident, essentially unchanged oil is still being released from the sediments. The presence of the same pollutant is demonstrated in whole oysters Crassostrea virginica and in the adductor muscle of the scallop Aequipecten irradians. A presumably biochemical modification leads to a gradual depletion of the straight chain and, to a lesser extent, of branched chain hydrocarbons. This does not result in detoxification, as the more toxic aromatic hydrocarbons are retained in the organisms several months after the accident. Scallops from an uncontaminated area contain hydrocarbons in lesser amounts and of very different molecular weight and type distribution; they are accountable entirely from biological sources.Contribution No. 2444 of the Woods Hole Oceanographic Institution.     

Heterozygosity and growth in transplanted mussels

Growth comparisons were made involving mussels (Mytilus spp.) collected from five different localities in Britain in 1980–1981. Two of the localities, Mumbles, South Wales, and Bude, Southwest England, have pure populations of M. edulis and M. galloprovincialis, respectively. The other three, Whitsand Bay, Southwest England, Croyde Bay, Southwest England and Robin Hood's Bay, Northeast England, have hybrid populations with both M. edulis and M. galloprovincialis ancestry. To make growth comparisons, mussels from different populations were mixed in oyster nets and transplanted to three different localities. After periods of transplantation varying between several months and one year, growth was assessed by measuring increase in shell length or dry body weight. Starch-gel electrophoresis was used to assay variation in the transplanted mussels at three allozyme loci partially diagnostic for M. edulis and M. galloprovincialis. The results provide evidence of small growth differences between populations and between allozyme genotypes within populations. These differences accounted for no more than a few percent of the total variation in growth between mussels. Statistically significant results were obtained, but were frequently found not to be reproducible. There is no clear evidence of a growth difference between M. edulis and M. galloprovincialis. Allozyme heterozygotes appear to have growth rates intermediate between allozyme homozygotes; this study thus fails to provide evidence for overdominance with respect to growth rate.     

Organic contaminants in sediments and mussels from Thermaikos gulf,Greece

Thirteen surficial sediment samples, two sediment cores and mussels (Mytilus galloprovincialis) from six locations were collected from Thermaikos gulf, Greece and analyzed for herbicides, organochlorine compounds and aliphatic hydrocarbons. Herbicide concentrations were low in surface sediments (not detected‐0.8 ng/g dry wt) and undetectable in subsurface samples. Organochlorine concentrations ranged between 0.8 and 88.2 ng/g dry wt for the polychlorinated biphenyls (PCBs) and between 1.7 and 22.8 ng/g dry wt for the SDDTs, while aliphatic hydrocarbon values varied from 28.8 to 965.4 ng/g dry wt. The highest concentrations for all the pollutants were measured in the vicinity of the city of Thessaloniki. Petroleum‐related hydrocarbons were dominant in surface sediments, while were still detected till about 1 m depth in the sediment core. Mussels presented low organochlorine concentrations (3.6–10.5 ng/g dry wt for the PCBs, 12.2–34.0 ng/g dry wt for the DDTs), but petroleum hydrocarbon concentrations were rather increased, mainly in the samples collected along the east coastline of the gulf (aliphatic hydrocarbons 52.3–224.3 μg/g dry wt).     

Evolutionary relationships of deep-sea mussels inferred by mitochondrial DNA sequences

In order to elucidate the evolutionary process of deep-sea Bathymodiolus mussels, we investigated the phylogenetic relationships of 16 species worldwide by analyzing nucleotide sequences of the mitochondrial COI and ND4 genes. Deep-sea mussels were clustered into three groups by basal trichotomous divergence. The first was composed of four species found in Japanese waters and one species from the Gulf of Mexico, which contain methanotrophic endosymbiotic bacteria. The second included nine species distributed in the West and East Pacific, Indian, and Atlantic Oceans. Members of the second group were trichotomously divided into the Indo-West Pacific, Atlantic, and East Pacific subclusters. The Indo-West Pacific subcluster was composed of three very closely related species with mutual genetic distances at the intraspecific level (av. 0.019 in COI and 0.009 in ND4 relative to av. 0.156 in COI and 0.265 in ND4 among Bathymodiolus species other than Cluster A species), suggesting some gene flow among these species. The third consisted of two West Pacific species. Species in the second and third groups contain mainly thioautotrophic endosymbionts, including some species harboring both methanotrophs and thioautotrophs.     

Phytoplankton patchiness and frontal regions

In the Chesapeake Bay estuary there are persistent seasonal frontal and interfrontal regions that serve to deliver and retain different phytoplankton populations. The patchiness of phytoplankton, both in total chlorophyll a concentrations and in species compositions and abundances, is shown to be causally related to density flow forcing which results in these frontal and interfrontal regions. The delineation of these regions by on-line, two-dimensional profiling of density isopleths serves to identify stations within these regions for biological and chemical sampling as opposed to sampling on an arbitrary geographical grid. It is possible, by superposition of nutrient and organism concentration isopleths upon salinity isopleths, to infer conservative and non-conservative features of the system.Contribution 1059 from the McCollum-Pratt Institute and Department of Biology. Research support by DOE contract DEASO2-76-EVO3278. The data in this paper have been presented at the Winter Meeting of the American Society of Limnology and Oceanography in Corpus Christi, Texas January 2–5, 1979 and the 42nd Annual Meeting of ASLO in Stony Brook, NY, USA June 18–21, 1979     

太湖浮游植物群落结构及其与水质指标间的关系

为了探讨太湖浮游植物群落结构时空分布特征、以及太湖浮游植物群落指标与水质指标间的关系,于2013年1月─2013年12月对太湖7个点位浮游植物群落结构和水质指标（水温、透明度、pH、溶解氧、电导率、总氮、总磷、氨氮、高锰酸盐指数、化学需氧量、氟化物、生化需氧量、硝酸盐氮、亚硝酸盐氮、溶解性磷酸盐和叶绿素a）进行月度调查,研究其浮游植物群落结构和湖泊水质的时空分布;并利用Pearson相关性分析浮游植物密度、浮游植物多样性与水质指标间的关系;找出影响太湖浮游植物群落结构的主要水质指标。结果表明：太湖7个点位共获得124种浮游植物物种,其中蓝藻门（Cyanophyta）30种、绿藻门（Bacillariophyta）47种、硅藻门（Chlorophyta）34种、隐藻门（Cryptophyta）3种、裸藻门（Euglenophyta）6种和甲藻门（Dinoflagellate）4种;其中蓝藻门的微囊藻属（Microcystis spp.）为绝对优势种群,优势度为80.8%;太湖浮游植物总密度与蓝藻门密度呈极显著正相关（r=1.000,P＜0.0001）;绿藻门和硅藻门占浮游植物总密度百分比分别和蓝藻门占浮游植物总密度百分比呈极显著负相关（r=-0.497,P＜0.0001;r=-0.814,P＜0.0001）。太湖7个点位水质首要污染物为总氮,其次是总磷和化学需氧量;西太湖污染物浓度最高。从空间上看,太湖浮游植物总密度最高值出现在贡湖湾（远离其入湖口处）,且贡湖湾浮游植物群落多样性相对低于太湖其他点位,同时贡湖湾微囊藻属密度百分比达90.1%,远高于太湖其他点位;从时间上看,太湖浮游植物总密度最高值出现在12月份、其次是6月份;通过浮游植物群落指标与水质指标相关性分析,水温、透明度、总氮、化学需氧量、叶绿素a是影响太湖浮游植物群落结构的主要水质指标。控制太湖入湖口水质污染物浓度排放和修?     

Non-random perch selection by cryptic lizards,Amphibolurus muricatus

Behavioral Ecology and Sociobiology - Animals are often confronted with more sensory stimuli than they can attend to, and so should pay attention to stimuli that are relevant to them and habituate...     

Influence of body size on the uptake,depuration, and bioaccumulation of polychlorinated biphenyl congeners by Baltic Sea blue mussels,Mytilus edulis

The present study was designed to examine the influence of body size on the bioaccumulation of polychlorinated biphenyl (PCB) congeners by Baltic Sea blue mussels, Mytilus edulis L. This was done, firstly, by establishing the relationship (as a power function: PCB tissue conc = a tissue dry wt^b) between tissue concentration and body weight for seven PCB congeners (International Union of Pure and Applied Chemistry No. 52, 101, 105, 118, 138, 153, and 180) in field sampled mussels; and, secondly, by assessing the influence of body weight on the uptake clearance coefficients, the depuration rate coefficients and the calculated bioaccumulation factors (BAFs) of three ¹⁴C-labelled PCB congeners (IUPAC No. 31, 49, 153) in mechanistic kinetic experiments. Both the background tissue concentrations of PCB 138, PCB 153 and PCB 180 and the predicted BAF values in the kinetic experiments correlated negatively with body weight (b=-0.17 and-0.31, respectively). Of the two kinetic rate coefficients examined, only the uptake clearance rate showed weight dependency (b=-0.32, i.e. negative correlation with body weight), whereas depuration rates were unaffectd by body weight. Uptake clearance rates and BAFs adjusted for body weight increased with the octanol/water partition coefficient (K_ow) of the congener, whereas depuration rates dectreased with K_ow. These observations suggest that size-dependent bioaccumulation of hydrophobic organic contaminants (HOCs) in suspension-feeding bivalves is driven by size-related differences in uptake rate, although several other mechanisms may also affect bioaccumulation in natural mussel beds (e.g. sizerelated differences in lipid content, production, and contaminant exposure). From an ecotoxicological perspective size-dependent bioaccumulation implies not only that variability due to body size differences has to be dealt with in experimental designs, but also that several ecological factors such as size-specific predation and shifts in population structure may affect HOC cycling by dense populations of bivalve suspensionfeeders.     

Phytoplankton sinking rates in oligotrophic waters off Hawaii,USA

The sinking rates in two size fractions of natural phytoplankton were measured over much of the photic zone in a subtropical environment. At 24, 40 and 71 m, sinking rates (± SD) of the 3 to 20 m fractions were 0.72±0.05, 0.83±0.05, and 0.34±0.04 m · d^-1, respectively, and rates for the 20 to 102 m fraction were 1.50±0.21, 1.65±0.14, and 0.95±0.22 m · d^-1, respectively. At all depths sampled, the 20 to 102 m size fraction was observed to sink significantly (P<0.01) faster than the 3 to 20 m fraction. considering vertical variability, both size fractions were observed to have significantly (P<0.01) lower sinking rates at 71 m than at more shallow depths. The finding of lower sinking rates in the region which lies just above the subsurface chlorophyll maximum provides empirical support for the hypothesis that variations in phytoplankton buoyancy may be related to the maintenance of this feature which is typical in oceanic environments. Analysis of sinking rate traces describes the distributions of specific sinking velocities for each size fraction and their variation.Oceanic Institute Contribution No. 162     

Flexible seed selection by individual harvester ants, Pogonomyrmex occidentalis

Summary Individual seed harvester ants (Pogonomyrmex) have been shown to specialize on specific seed types. We examined possible mechanisms for seed specialization and tested whether fidelity to food type limits the foraging decisions of individual western harvester ants, Pogonomyrmex occidentalis. The seed selection regimes of individually marked ants foraging at piles of two seed types were described and related to differences in seed quality and colonial dietary history. Individual foraging choices were affected by multiple factors, including seed caloric rewards, the previous seed selected, and the dietary history of the colony. Individual seed choices generally converged on the most energetically profitable species, suggesting that foragers exhibit labile preference. However, for a portion of the foragers, seed specialization was also partially due to constancy, defined as a tendency to select seed species that were previously collected. When colonies were presented with one seed type for 1 h and then were offered a mix of that seed and a novel seed type, individuals showed a strong preference for the novel seeds. Such rapid changes in seed preference argue strongly that individual P. occidentalis ants are highly flexible in seed choice and that resource assessment by these ants is more complex than simple maximization of net energetic return.Offprint requests to: J.H. Fewell at the current address     

Paralytic shellfish toxins sequestered by bivalves as a defense against siphon-nipping fish

Saxidomus giganteus (butter clams), are known to sequester diet-derived paralytic shellfish toxins (PST), highly potent neurotoxins, in their siphons. Captive staghorn sculpins (Leptocotus armatus), a marine fish species known to crop bivalve siphons, developed a significant aversion to siphons from toxic but not non-toxicS. giganteus following a single conditioning feeding of toxic siphon tissues. Control fish showed no aversive response to siphons from non-toxicS. giganteus during 11 feeding sessions over 56 d. Aversive and non-aversive behavior varied with the toxicity of the siphons, but not with the geographic origin of the clams. Both experimental and control fish ate freely and showed no aversion to siphons from toxic littleneck clams (Protothaca staminea). Littleneck clams, unlikeS. giganteus, retain PST in their visceral mass but not in their siphons. Both toxic and non-toxicS. giganteus extended their siphons significantly more often and higher above the sediment surface during dark hours, but toxicS. giganteus extended their siphons higher than non-toxic individuals. These results support the hypothesis that siphon-nipping by fish may have selected for the retention of PST in butter clam siphons as a chemical defense.     

Salt poisoning blindness in wistar rat,rabbit, and pig

The objective of this study was to determine the ocular changes leading to blindness following different salt concentration consumption in Wistar rats. Test groups consumed 1, 2, 3, 3.5, or 4% salt concentration respectively as potable water, while the control group used tap water. Ophthalmic examinations with a slit-lamp and fundus camera were performed. Lack of eye response to light and manual stimuli were considered as blindness criteria. Similar experimental conditions were performed in pigs and rabbits. Short duration consumption of 1 or 2% salt concentrations did not produce any detectable ocular signs, whereas long duration consumption of 2% salt concentration induced prominent ocular signs. After 3–4 days of consuming 3% and higher salt concentrations, retinal and vitreous hemorrhages, posterior vitreous detachment PVD, and retinal detachment were observed. The following ocular changes included conjunctivital injection, cornea and lens opacities, swelling of the optic disc, blindness, vitreous collapse, and finally phthisis bulbi. Salt poisoning led to hypernatremia and hyperosmolarity. Due to hyperosmolarity, the vitreous lost some of its fluid contents and vitreous shrinkage resulted in vascular damage, retinal and vitreous hemorrhage, PVD, and retinal detachment. In addition, conjunctivitis, corneal and lens opacities, swelling of the optic disc, blindness, vitreous collapse, and finally phthisis bulbi was evident.