The elucidation of the life cycle of Saccocoelioides nanii Szidat, 1954 (Digenea: Haploporidae) using molecular techniques.

In South America, the knowledge of trematode diversity parasitizing freshwater fishes is still scarce, as less than 5% of the freshwater fish fauna has been examined for parasites. A similar situation applies to studies on digenean life cycles, which have become increasingly rare. Among the digenean families parasitizing freshwater fishes in the region, Haploporidae is considered the richest in species diversity. However, information about the developmental stages of haploporid life cycles remains fragmentary. Particularly, in Argentina, nine cercariae attributed to the family Haploporidae have been described using morphological analysis, and only two life cycles of this family have been completely elucidated. In this study a new type of cercaria, morphologically assigned to the family Haploporidae and collected from the snail Heleobia parchappii (Cochliopidae) in Los Padres shallow lake, Buenos Aires province, was identified using morphological and molecular techniques. The molecular analysis, based on 28S and ITS2 sequences, revealed that the cercariae were 100% identical to adult specimens of Saccocoelioides nanii (Haploporidae) parasitizing the fish Prochilodus lineatus (Prochilodontidae) from Los Talas, Buenos Aires province. Our results not only provide information about the life cycle of S.nanii but also show that a molecular and morphological approach can be extremely useful in identifying the developmental stages of digeneans and elucidating their life cycles.

New perspectives on morphological and genetic variability of Corynosoma bullosum [Linstow, 1892] parasitizing southern elephant seals from the Antarctic Peninsula.

Previous descriptions of Corynosoma bullosum (Linstow, 1892) show that specimens vary greatly in the proportions of different body structures, measurements of females and males, number of rows of hooks, and egg measurements, among other features. We redescribe this species from specimens found in southern elephant seal faeces from King George Island. We also provide a molecular characterization, in addition to 5.8S and internal transcribed spacer (ITS) existing sequences. We examined 41 elephant seals, and 30 adult acanthocephalans were found in 15 of them. The specimens were identified as belonging to the genus Corynosoma due to each having a tubular body with an inflated anterior part forming a thorny disk and the posterior part bearing somatic spines on the ventral surface, and genital spines surrounding the genital pore. Individual morphology corresponded to C. bullosum: large size, marked sexual dimorphism, and proboscis with 16-18 rows of spines with 11-15 spines per row. The molecular profile of three specimens of C. bullosum was analysed using 18S rDNA. We inferred phylogenetic relationships of the family Polymorphidae using maximum likelihood and Bayesian inference. We provide an updated morphological redescription for C. bullosum including electron microscopy photographs and molecular data. The 18S gene sequences showed low genetic variation and supported that C. bullosum is a sister to Corynosoma australe.

Pathological conditions of the sentinel bivalve, the little mussel Brachidontes rodriguezii, from contaminated intertidal sites in the Southwestern Atlantic coast.

A survey of pathological conditions affecting the natural beds of the dominant bivalve species of the intertidal rocky coasts of the Southern Atlantic Ocean, was performed. Samples of the little mussel (n = 992), Brachidontes rodriguezii, which is considered a sentinel species, were collected from seven sites that present different anthropogenic activity (low anthropogenic activity, harbor, and sewage waste) along the Argentinean coast, and processed by standard histological techniques. Our results showed for the first time in a B. rodriguezii population from Mar del Plata harbor a bacterium causing cellular lysis of the spermatogenic cells (6.5% of prevalence) and in a population from Villa Gesell, a site with low anthropogenic activity, one little mussel presented cytoplasmic vacuoles in the oocytes's cytoplasm similar to a microsporidian (0.2% of prevalence). A ciliated protozoan parasitizing the cytoplasm of the epithelial digestive tubules and larval stages of two different digenean parasites, was found infecting the little mussel from both contaminated and uncontaminated sites. Sporocysts of the family Bucephalidae invaded the gonad, causing severe gonadal damage including castration, and gymnophallid metacercariae located between the mantle and shell or embedded in the mantle tissues, occupied the gonadal space. In Punta Carballido, a site located next to a sewage outfall, the highest prevalence of infection was found, for the ciliated protozoan in the digestive tubules (5.1%) and for the digenean parasites (bucephalid sporocyst 24.4%, and gymnophallid metacercariae 50%). Moreover, we found a positive correlation between shell length and overall prevalence of parasites.

Diseases and parasites of wild and cultivated mussels along the Patagonian coast of Argentina, southwest Atlantic Ocean.

A histological survey of the commercially edible mussels Mytilus platensis and M. chilensis from wild and cultivated populations along the coast of Patagonia, Argentina (42°00' to 54°47'S), was carried out to determine their health status. Diagnostic results included 3 types of inflammatory responses (infiltrative, nodular, and encapsulation), disseminated neoplasia disease, 2 abnormal reproductive conditions (gonadal atresia and intersex), prokaryotic inclusions, protozoans, and metazoans. Pathogen prevalence and infection intensity among mussels of different sampling sites and between those of wild and cultivated populations were compared. Inflammatory responses were recorded in all mussels from all sites, while disseminated neoplasia only occurred in the most southern cultivated M. chilensis. Intracellular prokaryotic inclusions were broadly distributed in the mussels from both northern and southern Patagonian coasts. Ciliates showed the highest prevalence among wild mussels from the colder waters of Bahía Brown. Turbellaria were recorded at higher prevalence in cultured mussels (41.7%), and trematode metacercariae occurred exclusively in intertidal wild mussels. None of the parasites found appears to be a problem to the fishery or farming, although disseminated neoplasia should be monitored. In addition, we found that mytilid species coexisting with M. platensis (Aulacomya atra and Perumytilus purpuratus) at one location shared the same pathological conditions and parasites, which differed from those of M. platensis at a distant locality. These results suggest that pathological conditions and parasites were influenced more by ecological habitat factors than by the species of mussels present, based on similar parasite assemblages found among closely related mytilid hosts in the same geographical area.

Survey of trematodes in intertidal snails from Patagonia, Argentina: new larval forms and diversity assessment.

Larval trematodes are the main parasites of snails, and they play a crucial role because they usually castrate their snail hosts and can thus alter their population and community dynamics. This study involved a survey of seven gastropod species (Crepipatella dilatata, Fissurella radiosa, Nacella magellanica, Pareuthria fuscata, Siphonaria lessonii, S. lateralis and Trophon geversianus) parasitized by 12 trematode species (one hemiurid, one gymnophallid, two lepocreadiids, two microphallids, one notocotylid, two renicolids, one philophtalmid, one schistosomatid and one zoogonid) from southern Patagonia (47°S, 65°W), Argentina. Only F. radiosa was free of parasites. The study included the description of five new larvae, based on morphological and molecular information, and a comparison of the parasite diversity with that of a northern locality (42°S, 64°W), characterized by a lower mollusc diversity. Species richness and diversity of parasites were higher in the southern site. This suggests a correlation between the level of parasitism and the diversity of molluscs (first intermediate hosts), which is higher at the high-latitude site and seems to attract shorebirds, which disperse the digenean eggs and facilitate the completion of their life cycles. These results support the notion that parasitism is influenced by large-scale factors such as biogeographical patterns, and small-scale factors such as diversity or abundance of intermediate and definitive hosts.

Morphology and molecular systematic of marine gregarines (Apicomplexa) from Southwestern Atlantic spionid polychaetes.

Gregarines are a common group of parasites that infect the intestines of marine invertebrates, and particularly polychaetes. Here, we describe for the first time four gregarine species that inhabit the intestines of three spionid species: Dipolydora cf. flava, Spio quadrisetosa and Boccardia proboscidea from the Patagonian coast, Argentina, using light and scanning electron microscopy and molecular phylogenetic analyses of small subunit (SSU) rDNA sequences. Even though the spionid species thrive in the same environments, our results showed a high host specificity of the gregarine species. Selenidium cf. axiferens and Polyrhabdina aff. polydorae were both identified from the intestine of D. cf. flava. The new species, Polyrhabdina madrynense sp. n. and Selenidium patagonica sp. n., were described from the intestines of S. quadrisetosa and the invasive species B. proboscidea, respectively. All specimens of D. cf. flava and S. quadrisetosa were infected by gregarines (P = 100%), recording the highest mean intensity values of infection (MI = 80; 60 respectively), in contrast to B. proboscidea (P = 60%; MI = 38). We associated this finding with the recent invasion of this host. It is expected that in the future, an increase of its population density might favour a rising intensity of this gregarine infection.

A new species of Procyrnea (Nematoda: Habronematidae) parasitic in Rhea pennata (Aves: Rheidae) from Patagonia, Argentina, with a key to species of the genus.

Procyrnea choique n. sp. is described from the lesser rhea, Rhea pennata d´Orbigny (Aves: Rheidae), from the Protected Natural Area Península Valdés, Chubut province, Argentina. The new species resembles P. ficheuri, P. murrayi, P. excisiformis, P. dollfusi, P. haliasturi, P. anterovulvata, P. graculae, P. brevicaudata, P. uncinipenis, P. javaensis, P. ameerae, P. ornata, P. aegotheles, P. spiralis, P. ruschii and P. aptera, mainly in the absence of lateral alae; but differs from its congeners by having a left spicule without barbs, the position of the vulva which is post-equatorial, the absence of lateral ridges, absent median precloacal papilla, asymmetrical caudal alae, males more than 6 mm long, left spicule 1 mm long and spicule ratio 1:3. Our results extend the taxonomy of Procyrnea Chabaud, 1958 and comprise the first report of a habronematid from R. pennata. A key to species of Procyrnea is presented.

Larval digenean preferences in two sympatric snail species at differing tidal levels off the Atlantic coast of Patagonia.

Larval stages of the trematodes Maritrema madrynense and Hemiuroidea fam. gen. et sp. differentially parasitized Siphonaria lessonii and S. lateralis, two sympatric pulmonate snail species on the rocky intertidal shore at Puerto Deseado, south-western Atlantic coast of Patagonia, Argentina. Snail specimens were collected at two sampling sites with contrasting physical-chemical characteristics. One site, in the upper intertidal, was exposed to sewage from fish-processing plants, greater hydrodynamic forcing and desiccation, a wider temperature range, longer exposure to ultraviolet radiation and higher abundance of birds. The second site, in the lower intertidal, was generally characterized by less stressful environmental conditions. At both sites, S. lateralis showed a markedly higher density than S. lessonii (55.13 vs. 5.87 snails/m2, respectively). Despite this, the prevalence of both digeneans was higher in S. lessonii (17.37% and 3.52% for M. madrynense and Hemiuroidea, respectively) than in S. lateralis (0.09% and 0% for M. madrynense and Hemiuroidea, respectively). This study demonstrates high parasite specificity for the host. Low densities of S. lessonii are attributed to castration of parasitized hosts and reduction of their physiological condition. The prevalence and intensity of infection of both digenean parasites were higher at the more stressful, upper intertidal site, thus suggesting that a higher abundance of birds and exposure to sewage pollution may promote the transmission of trematodes.

Diversity of helminth parasites in aquatic invertebrate hosts in Latin America: how much do we know?

Helminths in aquatic invertebrate hosts have been overlooked in comparison with vertebrate hosts. Therefore, the known diversity, ecology and distribution of these host-parasite systems are very limited in terms of their taxonomic diversity, habitat and geographic regions. In this study we examined the published literature on helminth parasites of aquatic invertebrates from Latin America and the Caribbean (LAC) to identify the state of the knowledge in the region and to identify patterns of helminth diversity. Results showed that 67% of the literature is from Argentina, Mexico and Brazil. We found records for 772 host-parasite associations. Most records relate to medically or economically important hosts. Molluscs were the most studied host group with 377 helminth records (80% trematodes). The lymnaeids and planorbids were the most studied molluscs across LAC. Arthropods were the second most studied host group with 78 helminth records (trematodes 38%, cestodes 24% and nematodes 20%), with shrimps and crabs being the most studied hosts. Host species with the largest number of helminth taxa were those with a larger sampling effort through time, usually in a small country region. No large geographical-scale studies were identified. In general, the knowledge is still too scarce to allow any zoogeographical or helminth diversity generalization, as most hosts have been studied locally and the studies on invertebrate hosts in LAC are substantially uneven among countries.

Pearls in the ribbed mussel Aulacomya atra caused by polydorin polychaetes (Spionidae) infestation.

Aulacomya atra populations of the San Jose gulf, Northern Patagonia, Southwestern Atlantic Ocean, are infested by two polydorin species, Polydora rickettsi and Dipolydora cf. giardi. The infestation by these boring polychaetes causes the formation of pearls which is evidenced by the presence of capsules containing polydorin tissue debris and the elemental composition of organic material inside the pearls. Moreover, a positive relationship between the abundance of perforations of polydorin polychaetes and abundance of pearls was found by applying generalized lineal model analysis. These results constitute the first evidence of pearls formation due to infestation by polychaete.

Stomach nematodes of three sympatric species of anatid birds off the coast of Patagonia.

The present study focuses on the nematode community in the stomachs of three sympatric anatid bird species from the Central Patagonian coast, Argentina. The bird species include the Chubut steamer duck, Tachyeres leucocephalus, the crested duck, Lophonetta specularioides, and the black-necked swan, Cygnus melancoryphus. Up to 138 nematodes representing five species were recovered from 10 of the 13 ducks examined, with an overall prevalence of 77% and a mean intensity of 13.8. Nematodes isolated from the gizzard were Streptocara formosensis and Sciadiocara legendrei (Acuariidae) in T. leucocephalus, and Epomidiostomum vogelsangi (Amidostomatidae) in C. melancoryphus, whereas Echinuria uncinata (Acuariidae) and Tetrameres (Petrowimeres) fissispina (Tetrameriidae) were found in the proventiculus of L. specularioides. In particular, S. legendrei was registered for the first time in South America and T. leucocephalus, whereas T. fissispina represents a new record in Argentina and L. specularioides. Moreover, E. vogelsangi and E. uncinata were isolated in Patagonia for the first time. The birds studied herein are sympatric in their distribution, and two of them are syntopic breeders; however, they were infected with different parasite species. This situation could be partially due to their diet or their seasonal movements, or a combination of both. Despite the low number of hosts examined, this work enhances our knowledge about parasites from a frequently occurring group of birds on the Patagonian coast, a subject that has not been studied extensively in South America.

Shell damage in the Tehuelche scallop Aequipecten tehuelchus caused by Polydora rickettsi (Polychaeta: Spionidae) infestation.

The different types of shell damage caused to the commercially valuable Tehuelche scallop (Aequipecten tehuelchus) by the polychaete Polydora rickettsi are described. X-rays, computerized tomography, shell sections, scanning electron microscopy, Energy Dispersive X-ray analysis (EDAX), mineralogical analyses and geometric morphometrics were applied to that end. Scallop shells presented three types of damage: (1) spots, (2) calcareous alterations, and (3) mud blisters. Microstructural alterations consisted of a simple conchiolin membranous layer in the case of spots, a series of interleaved layers of different degree of calcifications in calcareous alterations, and two different surface morphologies (muddy and mucous layers) in mud blisters. Damage was localized mainly along concentric growth rings, coincidentally with the location of most burrows, as shown by X-ray. Mineralogical analysis showed that in all cases (including non-infested shells) calcite was the calcium carbonate polymorph present. Geometric morphometrics showed that only 5% of shape variation was explained by infestation with P. rickettsi, irrespective of the type of damage. Number of worms per infested shell varied significantly among four beds. Left shells (upward-oriented) were significantly more affected than right shells, which are in closer contact with the bottom.

The life cycle and geographical distribution of the monorchiid Proctotrema bartolii (Digenea) in the clam Darina solenoides from the Patagonian coast, Argentina.

The life cycle of Proctotrema bartolii Carballo, Laurenti & Cremonte 2011 (Digenea: Monorchiidae) at Fracasso Beach (the type locality) (42°25'S, 64°07'W), Península Valdés, Argentina, was investigated. This digenean uses the clam Darina solenoides (Mactridae) as both the first and second intermediate hosts in the natural environment. The metacercariae were located mainly at the tip of the incurrent siphon, with an infection prevalence of 100%. Experimental infections in other macroinvertebrates, such as the clam Tellina petitiana and the polychaete Glycera americana, were successful, but these and other invertebrates are not naturally infected. Silversides Odontesthes smitti and Odontesthes nigricans (Pisces: Atherinopsidae) and the mullet Eleginops maclovinus (Eleginopidae) act as the definitive hosts of both experimentally and naturally obtained adults. Fish acquire infection by eating either the siphon or the entire clam. Proctotrema bartolii seems to be endemic to the Magellan Region and is distributed where its intermediate clam host is present, from the San José Gulf in Península Valdés to the southern tip of South America.

Similarity in parasite community structure may be used to trace latitudinal migrations of Odontesthes smitti along Argentinean coasts.

The aims of this study were to determine the existence of migratory movements and to identify ecological stocks of the silverside Odontesthes smitti along its distribution in the Southern Atlantic Ocean, using metazoan parasites as biological tags. Samples were obtained from San José Gulf (SJ) (42° 25' S; 64° 07' W) and Nuevo Gulf (NG) (42° 47' S; 65° 02' W) in north Patagonia during winter and summer and in waters off Mar del Plata (MDP) (38° 03' S; 57° 32' W), Bonaerense region, during winter. Fifteen parasite species were collected. Multivariate statistical procedures on parasite community data showed strong effect of host size on the structure of parasite assemblages. Taking into account the variations among samples due to host size, the differential structure of parasite communities between SJ and NG suggests that fish inhabiting these localities could belong to different ecological stocks. Fish from MDP and SJ caught in summer showed similar composition in their parasite assemblages, which is congruent with a migratory cycle that implies that fish caught in MDP during winter inhabit SJ during summer. Further evidence of the Patagonian origin of MDP O. smitti is the presence of the digenean Proctotrema bartolii in fish from both regions. Proctotrema bartolii is acquired by O. smitti only in the Magellanic province, where its intermediate host, Darina solenoides, is distributed. The analyses suggest that O. smitti inhabiting north Patagonian gulfs could belong to different ecological stocks and that O. smitti caught in MDP could have come from SJ.

Parasitic castration of Buccinanops cochlidium (Gastropoda: Nassariidae) caused by a lepocreadiid digenean in San José Gulf, Argentina.

Parasitic castration of Buccinanops cochlidium from San José Gulf, Argentina, caused by a lepocreadiid digenean, is reported for the first time. Rediae and ophtalmotrichocercous cercariae probably belonging to Opechona sp. were identified in the gonad and digestive gland. Opechona sp. has been reported previously parasitizing B. monilifer from a northern locality in the Argentine Sea. Overall prevalence of infection was 15.5%; it varied seasonally, rising during the warm months after the host oviposition period. Cercariae were expelled at the same time as the hatching of snail embryos (during the higher water temperature period). Rediae affected male and female snails equally, but prevalence increases along with host size. The parasite causes the complete castration of the host. Parasitized adult snails showed a reduction of penis size in comparison with healthy males. It remains to be confirmed whether the peak of cercariae emission coincides with the presence of jellyfishes and scombrid or other fishes in the area, which could act as second intermediate and definitive hosts, respectively.

Bartolius pierrei n. g., n. sp. (Digenea: Gymnophallidae) from the Península Valdés, Argentina.

Bartolius pierrei n. g., n. sp. (Digenea: Gymnophallidae) is described from metacercariae and naturally and cultivated obtained adults from southern Argentina. The second intermediate host is Darina solenoides (King) (Bivalvia: Mactridae) and the definitive host is Larus dominicanus Lichtenstein (Aves: Laridae). The diagnostic characters are as follows: Body small, oval. Oral sucker without lateral projections, twice size of ventral sucker (except in young metacercariae). Caeca short (in adults), without dorsal diverticula. Ventral sucker in posterior third of body. Ventral pit absent. Seminal vesicle bipartite. Ovary post-testicular. Vitelline glands paired, compact, close to ventral sucker. Uterus in fore- and hindbody. Genital atrium tubular. Genital pore inconspicuous, close to anterior margin of ventral sucker. Excretory vesicle Y-shaped with very short stem. Excretory formula: 2[(2+2)+(2+2)] = 16. Bartolius is distinguished from other genera of the Gymnophallidae by the post-testicular position of the ovary.

New host and locality records for Tetrameres (Gynaecophila) spirospiculum Pinto & Vicente, 1995 (Nematoda: Tetrameridae), with new morphological data.

We report the finding of Tetrameres spirospiculum Pinto & Vicente, 1995 from Theristicus melanopis melanopis (Threskiornithidae) from Patagonia, Argentina. These constitute new host and locality records. We propose the assignation of this species to the subgenus T. (Gynaecophila) Gubanov, 1950, based on the presence of labia and the absence of cuticular flanges at the anterior end. Some new morphological data are provided, such as the arrangement of cuticular spines and the presence of a pair of somatic papillae at beginning of posterior third of body length. T. (G.) spirospiculum may probably be regarded as specific to birds of the genus Theristicus.

Tetrameres (Tetrameres) megaphasmidiata n. sp. (Nematoda: Tetrameridae), a parasite of the two-banded plover, Charadrius falklandicus, and white-rumped sandpiper, Calidris fuscicollis, from Patagonia, Argentina.

Tetrameres (Tetrameres) megaphasmidiata n. sp. is described from the proventriculus of the two-banded plover, Charadrius falklandicus, and the white-rumped sandpiper, Calidris fuscicollis, from Patagonia, Argentina. The new species shares with T. (T.) nouveli, T. (T.) paradisea, T. (T.) prozeskyi, T. paraaraliensis, T. (T.) cladorhynchi, and T. lobybicis the absence of the right spicule and the presence of 4 rows of somatic spines. Tetrameres (T.) megaphasmidiata n. sp. differs from the first 4 species mainly by its longer left spicule. The new species can be distinguished from T. (T.) cladorhynchi by the extension of the lateral alae, the number and arrangement of the caudal papillae, and the absence of polar filaments in the eggs. Tetrameres lobybicis differs from the new species by having shorter rows of dorsal spines and a different number and arrangement of the caudal papillae. This report is the first record of a species of Tetrameres in C. falklandicus and C. fuscicollis.

Mantle-shell complex reactions elicited by digenean metacercariae in Gaimardia trapesina (Bivalvia: Gaimardiidae) from the Southwestern Atlantic Ocean and Magellan Strait.

The host response to 3 different larval digeneans affecting Southwestern Atlantic and Magellanic populations of the bivalve Gaimardia trapesina (Lamarck, 1819) (Gaimardiidae) is described. Unencysted metacercariae of 2 species of Gymnophallidae and 1 species of Lepocreadiidae co-exist in the peripheral and general extrapallial spaces. Differences in host responses to stimuli generated by each parasite are described. Infections by Gymnophallidae gen. sp. 1 metacercariae elicit a mantle-shell complex reaction involving both alteration of the mantle epithelium (hyperplasia and metaplasia) and calcium carbonate deposition to form an incomplete calcareous covering of single larvae, comprising individual crystallites in an organic matrix. The calcareous covering covers only the posterior two-thirds of the larval body, having its anterior end free in the extrapallial space, which ensures survival of the larvae. The peculiar features of the host response result from a successful process of adjustment to the parasite's life cycle. Metacercariae of Gymnophallidae gen. sp. 2 occur in one or more small groups of up to 30 individuals, either in the general or in the peripheral extrapallial spaces. The outer mantle reaction includes hyperplasia and metaplasia of the epithelium adjacent to the larvae. Deposition of calcium carbonate did not occur. Lepocreadiidae gen. sp. metacercariae were observed exclusively in the general extrapallial space lodged in single shallow pits whose shape fit the parasite's body shape and size. In simultaneous infections, metacercariae of Gymnophallidae and Lepocreadiidae elicited different responses in an individual, which suggests that each parasitic entity involves a different stimulus to the host.

Contracaecum multipapillatum (Nematoda: Anisakidae) from Egretta alba (Aves: Ardeidae) and comments on other species of this genus in Argentina.

During a parasitological survey of aquatic birds in Mar Chiquita coastal lagoon (Buenos Aires province, Argentina), specimens of Contracaecum Raillet & Henry, 1912 sp. were found in the esophagus and stomach of the great egret Egretta alba (Gmelin, 1789) (Aves: Ardeidae). The nematodes were identified as Contracaecum multipapillatum (Drasche, 1882) on the basis of the following features: ratio of ventricular appendage length to esophagus length, ratio of intestinal caecum length to esophagus length, spicule lengths, number and arrangement of postcloacal papillae (3-4 pairs of adanal papillae, 1 double pair, and a subterminal group of 2 subventral and 3 sublateral pairs). A complete description of the adult specimens of C. multipapillatum is given in the present paper, together with a discussion regarding the validity of other species of Contracaecum reported from Argentina. Contracaecum philomultipapillatum Labriola and Suriano, 1996 is reduced to a junior synonym of C. multipapillatum, and Contracaecum longicaecum Schuurmans Sterkhoven, 1952 is considered a species inquirenda. A revision of Contracaecum spheniscus Boero and Led, 1970 based on appropriate material is necessary.