Infectivity of Paranosema locustae (Microsporidia) against gregarious-phase South American locust (Orthoptera) when treated en masse.

En masse inoculations with Paranosema locustae, an intracellular parasite of adipose tissue of grasshoppers and locusts and the only microsporidium registered as a biocontrol agent, were conducted against crowded fourth-instar nymphs of the South American locust Schistocerca cancellata and the grasshoppers Dichroplus schulzi and Ronderosia bergii. Infection did not develop in the locust, but was highly prevalent in the two grasshopper species. We hypothesize that absolute absence of infection in S. cancellata may constitute a case of density-dependent prophylactic resistance, an elevation of the baseline immunity of an organism in order to cope with disease that is prevalent in species exhibiting phase polyphenism.

Beauveria bassiana (Ascomycota: Hypocreales) Introduced as an Endophyte in Corn Plants and Its Effects on Consumption, Reproductive Capacity, and Food Preference of Dichroplus maculipennis (Orthoptera: Acrididae: Melanoplinae).

In this study, the effects of strain Beauveria bassiana (LPSC 1067) as an endophyte in corn plants on consumption, fecundity, and food preference of Dichroplus maculipennis were examined. We observed that the daily consumption by grasshoppers fed with control plants was almost twice that of those that were fed treated plants. Significant differences in fecundity of grasshoppers that were fed with treated plants compared with those that only fed on control plants were also observed. The number of eggs laid per female fed with control plants was 27.2, while the number of eggs laid per female that were fed during 15 d with treated plants was 17.7. Similar results were observed when the number of embryonated eggs was evaluated. The highest number of embryonated eggs were recorded in those females that only fed on control plants (96%) while fewer embryonated eggs were recorded in grasshoppers fed for 15 d with treated plants only (25%). In relation to food preference the average consumption rate for D. maculipennis females on control corn plants was 303.8 ± 24.5 mg while it was only 25 ± 2.1 mg on plants treated with B. bassiana as an endophyte. In summary, we observed that B. bassiana as a corn plant endophyte negatively affected the daily consumption rate, fecundity and food preference of D. maculipennis.

Tubulinosema pampeana sp. n. (Microsporidia, Tubulinosematidae), a pathogen of the South American bumble bee Bombus atratus.

An undescribed microsporidium was detected and isolated from the South American bumble bee Bombus atratus collected in the Pampas region of Argentina. Infection intensity in workers averaged 8.2 × 10(7)spores/bee. The main site of infection was adipose tissue where hypertrophy of adipocytes resulted in cyst-like body formation. Mature spores were ovoid and monomorphic. They measured 4.00 µm × 2.37 µm (fresh) or 3.98 µm × 1.88 µm (fixed). All stages were diplokariotic and developed in direct contact with host cytoplasm. Isofilar polar filament was arranged in 16 coils in one or, posteriorly, two layers. Coiling angle was variable, between perpendicular and almost parallel to major spore axis. Late meronts and sporogonial stages were surrounded by vesicles of approximately 60 nm in diameter. Based on both new and already designed primers, a 1827 bp (SSUrRNA, ITS, LSUrRNA) sequence was obtained. Data analyses suggest that this microsporidium is a new species of the genus Tubulinosema. The name Tubulinosema pampeana sp. n. is proposed.

Spore loads of Paranosema locustae (Microsporidia) in heavily infected grasshoppers (Orthoptera: Acridoidea) of the Argentine Pampas and Patagonia.

Paranosema locustae, an entomopathogen of grasshoppers and locusts, remains the only microsporidium registered as a biocontrol agent. After introductions from North America, it became established in grasshopper communities of Argentina. We measured the infection intensity of field collected, heavily infected male and female adults of individuals belonging to six grasshopper species, five melanoplines (Melanoplinae) (Baeacris pseudopunctulatus, Dichroplus maculipennis, Dichroplus vittatus, Neopedies brunneri, Scotussa lemniscata), and one gomphocerine (Gomphocerinae) (Staurorhectus longicornis). Average spore load among heavily infected grasshoppers ranged from 8.7±0.5×10(7) to 1.1±0.7×10(9). Only females of B. pseudopunctulatus and S. longicornis showed significantly higher spore loads than the males.

Relationships and taxonomy of the genus Diponthus Stl (Orthoptera: Acridoidea: Romaleidae).

Diponthus Stl, one of the most diversified and widely distributed Romaleinae genera, is endemic to southern South America. This colorful grasshopper genus is placed within Romaleini and currently includes 16 valid species from a total of 22 nominal ones, most of them only known from their original descriptions. The aims of this study were to propose a morphology-based phylogenetic hypothesis of relationships among the species of Diponthus, and to conduct a taxonomic revision, including the description of four previously unknown species. Parsimony analysis of 79 morphological characters strongly recovered the monophyly of genus Diponthus, and Gurneyacris as its sister group. Internal clades within Diponthus were supported by external morphology and coloration patterns, while characters from male genitalia were shown to be more useful for species differentiation. Based on the results of the taxonomic review, Diponthus is constituted by 16 valid species, with the following nomenclatural changes proposed: Diponthus nigroconspersus (Stl) is considered a valid name; D. invidus Carl and D. bilineatus Rehn are synonymized under D. virgatus (Gerstaecker); Diponthus clarazianus Pictet & Saussure is synonymized under D. cribratus (Serville); D. paulista Rehn is synonymized under D. porphyreus (Gerstaecker); D. maculiferus (Walker) is synonymized under D. electus (Serville). New names are proposed for four undescribed species from Bolivia (D. colorbellus sp. nov. and D. dilatatus sp. nov.), Argentina (D. paranaensis sp. nov.) and Brazil (D. salvadorii sp. nov.).

Sphaerularia bombi (Nematoda: Sphaerulariidae) parasitizing Bombus atratus (Hymenoptera: Apidae) in southern South America.

Bumble bees are some of the most important insect pollinators. However, knowledge on parasites associated to bumble bees in South America is very limited. This study reports the first isolation of a sphaerularid nematode parasitizing queens of the native bumble bee Bombus atratus in Argentina. Measurements and morphological characters of eggs, juveniles, and adults strongly suggest that the species is Sphaerularia bombi, a parasite that affects the reproduction and foraging behavior of the host. The nematode was detected in bumble bees of San Carlos de Bariloche, northwestern Patagonia region, and the surroundings of La Plata, northeastern Pampas region. Prevalence varied between 8% and 20%.

[Postembryonic development, fecundity and food consumption of Dichroplus exilis (Orthoptera: Acrididae) under controlled conditions]. / Desarrollo post-embrionario, fecundidad y consumo de alimento de Dichroplus exilis (Orthoptera: Acrididae) bajo condiciones controladas.

Dichroplus exilis is a widely distributed species in Southern South America. Although there have been reports of D. exilis as an agricultural pest, some recent observations suggest that the damage attributed to D. elongatus may actually have been caused by D. exilis. This study was conducted to determine the postembryonic life cycle stages, fertility and food consumption of this species under controlled conditions (30 degrees C, 14L-10D, 40% RH). Individuals employed belong to the laboratory-hatched first generation (F1), from adults (n = 64, female = 28, male = 36) collected in natural grasslands near Rafaela, Santa Fe province in North-Eastern Argentina. Three cohorts of 16, 17 and 20 individuals were monitored independently in acetate tubes on a daily basis, until death of the last insect. Average fecundity was 381.84, 38.54 eggs per female. Egg-pod incubation time was 14.4, 1.08 days and six nymphal instars were recorded. Nymphal development time was 41.38, 0.71 days (I = 8.73, 0.20; II = 6.38, 0.24; III = 5.64, 0.33; IV = 7.15; 0.43; V=9.76, 0.54; IV = 7.85, 0.95). The recorded food consumption was 9.89, 1.08 (mg/ind/day) for nymphs IV, 18.04, 0.73 (mg/ind/day) for nymphs V-IV, 16.76, 1.06 (mg/ind/day) for pre-reproductive males, 28.09, 1.81 (mg/ind/day) for pre-reproductive females, 7.71,0.91 (mg/ind/day) for reproductive males and 13.06, 0.71 (mg/ind/day) for reproductive females, while the average adult food consumption, regardless of sex and reproductive status, was 16.41, 4.32 mg/day. Average food consumption of adult females was 17.47, 1.15 mg, and was significantly higher than that of males (10.83, 0.91mg). Data obtained in this study showed that D. exilis exhibits at least some of the biological attributes needed to configure an actual or potential agricultural pest, albeit not yet recognized as such. Field monitoring of grasshopper communities in areas where damage by D. exilis is suspected is envisaged in order to determine its possible status as a pest.

Invasive Bombus terrestris (Hymenoptera: Apidae) parasitized by a flagellate (Euglenozoa: Kinetoplastea) and a neogregarine (Apicomplexa: Neogregarinorida).

The flagellate Crithidia bombi and the neogregarine Apicystis bombi have been found in individuals of Bombus terrestris, a Palaearctic species of bumble bee commercially reared and shipped worldwide for pollination services. B. terrestris has recently entered into the northwestern Patagonia region of Argentina from Chile, where it was introduced in 1998. Prevalence was 21.6% for C. bombi and 3.6% for A. bombi (n=111). The pathogens were not detected in 441 bumble bees belonging to five of the eight known Argentine native species (Bombus atratus, Bombus morio, Bombus bellicosus, Bombus opifex, Bombus tucumanus) collected elsewhere in the country. Although the absence of natural occurrence of C. bombi and A. bombi in Argentine native bumble bees cannot be ascertained at present due to the limited surveys performed, it is important to report their detection in invasive B. terrestris. The invasion event is relatively recent and the accompanying pathogens are not species specific within the genus Bombus.

Morphology and taxonomy of the microsporidium Liebermannia covasacrae n. sp. from the grasshopper Covasacris pallidinota (Orthoptera, Acrididae).

During a survey for grasshopper pathogens in Argentina in 2005-2006, individual Covasacris pallidinota from halophylous grasslands in Laprida, Buenos Aires province were found to be infected with a microsporidium. Infection was restricted to the salivary gland epithelial cells. The microsporidium produced ovocylindrical spores averaging 2.6+/-0.28 x 1.4+/-0.12 microm (range 2.2-3.4 x 1.1-1.7 microm), which resembled in size and shape the spores of Liebermannia patagonica and L. dichroplusae, two recently described species that also parasitize Argentine grasshoppers. The life cycle of the microsporidium included the formation of polynucleate, diplokaryotic, moniliform, merogonial plasmodia wrapped in flattened cisterns of the host endoplasmic reticulum (ER). Plasmodia divided to produce diplokaryotic cells. The latter underwent elongation, dissociation of diplokarya counterparts, vacuolization, dismantling of the host ER envelope, and deposition of electron-dense material outside the plasma membrane. The resultant binucleate sporogonial plasmodia divided into two uninucleate sporoblasts, which eventually transformed into spores. Uninucleate spores contained a lamellar polaroplast, embraced by an elongated polar sac, anchoring disc, 3-5 polar filament coils, and a cluster of anastomizing tubules (sporoblast trans-Golgi, posterosome) at the posterior end. Sequence similarity of the SSU rDNA of the newly discovered microsporidium (Genbank accession no. EU709818) to L. patagonica and L. dichroplusae was 99% and 97%, respectively, suggesting that the three species belong to one genus. All three species fell into one clade in SSU rDNA-based phylogenetic trees produced by neighbor joining, maximum parsimony, and maximum likelihood analyses with 100% statistical support. We assign the name Liebermannia covasacrae to this microsporidium. It can be easily differentiated from both congeners by host species, tissue tropism, type of sporogony, and several features of morphology. Comparison of the three Liebermannia spp. demonstrates that the nuclear phase (dikaryotic versus monokaryotic spores) and type of sporogony (polysporous versus disporous) may vary in closely related species.

New case of long-term persistence of Paranosema locustae (Microsporidia) in melanopline grasshoppers (Orthoptera: Acrididae: Melanoplinae) of Argentina.

We report an additional case of long-term persistence of Paranosema locustae in grasshoppers of Argentina. The pathogen was introduced from North America on rangeland at Loncopué, Neuquén province. Microsporidia were not detected in pre-introduction samples whereas infected grasshoppers were found 11 years after introduction. Affected grasshoppers were the melanoplines Dichroplus elongatus, Dichroplus maculipennis, and Scotussa lemniscata, some of them with high spore loads. The case highlights the ability of P. locustae to recycle in local grasshopper communities by parasitizing susceptible species other than the natural hosts.

Phylogenetic relationships of Heterovesicula cowani, a microsporidian pathogen of Mormon crickets, Anabrus simplex (Orthoptera: Tettigoniidae), based on SSU rDNA-sequence analyses.

The microsporidium Heterovesicula cowani, discovered in 1985, was initially identified as Vairimorpha sp. because it produces two types of spores: Nosema-like diplokaryotic spores and Thelohania-like mononuclear meiospores. However, light and electron microscopy studies revealed characters that did not fit any known microsporidian genera, and a new monotypic genus Heterovesicula was erected. The goal of this study was to test the validity of the genus Heterovesicula by molecular characterization of H. cowani and to assess its phylogenetic relationships to other microsporidia from insects. DNA from spores stored at -32 degrees C since 1992 was isolated and PCR-amplified with V1-1492 primers to obtain a partial small subunit ribosomal RNA gene sequence of 1165bp, which was submitted to GenBank (Accession No. EU275200). Neighbor joining, maximum parsimony and maximum likelihood analyses performed against 18 microsporidia sequences, placed H. cowani as a sister taxon to the Nosema-Vairimorpha clade. The consensus of these analyses suggests that the Heterovesicula-Nosema-Vairimorpha group forms a dichotomy with the Encephalitozoon spp. branch. Other microsporidia parasitizing Orthoptera fell into two unrelated (or distantly related) lineages of terrestrial microsporidia: the Liebermannia spp. branch forms a dichotomy with Orthosomella operophterae within the Endoreticulatus-Orthosomella-Liebermannia group; and the Paranosema spp. branch clusters together with the Tubulinosema-Systenostrema lineage. The minimum pairwise distance in Kimura-2-Parameter analysis among 18 analyzed sequences was 0.37, which supports well the generic status for Heterovesicula. The obtained phylogenetic trees suggest that H. cowani is related to the Vairimorpha necatrix group, but not to other insect microsporidia producing octospores.

New insect system for testing antibiotics.

New and efficient methods to screen antibiotics are needed to counter increased antibiotic resistance in pathogens and the emergence of new diseases. Here we report a new insect model for screening antibiotics in vivo using the grasshopper Romalea microptera. The system is inexpensive, efficient, and flexible, avoids animal-welfare problems, and can be used to test against most major pathogenic groups. We employed this system to test 11 commercial antibiotics against a pathogenic Encephalitozoon species (Microsporidia). Oral treatment with fumagillin or thiabendazole significantly reduced pathogen spore counts, whereas spore counts of grasshoppers fed with albendazole, ampicillin, chloramphenicol, griseofulvin, metronidazole, sulfadimethoxine, or tetracycline were not significantly different from the infected controls. Quinine produced a distinct, but nonsignificant, reduction in spores, and streptomycin a nonsignificant increase in spores. Although 2 antibiotics significantly reduced spore counts, in no case was the pathogen totally eliminated. This study demonstrates the validity of this system as a method to screen antibiotics. It also corroborates the difficulty researchers and physicians have had in treating microsporidia infections, and suggests that quinine and related alkaloid compounds should be further examined as possible therapeutic agents against this group of ubiquitous pathogens. In addition, streptomycin and related compounds should be tested to determine if this widely used antibiotic enhances microsporidiosis.

Long-term prevalence of the protists Crithidia bombi and Apicystis bombi and detection of the microsporidium Nosema bombi in invasive bumble bees.

An initial survey in 2009 carried out at a site in northwestern Patagonia region, Argentina, revealed for the first time in South America the presence of the flagellate Crithidia bombi and the neogregarine Apicystis bombi, two pathogens associated with the Palaearctic invasive bumble bee Bombus terrestris. In order to determine the long-term persistence and dynamics of this microparasite complex, four additional collections at the same site (San Carlos de Bariloche) were conducted along the following seven years. Both protists were detected in all collections: prevalence was 2%-21.6% for C. bombi and 1.2%-14% for A. bombi. In addition, the microsporidium Nosema bombi was recorded for the first time in the country in the last two collections, at prevalences of 12.4% and 2.4% and unusually high infection intensities (Average = 6.56 × 107 spores per individual). Due to the exceptional dispersal ability of the exotic B. terrestris, these three multihost pathogens should be considered as potential threats to South American native bumble bees.

Phylogenetic relationships in Dichroplus Stål (Orthoptera: Acrididae: Melanoplinae) inferred from molecular and morphological data: testing karyotype diversification.

The neotropical genus Dichroplus and related genera are characterized by a relatively uniform external morphology and a remarkably divergent male genitalia and hence its taxonomy is controversial. It also shows an extreme karyotypic diversification. In this study we used molecular and morphological characters to test the monophyly of the genus and to evaluate chromosome evolution. Twenty-seven species from Dichroplus and related genera were included in the analysis. Morphological characters refer to the general morphology, male genitalia and female structures. Molecular studies were performed, sequencing part of two mitochondrial genes, cytochrome oxidase I and II. Independent and combined phylogenetic analyses of the data were performed under maximum parsimony. The karyotypic characters (rearrangements) were either mapped onto the combined topology or combined with the other data sets. While the molecular analysis confirms some results attained with morphology, some others do not. All point towards the paraphyly of the genus. Our results show the relevance of morphological data in phylogenetic studies because morphology and molecules supply complementary evidence. The mapping of chromosome characters on the combined tree shows that the most extreme karyotype, in D. silveiraguidoi, is a derived condition, probably reached through several centric fusions, and that X-autosome centric fusions were recurrently fixed during the evolution of the group.

The tracheal mite Locustacarus buchneri in South American native bumble bees (Hymenoptera: Apidae).

As in other regions of the world, bumble bees (Bombus spp.) are important pollinators in the neotropics. Despite its relevance, knowledge on their health is still limited in the region. While external acari are known to occur in these insects, presence of the internal, tracheal mite Locustacarus buchneri is here reported for first time. After the examination of 2,508 individuals of eight Bombus species from Argentina, two workers of Bombus bellicosus and one of Bombus atratus were found parasitized by L. buchneri in localities within San Luis and Buenos Aires provinces, respectively. The rare occurrence recorded agrees with findings from elsewhere in the world.

Genetic variability of the neogregarine Apicystis bombi, an etiological agent of an emergent bumblebee disease.

The worldwide spread of diseases is considered a major threat to biodiversity and a possible driver of the decline of pollinator populations, particularly when novel species or strains of parasites emerge. Previous studies have suggested that populations of introduced European honeybee (Apis mellifera) and bumblebee species (Bombus terrestris and Bombus ruderatus) in Argentina share the neogregarine parasite Apicystis bombi with the native bumblebee (Bombus dahlbomii). In this study we investigated whether A. bombi is acting as an emergent parasite in the non-native populations. Specifically, we asked whether A. bombi, recently identified in Argentina, was introduced by European, non-native bees. Using ITS1 and ITS2 to assess the parasite's intraspecific genetic variation in bees from Argentina and Europe, we found a largely unstructured parasite population, with only 15% of the genetic variation being explained by geographic location. The most abundant haplotype in Argentina (found in all 9 specimens of non-native species) was identical to the most abundant haplotype in Europe (found in 6 out of 8 specimens). Similarly, there was no evidence of structuring by host species, with this factor explaining only 17% of the genetic variation. Interestingly, parasites in native Bombus ephippiatus from Mexico were genetically distant from the Argentine and European samples, suggesting that sufficient variability does exist in the ITS region to identify continent-level genetic structure in the parasite. Thus, the data suggest that A. bombi from Argentina and Europe share a common, relatively recent origin. Although our data did not provide information on the direction of transfer, the absence of genetic structure across space and host species suggests that A. bombi may be acting as an emergent infectious disease across bee taxa and continents.

Apicystis bombi (Apicomplexa: Neogregarinorida) parasitizing Apis mellifera and Bombus terrestris (Hymenoptera: Apidae) in Argentina.

The neogregarine Apicystis bombi is considered a low prevalence parasite of Bombus spp. Before our work it has only once been detected in one single specimen of the Western honeybee Apis mellifera. This contribution reports the presence of A. bombi parasitizing both A. mellifera and Bombus terrestris at a site in Northwestern Argentine Patagonia (Bariloche, close to the border with Chile) and analyses its possible absence in the Pampas region, the most important beekeeping region of the country. In Bariloche, prevalence of A. bombi in A. mellifera was 7.6% in 2009, and 13.6% in 2010, whereas in B. terrestris it was 12.1%. Infections were not detected in 302 bee hives periodically prospected along 3 years (almost 400 000 honeybee specimens) in the Pampas. Analysis with the probability program FreeCalc2 suggested a possible absence of A. bombi in this area. Because of high virulence showed in several species of Bombus in the Northern hemisphere, A. bombi should be closely monitored in A. mellifera and in native Bombus species or other Apidae.

A new Encephalitozoon species (Microsporidia) isolated from the lubber grasshopper, Romalea microptera (Beauvois) (Orthoptera: Romaleidae).

We describe a new microsporidian species, Encephalitozoon romaleae n. sp., isolated from an invertebrate host, the grasshopper Romalea microptera, collected near Weeks Island, Louisiana, and Jacksonville, Florida. This microsporidian is characterized by specificity to the gastric caecae and midgut tissues of the host and a life cycle that is nearly identical to that of Encephalitozoon hellem and Encephalitozoon cuniculi. Mature spores are larger (3.97 x 1.95 microm) than those of other Encephalitozoon species. Polar filament coils number 7 to 8 in a single row. Analysis of the small subunit (SSU) rDNA shows that E. romaleae fits well into the Encephalitozoon group and is a sister taxon to E. hellem. This is the first Encephalitozoon species that has been shown to complete its life cycle in an invertebrate host.

Establishment of Liebermannia dichroplusae n. comb. on the basis of molecular characterization of Perezia dichroplusae Lange, 1987 (Microsporidia).

Perezia dichroplusae Lange, 1987 is a parasite of the Malpighian tubules of an Argentine grasshopper, Dichroplus elongatus (Orthoptera, Acrididae, Melanoplinae). In order to determine relationships of this microsporidium with Perezia nelsoni and with other microsporidia, we sequenced its small subunit ribosomal RNA gene (SSU rDNA) (GenBank Accession No. EF016249) and performed phylogenetic analysis of the novel sequence against 17 microsporidian SSU rDNA sequences from GenBank, using neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) methods. This analysis revealed the highest similarity (96%) of the new sequence to Liebermannia patagonica, a parasite of gut epithelium cells of another grasshopper from Argentina, versus only 65% similarity to P. nelsoni, a parasite of muscles of paenaeid shrimps. In phylogenetic trees inferred from SSU rDNA sequences, the microsporidium from D. elongatus is sister taxon to L. patagonica and both cluster with Orthosomella operophterae. At the higher hierarchical level, the Liebermania-Orthosomella branch forms a clade with the Endoreticulatus-Cystosporogenus-Vittaforma group and with Enterocytozoon bieneusi. Perezia nelsoni falls into another large clade together with Nosema and Ameson species. We propose transferring P. dichroplusae to the genus Liebermannia and creating a new combination Liebermannia dichroplusae n. comb., based both on SSU rDNA sequence analysis and on common characters between P. dichroplusae and L. patagonica, which include the presence of elongated multinuclear sporonts, sporoblastogenesis by a similar process of sequentially splitting off sporoblasts, ovocylindrical spores of variable size, tissue tropism limited to epithelial cells, Orthoptera as hosts, and geographical distribution of hosts in the southern temperate region of Argentina. We argue that the condition of the nuclei in spores (i.e. diplokaryotic in L. patagonica or monokaryotic in L. dichroplusae) cannot be used to distinguish genera. Therefore, we remove the statement about the presence of diplokaryotic spores from the revised diagnosis of the genus Liebermannia.

Development, ultrastructure, natural occurrence, and molecular characterization of Liebermania patagonica n. g., n. sp., a microsporidian parasite ot the grasshopper Tristira magellanica (Orthoptera: Tristiridae).

A new microsporidium, Liebermannia patagonica n. gen., n. sp., is described from midgut and gastric caecum epithelial cells of Tristira magellanica, an apterous grasshopper species of southern Patagonia, Argentina. L.patagonica is diplokaryotic, apansporoblastic, homosporous, and polysporoblastic. Transitional (from merogony to sporogony) stages and sporonts of L. patagonica were surrounded by host rough endoplasmic reticulum. The ovocylindrical spores measured 2.9 +/- 0.09 x 1.2 +/- 0.04 microm (fresh, n = 50), and they had an isofilar polar filament of only three coils and a cluster of tubules instead of a classical posterior vacuole. Prevalence was high (up to 80.6%) at the type locality for the four years sampled . Maximum likelihood , neighbor joining, maximum parismony analyses of the small submit rDNA all placed L.patagonica(Accession No. DQ 239917) in one with Orthosomella operophterae.