Attini; Formicidae: Hymenoptera) and their fungal cultivars . In recent years, this mutualism has emerged as a model system for studying coevolution, speciation, and multitrophic interactions. Ubiquitous in this ant-fungal symbiosis is the “weedy” fungus Escovopsis , known only as a mycoparasite of attine fungal gardens. Despite interest in its biology, ecology and molecular phylogeny—noting, especially, the high genetic diversity encountered—which has led to a steady flow of publications over the past decade, only two species of Escovopsis have formally been described.Symbiotic relationships modulate the evolution of living organisms in all levels of biological organization. A notable example of symbiosis is that of attine ants  of nests of the leaf-cutting ant genus Escovopsis and its relatives associated with nests of both the lower and higher Attini throughout their neotropical range, as suggested in previous studies.The present study makes a start at assigning names and formal descriptions to these specific fungal parasites of attine nests. Based on the results of this exploratory and geographically-restricted survey, we expect there to be many more species of the genus  There is increasing evidence that actual and predicted levels of biodiversity in the kingdoms of life have been greatly underestimated, especially in the Kingdom Fungi Ophiocordyceps unilateralis (Tul.) Petch sensu lato Camponotini could be parasitized by a unique species of the O. unilateralis clade O. unilateralis complex, each associated with a different species of carpenter ant (Camponotus) in Thailand Attini in the same fragment of Atlantic rainforest.Currently, we are assessing the diversity of fungal parasites associated with ant societies in a fragment of the highly-threatened Atlantic rainforest in the south-eastern region of the Brazilian state of Minas Gerais. Our work, thus far, has revealed an unexpectedly high level of speciation in the zombie-ant fungus Acromyrmex, Atta), the mutualistic cultivars in the fungal gardens are highly derived species of the basidiomycete genus Leucoagaricus Escovopsis  In insect societies, leaf-cutting ants are unique in their habit of harvesting fresh vegetation. This serves as a substrate for the gardens in which the ants tend their fungal cultivars, the main food source of the colonies. These fungal gardens, built in subterranean chambers, provide a stable habitat with few competitors. Such highly evolved traits have made leaf-cutting ants the dominant herbivores in the Neotropics, according to Hölldobler and Wilson Escovopsis externally, on their cuticles, or internally, in the incipient mutualistic fungal pellet that provides nourishment during the claustral founding stage of the colonies, it has been concluded that transmission of the mycoparasite between leaf-cutting ant colonies is horizontal Although frequently found in leaf-cutting ant colonies Escovopsis was erected by Muchovej and Della Lucia in 1990 to replace Phialocladus Kreisel (1972), which they considered to be an invalid genus since it had been described without designating a holotype species Escovopsis weberii Muchovej & Della Lucia Index Fungorum as E. weberi, presumably because the original name was an orthographic error. The authors assumed this to be identical to Phialocladus zsoltii Kreisel, isolated from a fungal garden of Atta insularis Guérin in Cuba P. zsoltii being signficantly larger than those of E. weberiPhialocladus is a nomen invalidum—and that this warranted a name change of both genus and species—they stressed that the sporogenous cells are not phialidic, and, therefore, that “it is troublesome to validate the name Phialocladus since it would be understood from the root of the name that sporogenesis is phialidic” The genus E. aspergilloides Seifert, Samson & Chapela—it was concluded that: “We find no reason to believe that the conidiogenous cells of Escovopsis species are not phialides” Aspergillus-like vesicles, rather than the clavate to cylindrical, phialide-bearing vesicles of the type species. There may be some counter claims, therefore, for resurrecting the genus Phialocladus which was illustrated beautifully by Carmichael et al. Escovopsis is now in such common usage—especially in the many disciplines involved in studying leaf-cutting ants—the name is conserved here.On describing a second species in the genus—E. weberi and E. aspergilloides vesicle forms, based on material from Brazil E. weberi’ as the dominant (“starke” or ‘strong’) conidial form associated with Atta nests and ‘E. aspergilloides’ as exclusive to colonies of the lower Attini genus Apterostigma (“Haarameisen” or ‘hairy ant’). Similarly, nearly 50 years later, Stahel and Geijskes (1941) also illustrated this distinctive but still unnamed fungal genus overgrowing a colony of Atta sexdens in Surinam Trachymyrmex septentrionalis (McCook) Escovopsis might be important symbionts in the fungal gardens of leaf-cutting ants, but they do not represent anamorphs of the associated symbiotic basidiomycetes” Attini nests, it was not classified taxonomically until almost 80 years after its first description Much earlier, however, Moeller in 1893 provided the first descriptions and illustrations of both the Attini—and thus of their ecological and evolutionary significance—and, in particular, of the discovery that Escovopsis is a coevolved pathogen of the fungal gardens HypocrealesEscovopsisEscovopsis from attine nests in Panama alone, but these have never been described, and publications since then have confirmed the high genetic diversity amongst isolates of Escovopsis from fungal gardens Attini and each may also interact differently with their host. The present study makes a start at assigning names and formal descriptions to at least a small number of these taxa isolated from the fungal gardens—as well as from the garden waste or middens—of ants of the genus Acromyrmex collected during exploratory surveys in the Atlantic rainforest region of Minas Gerais, Brazil. It also highlights morphological features of the fungi that provide insights into their strategies of host exploitation, as well as their dispersal between hosts.There has been increasing awareness about the impact of mycoparasites on nests of the Escovopsis and two distinct forms can be delimited: the E. weberi-type with clavate-cylindrical vesicles ”—have meant that this is no longer a requirement. Nevertheless, it is recommended that a diagnosis (a short statement on how the new taxon differs from related taxa), which may be in Latin or English, should still precede the main description Escovopsis moelleri H.C. Evans & J.O. Augustin sp. nov.MycoBank: MB800440GenBank: JQ815077; JQ855712–JQ855715Escovopsis.Etymology: Named in honour of A.F.W. Moeller who pioneered the work on fungal gardens of ants and provided the first illustrations of Escovopsis weberi, in spore morphology—conidia of E. moelleri are much larger and heavily ornamented—and from E. aspergilloides in the clavate to cylindrical vesicles, and from both by DNA sequences.Differs from the type species, J.O. Augustin & H.C. Evans, isolated from fungal garden of Acromyrmex subterraneus molestans Forel .Type: BRAZIL. Minas Gerais: Viçosa, Mata do Paraíso, 700 m, 9 Mar 2010, Colonies fast-growing, 5–6 cm diam after 7 days on PCA at 25°C, covering a 9-cm diam plate within 14 days; greyish-white becoming brown as conidia mature; but slower, more compact growth on OA   : those oapically . Other sEscovopsis weberi produces similarly-shaped vesicles, which are more cylindrical, less variable in form and longer (43–58×11–14 µm) than E. moelleriE. weberi was described as slow, but with no quantitative data Escovopsis microspora H.C. Evans & J.O. Augustin sp. nov.MycoBank: MB800442GenBank: JQ815076; KF293284Etymology: With reference to the small conidia.Escovopsis moelleri and E. weberi by the similarly ornamented but smaller conidia; and from both by DNA sequences.Distinguished from J.O. Augustin & H.C. Evans, isolated from fungal garden of Acromyrmex subterraneus molestans .Type: BRAZIL. Minas Gerais: Viçosa, Mata do Paraíso, 700 m, Apr 2010, Colonies fast-growing, on PCA and OA attaining 4.5–5.5 cm diam after 7 days at 25°C, relatively low with sparse aerial mycelium; khaki brown centrally with a distinct snow-white periphery  needle-like neck, 1–3 µm in length   .E. moelleri, such as the absence of rhizoids, presence of brown exudate and the distinctive white and even periphery around the colony edge of E. microspora  revealed that this description is erroneous, being based on immature spores, and that spore wall pigmentation and ornamentation develop later: a characteristic shared by all species of the genus due to the development of a distinct outer wall or mucilaginous sheath .Type: BRAZIL. Minas Gerais: Viçosa, Mata do Paraíso, 700 m, Apr 2010, Colonies on PCA and OA very slow growing, no recordable growth after 7 days  18–28 µm diam  µm, with a short, abrupt, spike-like neck; aberrant structures also produced on some vesicles, extending to form ampulliform to cylindrical phialides with long tapering necks. Conidia in basipetal chains, ovoid to subglobose, 3.0–4.0×2.0–2.5 µm; becoming brown and echinulate to verruculose, older conidia with a loose, dark outer covering or veil .BRAZIL. Minas Gerais: Viçosa, Mata do Paraíso, Jan 2011, A. subterraneus subterraneus. The conidia of the new species are slightly larger than E. aspergilloides (2.5–3.7×1.5–2.0 µm). However, as with E. weberi, there are discrepancies concerning conidial descriptions; in the original diagnosis of E. aspergilloidesEscovopsis spores–pigmentation and ornamentation–had been overlooked since these were found when the type of E. aspergilloides (CBS 423.93) was examined in this study.The paratype is slightly faster growing, 1.1–1.3 cm diam after 14 days; occasionally producing intercalary chlamydospores, globose (10–15 µm) to subglobose (15×10 µm). This species is also common on the middens of Escovopsioides H.C. Evans & J.O. Augustin gen. nov.MycoBank: MB800474Escovopsis in producing a brush-like anamorph with phialides on well-defined vesicles.Etymology: In reference to similarities with the genus Escovopsis by: the absence of pigmentation; the lageniform phialides produced on terminal and intercalary, globose vesicles; the hyaline, smooth conidia in long chains, and the DNA sequences.Differs from Escovopsioides nivea H.C. Evans & J.O. Augustin sp. nov.Type species: MycoBank: MB800475GenBank: JQ815078; JQ855713–JQ855716Etymology: In reference to the characteristic snow-white colonies both in culture and on the fungal gardens and middens.H.C. Evans, growing over and isolated from midden of Acromyrmex subterraneus subterraneus .Type: BRAZIL. Minas Gerais: Viçosa, Mata do Paraíso, 700 m, Jan 2011, Colonies on PCA and OA growing rapidly, up to 6–7 cm diam after 7 days at 25°C and reaching the edge of a 9-cm diam plate within 10 days; white, cotton-like aerial mycelium with abundant production of chains of chlamydospores s. l., uniting into snow-white, silvery ropes or strands  µm in length, 5 µm diam at base narrowing towards apex before swelling into a globose vesicle, 8–10 µm diam, often proliferating terminally to produce several intercalary vesicles in succession  on vesicles, but also singly on discrete lateral swellings  5–8×(1.5-) 2–3 µm, ending abruptly in or tapering to a truncate base .BRAZIL. Minas Gerais: Viçosa, Mata do Paraíso, Apr 2010, Escovopsis, apart from producing phialides on vesicles. Phialide form and conidial morphology, especially the long chains  Arx. We refer to the swollen spores produced in monilioid chains as chlamydospores s. l., because these are not thick-walled perennating cells produced endogenously Escovopsis  isolate from Cuba, as “makroconidien”, which were not found in the supposed “cospecific” type from Brazil As mentioned previously, the first mycologist to observe this fungus was Moeller in 1893 and his monograph presents accurate drawings of the new genus, including all three spore types ains see , share mLSU (ITS (EF1-αwww.treebase.org). The LSU tree  with the novel genus, represented by Escovopsioides nivea, as a distantly related sister genus. Escovopsis and Escovopsioides are included in the Hypocreaceae with a posterior probability (PP) value of 1 and the parsimony bootstrap (PB) value of this Hypocreaceae clade was 62%. However, the sister relationship amongst Escovopsioides and the Escovopsis-clade and “Hypocrea/Trichoderma”-clade has a PP value of only 0.63 and this association is not resolved in the parsimony bootstrap. Nevertheless, the Escovopsis clade itself (excluding Escovopsioides) is supported by PP = 1 and PB = 100%. For LSU, the Bayesian trees identified—with high posterior probability support—two sub-groups within the Escovopsis clade made up of Escovopsis weberi-like isolates, comprising E. weberi and E. microspora; and, Escovopsis aspergilloides-like isolates, comprising E. aspergilloides and E. lentecrescens; and, finally, placement of E. moelleri intermediate between these two groups. A similar association between E. weberi and E. microspora was observed in the ITS phylogeny  nest in Brazil) but which in fact could represent an additional species of Escovopsioides. Megablast searches of NCBI's GenBank nucleotide database using the obtained EF1-α sequences yielded no high identity hits with other sequences in GenBank but did reveal significant similarity (up to 97% identity) with other Escovopsis sequences or species of Hypocreales, confirming the affinity of our sequences with related sequences in GenBank. However, preliminary phylogenetic analyses using two different lengths of these sequences  and consistent with previous reported patterns of high incidence of Escovopsis among Acromyrmex ants E. lentecrescens was isolated in only 8% of the 25 samples. This may be due to its slow-growing habit, enabling faster-growing species to outcompete E. lentecrescens in the isolation plates. Conversely, the incidence of Escovopsioides nivea was high, and reached 72% of the 25 sub-colonies sampled.Infection of fungal gardens by Escovopsis among the leaf-cutting ants from Central America can vary between 42.9–51.4%, out of 49 and 29 colonies of Atta and Acromyrmex, respectively Acromyrmex species were positively sampled for EscovopsisE. weberi was found in 42% of 20 sub-colonies of A. sexdens rubropilosa Forel from south-east Brazil Syncephalastrum J. Schröt. and Trichoderma Pers.—came out from single garden pieces (data not shown). This means that ‘stronger’ fungal isolates probably outcompeted ‘weaker’ ones with the methodology used, which may have underestimated fungal diversity, as well as the incidence of species such as the slow-growing E. lentecrescens, for example. This species may be a more prevalent symbiont in the in vivo system—perhaps using other competitive strategies, such as antibiosis—but a poor competitor in vitro. It also suggests that a more intricate coevolutionary history exists between E. lentecrescens and Leucoagaricus, so that the parasite is nutritionally more dependent on its fungal host. Alternatively, fast-growing species, such as Escovopsioides nivea, could have been favored by the sampling method used in this study, essentially, based on a single, nutrient-rich medium. Potentially, therefore, the use of selective media could reveal an even higher diversity of Escovopsis-like mycoparasites.Previous work has shown that infection rates of Escovopsis strains and in vitro bioassays showed no apparent antagonistic interaction between them Escovopsis and Escovopsioides species isolated from individual Acromyrmex colonies in our study also did not appear to inhibit each other, and we observed that up to three different Escovopsis species emerged from individual garden pieces on agar . In addition, the four new species described here showed no apparent antagonistic interactions in dual cultures .It has previously been shown that individual colonies of leaf-cutting ants from Central America harbor genetically different Escovopsis and attine ants Genetic studies have demonstrated the long evolutionary history of E. moelleri, for example, the mature conidia become heavily pigmented with distinctly rugose walls and a well-defined apical cap  24–45 (-50)×8–16 (-18) µm; conidia ovoid to ellipsoidal, 3.5–4.5×2.5–3.5 µm, darkening with age due to a densely pigmented outer wall or sheath , differing from the above; whilst the vesicles are longer and slightly narrower (43–58×11.5–14 µm) than those reported here. Moreover, the growth rate cannot be described as slow (see E. microspora with a similar colony form grows faster (E. weberi (CBS 810.71) covered a 9-cm plate within 5 days at 25°C.Note: This re-description is based on the type culture deposited in the American Type Culture Collection (ATCC 64542), said to be isolated from “Carpenter ant fungal mass”, although this differs from the original publication which states that the fungus was isolated “during biosociological studies of slow see —E. micros faster —but not Escovopsis aspergilloides: Conidia ellipsoidal, 2.5–3.5×1.5–2 µm, becoming pale to tan brown with an outer pigmented wall or sheath (r sheath .Note: The above is based on an examination of the type culture (CBS 423.93) as described by Seifert et al. Escovopsioides nivea—characterized by its non-pigmented conidiophores and vesicles, producing relatively few phialides and hyaline, thin-walled spores in long chains—has no obvious phoretic or dormancy traits. The role of the phialoconidia, therefore, in the fungal life-cycle remains obscure. However, could the functions of horizontal dispersal and dormancy have been transferred to the other two spore forms? Intriguingly, most of the fungal resources appear to be channeled into producing the snow-white ropes or chains of chlamydospores s. l. over the fungal gardens in decline  have been found. It is possible that the teleomorph has been lost from the life-cycle, once the fungi entered and adapted to the attine ant symbiosis. However, this is considered unlikely given that genetic variation helps parasites keep up with their hosts over evolutionary time scales Escovopsis within the Hypocreales (Ascomycota) Bionectriaceae and Nectriaceae) to animal and fungal-based (Clavicipitaceae sensu lato and Hypocreaceae) Escovopsis lies within the Hypocreaceae and is closely related to mycoparasites such as Hypomyces, Hypocrea and Trichoderma . No endangered or protected species were involved in the studies. No permits were required for the described study, which complied with all relevant regulations. The subject of the photograph included in Acromyrmex: A. subterraneus molestans Santschi, A. subterraneus subterraneus Forel and A. niger F. Smith, between March and May 2010 at sites in the hosts' sympatric range in the Atlantic rainforest of Minas Gerais (Zona da Mata Mineira): all located in the municipality of Viçosa, within the campus of the Universidade Federal de Viçosa or in a nearby forest reserve (Mata do Paraíso), 20°44′31.71″S–42°52′43.83″W, 650–700 m a.s.l., belonging to the university. Acromyrmex nests were chosen because they are abundant and easily accessible in the study area, being located relatively close to the soil surface  from each sub-colony were transferred to plates containing potato dextrose agar (PDA) Escovopsis emerged from a garden piece, which typically occurred within 4 days of initial isolation, the colony was scored as infected. Escovopsis mycelium was then subcultured on potato carrot agar (PCA) Escovopsis-like overgrowths were observed  from three species of leaf-cutting ants in the genus  surface –C, or buobserved .Colony morphology and growth rates of isolates were compared on oatmeal agar (OA), PDA,PCA, as well as on malt extract agar (MA) Specimens were deposited in the IMI Culture Collection , as well as in the CBS-KNAW Fungal Biodiversity Centre  culture collection , and permanent slides were also deposited in Herb IMI . Isotype collections are held in Herbarium VIC .The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: .In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix 2PO4, 0.1 g MgSO4.7H2O, 0.44 mg ZnSO4.7H2O, 0.48 mg FeCl3.6H2O and 0.36 mg MnCl2.H2O) for 5 days at 26°C on a shaker (170 rpm). The resulting mycelium was washed out with distilled water and placed on sterile filter paper to dry. DNA extraction followed a CTAB extraction protocol modified from Doyle & Doyle Fungi were grown in Erlenmeyer flasks containing 100 mL of liquid medium , LSU rDNA  and part of the second half of EF1-α  were conducted with primers ITS1-F (CTTGGTCATTTAGAGGAAGTAA) TCCTCCGCTTATTGATATGC) 5′ GCATATCAATAAGCGGAGGA 3′), CLA-R (5′ GACTCCTTGGTCCGTGTTTCA 3′) 5′ GCYCCYGGHCAYCGTGAYTTYAT 3′), EF1-2218R (5′ GACTTGACTTCRGTVGTGAC 3′) Amplification of PCR products of three genomic regions, All PCR reactions were performed in a total volume of 50 µL and contained 20 to 100 ng of genomic DNA, with buffer 1× ; 1.5 mM MgCl2; 0.2 µM of each dNTP; 1 U of Taq DNA polymerase and 0.2 µM of the relevant primer. All reactions were done in a MJ Research PTC 100 thermocycler. For the ITS regions, PCR conditions were as follow: 5 min of denaturation at 95°C, followed by 30 cycles consisting of 30 s at 95°C, 30 s at 60°C and 90 s at 72°C and finally 10 min of extension at 72°C. The CLA reactions were done starting with 2 min of denaturation at 95°C, followed by 40 cycles consisting of 30 s at 95°C, 60 s at 62°C, 90 s at 72°C and finally 5 min of extension at 72°C. Meanwhile, the EF1-α reactions started with 2 min of denaturation at 95°C, followed by 40 cycles consisting of 30 s at 95°C, 60 s at 60°C and 90 s at 72°C and then 5 min of extension at 72°C. PCR products were then purified using minicolumns according to the manufacturer's protocols (Roche-High Pure PCR Product Purification Kit).5′ GGAAGTAAAGTCGTAACAAGG 3′) 5′ TCCTCCGCTTATTGATATGC 3′) 5′ GTACCCGCTGAACTTAAGC 3′) 5′ TCCTGAGGGAAACTTCG 3′) and EF1-728F (5′ CATCGAGAAGTTCGAGAAGG 3′) 5′ GGARGTACCAGTSATCATGTT 3′) 2; 0.1 µM of each dNTP; 0.5 U of BioTaq DNA polymerase and 0.2 µM of the relevant primer. PCR conditions were as follow: 5 min of denaturation at 94°C, followed by 40 cycles consisting of 45 s at 94°C, 30 s at 48°C and 90 s at 72°C and finally 6 min of extension at 72°C. The annealing temperature was increased to 52°C for EF1-α. Amplification products were sequenced directly without post-PCR cleanup.Amplification of PCR products of three genomic regions, ITS, LSU and part of the first half of EF1-α were conducted with primers ITS5 . Representatives from five families , as well as the type strain sequences of E. weberi and E. aspergilloides, were sampled for a total of 49 hypocrealean taxa, excluding one Glomerella cingulata isolate of the Phyllachorales for rooting the Hypocreales  and therefore the ITS phylogeny was focused on Escovopsis and Escovopsioides.Sequencing for all strains was carried out directly from purified PCR-amplified products using the automatic sequencer ABI Prism 3100. In order to determine the generic placement of the ales see . GenBankhttp://www.ebi.ac.uk/clustalw) MEGA 5 All sequences were aligned using ClustalW  and likelihood ratio test implemented in ModelTest 3.7 Figure S1Escovopsioides nivea. (A–D) Details of spore forms in paratype variant that initially produced only the chlamydospore sensu lato stage (D); Other spore forms develop intermittently, often producing solitary, lateral phialides . All scale bars = 10 µm.(TIF)Click here for additional data file.Figure S2Images supporting hypotheses relating to fungal transmission. (A)–(B) Structures found in ant-fungal gardens: (A) Ropes of iridescent chlamydospores sensu lato of Escovopsioides nivea over-growing the fungal garden of an abandoned attine nest; (B) Close-up of chlamydospores sensu lato of Escovopsioides nivea within garden; (C) Endophytic ability: Escovopsis microspora emerging from surface-sterilized leaf of privet —after 7 days on tap water agar—inoculated 2-months previously with conidia of E. microspora ; (D) Gongylidia of Leucoagaricus symbiont within garden.(TIF)Click here for additional data file.Figure S3Fungal sampling, Mata do Paraíso, Viçosa, Minas Gerais, Brazil. (A) Sampling of Acromyrmex nest—note, to the right, the mattock used for excavation and the nest entrance (arrow)—to expose the healthy (B) or diseased (C) fungal garden; (D) Close-up of ‘infected’ middens with blooms of Escovopsis microspora around periphery (arrows), inset (E) with detail of sporulation onto surrounding litter.(TIF)Click here for additional data file.Figure S4Distance analysis using neighbor joining with the HKY85 substitution model on 264 aligned EF1-α sequences spanning 716 characters downloaded from GenBank. A parsimony analysis on the same dataset was limited to 1000 equally most parsimonious trees and the derived consensus tree had the same overall topology for the terminal clades compared to the presented distance tree . However, the more basal nodes and the overall backbone structure changed between the different analyses. In both of these analyses, lineages within families were interspersed between families and therefore the clustering presented here is not an artifact of the analysis algorithm. Distance-based bootstrap support values are shown at the nodes and thickened branches indicate the parsimony bootstrap support values .(RAR)Click here for additional data file.Figure S5Distance analysis using neighbor joining with the HKY85 substitution model on 267 aligned EF1-α sequences spanning 373 characters downloaded from GenBank and including the novel species (in bold face) described in this study. A parsimony analysis on the same dataset was limited to 1000 equally most parsimonious trees and the derived consensus tree had the same overall topology for the terminal clades compared to the presented distance tree . However, the more basal nodes and the overall backbone structure changed between the different analyses. In both of these analyses, lineages within families were interspersed between families and therefore the clustering presented here is not an artifact of the analysis algorithm. Distance-based bootstrap support values are shown at the nodes and thickened branches indicate the parsimony bootstrap support values .(RAR)Click here for additional data file.Table S1Sources of fungal species and GenBank sequences for which novel sequences were generated in this study.(PDF)Click here for additional data file.Table S2Escovopsis and Escovopsioides species isolated from individual Acromyrmex colonies. MB = Mata da Biologia (UFV campus); Rep = Represa UFV (UFV campus); MP = Mata do Paraíso.Number of (PDF)Click here for additional data file.


Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a very rare form of skin lymphoma that is localized primarily to the subcutaneous adipose tissue without palpable involvement of the lymph nodes. Diagnosis of SPTCL is a challenge, especially during its early phases when symptoms mimic other, more common conditions, such as benign panniculitis, eczema, dermatitis, psoriasis and cellulitis. Clinical and systemic features are nonspecific and can include fever, chills, and weight loss. Further complicating diagnosis is the high number of false negatives provided by biopsy. Here we present a case of SPTCL that illustrates the full course of the disease, from presentation and multiple misdiagnoses to correct disease recognition and successful treatment. A review of the challenges of diagnosis is provided with recommendations for more accurate and timely recognition of SPTCL. Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a very rare form of skin lymphoma that is localized primarily to the subcutaneous adipose tissue without palpable involvement of the lymph nodes. It was first described in 1991 in an 8-case series . It is eHere we describe a case of a middle-aged woman who presented with recurring fevers of unknown origin and an 8-month history of thigh and leg swelling and rash. After several in-patient and out-patient visits, she was diagnosed with SPTCL based on clinical history and results of surgical pathology. Challenges of diagnosis are discussed.A 44-year-old morbidly obese female presented to the emergency department with fever of unknown origin (101.8°F at admission) and chills for 10 days that were unresponsive to antibiotics . She had a history of bulimia, depression, anemia, gastritis, hypertension, and rheumatic fever. She also had chronic skin lesions on the left lower lateral thigh and left upper calf for eight months but reported increased pain in those areas with development of yellow discharge, light bleeding, and numbness over the past 10 days. The rash showed multiple areas of activity and remission presenting as spreading rings . NumerouStreptococcus agalactiae. Deep tissue biopsy was performed. HIV and Lyme tests were negative. There was progressive worsening of neutropenia (WBC = 2.4 K/uL) and LFT . Hepatitis B and C tests were negative. Fever improved with antibiotics and patient was discharged home on oral levofloxacin. At admission, labs were within normal limits with the exception of low WBC  and abnormal liver function tests (LFT) . Vancomycin and Zosyn were started. Wound culture was positive for Pantoea Agglomerans. Due to clinical presentation and positive culture results, infectious etiology was suspected. A four-week course of ciprofloxacin was prescribed.Results of biopsy of left upper thigh showed interface/lichenoid dermatitis with granulomatous features and lobular fat necrosis. Left lower leg biopsy showed similar, but more subtle, results. At this time, the pathologist differential diagnoses included collagen vascular disease , foreign body reaction/unusual drug eruption, sarcoidosis, and Kikuchi's disease. Culture from biopsy grew Three months later, the patient was readmitted with periodic high fever, chills, nausea, night sweats, and vomiting. She continued to be neutropenic (WBC = 2.9) with slightly elevated LFT . Lesions were grossly unresponsive to previous antibiotics. Wound cultures and cancer antigen 125 were normal. After excisional biopsy from the left lower leg and the left groin area, including a lymph node, she was discharged home on antibiotics. The biopsy samples were submitted to an outside lab for review. Surgical pathology for the left lower leg was consistent with mild perivascular small lymphocytic infiltrate without specificity. On low-power field, results for the left groin area sample showed extranodal fibroadipose tissue containing a lymphoid infiltrate with a pattern similar to lobular and septal panniculitis (at 10x magnification) . On highαβ. The patient was referred to oncology and began CHOP  therapy regimen.Immunohistochemical stains for the left groin area sample were positive for CD3 and CD8 but negative for CD4 and CD56 . CD4 wasPatient was admitted 5 months later for complicated skin and skin structure infection (CSSSI) of the lower abdomen and mons pubis, right groin, and upper thigh, due to culture-proven MRSA. The areas that had previously showed signs of SPTCL were responding very favorably to CHOP therapy . CSSSI wαβ, is characterized by an indolent, protracted course and is usually CD4−, CD8+, and CD56−. The second, TCRγδ, is associated with rapid clinical deterioration and coexisting hemophagocytosis syndrome (HPS); it is usually CD4−, CD8−, and CD56+ [αβ, whereas TCRγδ is designated as cutaneous gamma/delta positive T-cell lymphoma (Cγδ-TCR) [γδ or Cγδ-TCR, while the individual phenotypes are being used to describe the specific phenotypes. There are two distinct types of SPTCL, classified by the T-cell receptor (TCR) phenotype and immunophenotypic characteristics. The first, TCRγδ-TCL phenotype (45% versus 6% in TCRαβ) [Diagnosis of SPTCL is based on pathological examination of skin and subcutaneous tissue, immunohistochemical staining patterns, molecular analysis, and clinical characteristics. Patients with SPTCL present with nodules or plaques; ulcerations may also develop, but this occurs in a significantly larger proportion of patients with the Cγδ-TCL, involvement of the epidermal/dermal tissue may also exist. Lymphocytes exhibit slight atypical features, including hyperchromatic, angulated nuclei, and indistinct cell borders. Admixed benign histiocytes, plasma cells, and neutrophils are present, mimicking benign panniculitis [As described and illustrated by Parveen and Thompson  and Pauliculitis . Scattericulitis –11. Histologically, SPTCL can be misinterpreted as other, more common diseases and conditions. Rimming of fat cells can be seen in primary and secondary cutaneous lymphomas and in lobular panniculitis . Some ca+ and CD4−. TCRαβ cells are CD8+ and usually CD30− and CD56− whereas Cγδ-TCL is usually CD8−, CD56+, and CD30+ [− B-cells mixed with CD3− cells that are both CD4− and CD8− [+ without CD8+ T cells [+ natural killer-like T-cell lymphoma is both CD30+ and CD56+, and unlike SPTCL, is often positive for Epstein-Barr [αβ, including abnormal lymphoid cells from extranodal tissues that were CD3+, CD4−, CD8+, and CD56−. Additionally, scattered B-cells were CD20+. Taken together, these findings rule-out most differential diagnoses. Immunophenotyping is very helpful for diagnosing SPTCL. The neoplastic cells in SPTCL are cytotoxic T cells that are CD3nd CD30+ . In term T cells . Primaryein-Barr . In our αβ, misidentification of the disease becomes more of a possibility. One differential diagnosis that is of particular interest is Pfeifer-Weber-Christian Disease (PWCD), which is a rare inflammatory disorder of the subcutaneous adipose tissue. PWCD is characterized by recurrent subcutaneous inflammatory nodules paired with systemic symptoms, such as fever and malaise. It has been theorized that it is a T-cell mediated autoinflammatory condition . Since PImaging technology has begun to be utilized in the diagnostic process; it is also being used for monitoring disease response to therapy. Positron emission tomography (PET) scanning may be useful for staging SPTCL and also for monitoring treatment responses while a patient is undergoing chemotherapy . Whole bαβ initially respond well to treatment with steroids, but more than 70% of responders achieve only short remission periods. Further research regarding the use of ciclosporins for treatment of SPTCL is ongoing [α, have been used for a limited number of cases without strong evidence of effectiveness [As described by Go and Wester , lesions ongoing . Exclusitiveness .+ SPTCL [Treatment of aggressive cases of SPTCL with anthracycline-containing chemotherapy has generally had poor outcomes. There has been one report of successful use of purine analog fludarabine in combination with mitoxantrone and dexamethasone (FND) for treating a multifocal and aggressive CD8+ SPTCL ; the pat+ SPTCL . SPTCL iαβ exceeds 80%. The presence of HPS, however, significantly decreases survival rates; the five-year survival rates for TCRαβ without HPS are around 90%, whereas if HPS is present the rate falls to less than 50%. On the other hand, there is rapid clinical deterioration in Cγδ-TCL whether HPS is present or not, with a 5-year survival rate of less than 20% in either group [αβ SPTCL is very rare. Metastasis is more common in Cγδ-TCL, and metastatic sites have included the lungs, liver, kidneys, CNS, and oral mucosa.The overall five-year survival rate for TCRer group , 4. Even SPTCL is a rare disease that can be a challenge to diagnose. When a patient presents with a long history of panniculitis-like lesions that have been unresponsive to topical and antibiotic therapy and are accompanied by nonspecific clinical symptoms, SPTCL should be considered. Neutropenia that does not respond to antibiotics is another clue for diagnosis. Biopsy with a sample deep enough to include subcutaneous tissue is essential; additionally, high power fields to analyze samples should be used in order to make an accurate differential diagnosis. Immunochemistry should be completed since it is the best way to differentiate between SPTCL and other, more common diagnoses, as well as to guide treatment decisions. Delayed diagnosis can lead to worsening of symptoms and higher probability of morbidity, such as severe skin infections.


Studies show that patients with schizophrenia exhibit impaired responses to sensory stimuli, especially at the early stages of neural processing. In particular, patients’ alpha-band (8–14 Hz) event-related desynchronization (ERD) and visual P1 event-related potential (ERP) component tend to be significantly reduced, with P1 ERP deficits greater for visual stimuli biased towards the magnocellular system. In healthy controls, studies show that pre-stimulus alpha  plays a pivotal role in sensory processing and behavior, largely by shaping the neural responses to incoming stimuli. Here, we address whether patients’ ERD and P1 deficits stem from impairments in pre-stimulus alpha mechanisms. To address this question we recorded electrophysiological activity in patients with schizophrenia and healthy controls while they engaged in a visual discrimination task with low, medium, and high contrast stimuli. The results revealed a significant decrease in patients’ ERDs, which was largely driven by reductions in pre-stimulus alpha. These reductions were most prominent in right-hemispheric areas. We also observed a systematic relationship between pre-stimulus alpha and the P1 component across different contrast levels. However, this relationship was only observed in healthy controls. Taken together, these findings highlight a substantial anomaly in patients’ amplitude-based alpha background activity over visual areas. The results provide further support that pre-stimulus alpha activity plays an active role in perception by modulating the neural responses to incoming sensory inputs, a mechanism that seems to be compromised in schizophrenia. Neural activity can exhibit temporally structured patterns in response to external sensory inputs, motor actions, cognitive states, but also to the absence of sensory stimulation during resting state conditions Alpha rhythms can be classified based on their frequency speed , is often computed in the period preceding a cue or target stimulus, and as such, it is not strictly associated with the presentation of a sensory stimulus (unlike the ERD) or an active deployment of attention . Rather, background alpha seems to represent a general state of sustained attention and task engagement, which usually displays a different topographical distribution, relative to phasic-alpha, that is concentrated over central posterior regions Anomalies in alpha-band activity of schizophrenia patients have also been found in stimulus-related responses, with the majority of studies reporting a significant decrease in patients’ alpha ERD In addition to impaired responses in post-stimulus alpha activity, patients with schizophrenia also manifest acute deficits in broadband responses to sensory stimuli. Particularly, studies have shown decreases in patients’ visual P1 event-related potential (ERP) component The goal of this study was to investigate the relationship between background alpha activity, the ERD and visual P1 stimulus-related responses in patients with schizophrenia. While many studies have reported deficits in patients’ background alpha, these impairments have predominately been assessed during resting state conditions. Here, we investigate background alpha-band activity during an active task, but more importantly, we query its role in regulating patients’ sensory processing, including stimuli biased towards the magnocellular pathway.The Nathan Kline Institute and Rockland County Department of Mental Health Institutional Review Boards (IRB) approved the study, and all participants provided written informed consent in accordance with the principles of the Declaration of Helsinki. Consent was obtained by research personnel who were trained and demonstrated competency in understanding the ethical obligations of the informed consent process. The person obtaining informed consent discussed the study with the participant in detail, providing an explanation of the study, its risks, benefits, procedures, what would be required of the participant during the study, and assessed the participant’s comprehension of the material. Participants were included in the study if they had full capacity to consent. Participants were not disadvantaged in any way by not participating in the study.Participants were 28 patients meeting DSM-IV criteria for schizophrenia or schizoaffective disorder and 25 age-matched healthy volunteers. Patients were recruited from inpatient and outpatient facilities associated with the Nathan Kline Institute for Psychiatric Research (NKI). Diagnoses were obtained using the Structured Clinical Interview for DSM-IV (SCID) and other available clinical information. Healthy volunteers with a history of SCID-defined Axis I psychiatric disorder were excluded. In addition, participants with history of any neurological or ophthalmologic disorder that might affect performance, substance dependence within the last 6 months, or abuse within the last month were also excluded. All participants had 20/32 or better corrected visual acuity on the Logarithmic Visual Acuity Chart , and received a moderate fee for their participation. For analyses of the neurophysiology data we excluded a subset of participants due to noisy ERP data . Thus, there were 21 participants per subject-group in all analyses. Clinical and demographic information for these participants are included in Stimuli consisted of faces depicting fearful, happy, sad or neutral expressions from 11 different individuals extracted from the Ekman and Friesen database A typical sequence of events is illustrated in High-density continuous EEG was acquired from 64 surface electrodes arranged geodesically, using the BioSemi Active II system . Data were digitized online at 512 Hz, and recorded relative to a common-reference during acquisition. Data were re-referenced offline to the average of all electrodes. Only EEG data associated with the standard stimuli were analyzed.Analyses of the data were carried out using in-house analysis scripts in Matlab . Data were bandpass filtered (.05–110 Hz) using a standard Butterworth filter, and downsampled to 256 Hz. EEG epochs were derived from −500 to 700 ms relative to the onset of the standard stimulus. A baseline measure defined as −100 to 0 ms was applied to each individual epoch before performing the wavelet time-frequency decomposition method. For each epoch, individual ‘noisy’ time points in a channel were splined-interpolated if the average signal from that channel exceeded ±2 standard deviations from the average of the 3 most-neighboring electrodes. However, if more than 25% of the channel’s data points exceeded this threshold, the whole epoch was reconstructed by spline-interpolating the data from the 3 most-neighboring electrodes. This was done by implementing these steps: (1) averaging the data from the neighboring electrodes, (2) randomly removing 25% of the points, and (3) applying the spline-interpolation method. Only neighboring channels that were below the artifact threshold were used. On average, less than 1% of trials were interpolated in each group. These values were not statistically significant across groups (t(40) = 1.63, p = 0.11). An artifact rejection criterion of ±125 microvolts (μV) was used at all other electrode sites to exclude periods of high EMG and other noise-transients. Trials with eye blinks and large eye movements were rejected offline. This was defined as continuous deviations of at least ±10 μV for more than ∼97 ms on both eye channels relative to a 10 ms baseline period. We excluded a subset of participants (4 controls and 7 patients) because their percentage of accepted trials was below 70%.Instantaneous oscillatory amplitude was characterized by applying a Morlet wavelet decomposition method on single trials Statistical analyses of the instantaneous alpha-power were carried out on the pre-stimulus and post-stimulus time periods of the epoch. Statistical testing was also performed on the ERD, which was derived using Preα and Postα equal the mean activity in alpha during the pre- and post-stimulus period, respectively where Separate mixed-models Analysis of Variance (ANOVA) were conducted for the pre-stimulus, post-stimulus, and ERD alpha periods. Each ANOVA had a between-subjects factor of Group (patient vs. control) and two within-subjects factors of Contrast  and Hemisphere (left vs. right). Post-hoc planned comparison t-tests were conducted when appropriate. Correlation analyses between the CPZ equivalents, symptoms, and the alpha-band measures were computed to examine the relationship between the symptomatology and neurophysiology.Separate analyses were performed to assess whether changes in pre-stimulus alpha lead to modulations in the P1 ERP component and alpha ERD. For each accepted trial, in each participant, the amplitude of the P1 and ERD were sorted with respect to the pre-stimulus alpha amplitude. Pre and post-stimulus alpha outliers consisting of values outside ±2 standard deviations from the mean were removed. The P1 and ERD values were then divided into 20 bins and the values in each bin were averaged to smooth out noisy data. Bin 1 and 20 consisted of P1 or ERD values associated with the 5% smallest and largest pre-stimulus alpha values, respectively. Given that stimuli with different contrasts modulate P1 amplitudes nonlinearly Alpha-band waveform trajectories, collapsed across contrast conditions, are plotted in 1,40 = 5.0, p = .03), with patients having lower pre-stimulus alpha activity relative to controls . Follow-up independent samples t-tests revealed that this effect was largely driven by a greater alpha amplitude difference over the right vs. left hemisphere in controls relative to patients , whereas patients showed a trend for greater alpha power in the left vs. right hemisphere   or Hemisphere . However, the ANOVA revealed a significant Group x Hemisphere interaction . Follow-up paired-samples t-tests revealed that this interaction was driven by patients having greater alpha-band amplitude in the left vs. right hemisphere , such that greater ERD was observed over the right vs. left hemisphere. No significant interactions were observed.There was a significant main effect of Group , demonstrating that patients exhibit an ERD. In summary, these findings support the hypothesis that patients’ ERD deficits arise from lower levels in background alpha, which in turn truncate the range over which alpha can desynchronize before reaching floor levels (We examined whether the Group main effect in the ERD was a result of patients’ inability to produce the ERD effect itself. This was done by computing a one-sample t-test (against zero) on the ERD of patients, averaged across left and right hemispheres. The results revealed a significant effect  and compared them to the ERDs of controls whose top pre-stimulus alpha bin values were not significantly different from the top 30% of patients’ pre-stimulus alpha. This was done by removing the top binned pre-stimulus alpha values of controls until the values between the groups were not significantly different from each other. We then computed independent samples t-tests on the associated ERDs to test for statistical significance. The t-test revealed no significant differences in ERDs across groups , their ERD values are also greater as compared to controls .We performed an additional analysis in which we compared the ERD values of patients vs. controls when the background alpha values of patients were significantly larger than those of controls. This analysis was performed to test whether deficits in patients’ ERD are solely due to a truncation in the range of pre-stimulus alpha levels, or whether they also arise from impairments in gain mechanisms. The assumption is that if patients have higher pre-stimulus alpha levels than controls, they should have a higher range over which alpha can desynchronize, or conversely controls have a lower range over which their alpha can desynchronize. This analysis was performed using a similar method as above. Specifically, each single-trial ERD value in both groups was sorted with respect to pre-stimulus alpha and grouped into 20 different bins. We selected the ERD values associated with the top 30% pre-stimulus alpha values in patients and compared them to the ERDs of controls whose top pre-stimulus alpha bin values were significantly lower than the top 30% of patients’ pre-stimulus alpha. This was done by removing the top binned pre-stimulus alpha values of controls until the values of patients were significantly greater than controls. We then computed independent samples t-tests on the ERDs to test for statistical significance. These data are presented in the right graph of 2,39 = .62, p = .54), nor were the Group x Contrast , or Hemisphere x Contrast  interactions , whereby greater increases in contrast led to greater decreases in alpha-amplitude , with greater contrast resulting in a greater ERD , and 57%  = 28.54, p = 1.51×−710; R2 = .09) contrast conditions and a trend towards significance for the 2% contrast condition  = 3.67, p = 0.056; R2 = .01). In patients, we did not observe an effect for the 2%  = 0.023, p = 0.631; R2 = .0001), 8%  = 2.73, p = 0.0987; R2 = .006) or 57%  = 2.57, p = 0.1091; R2 = .006) contrast conditions.Amplitude values of the P1 ERP component plotted as a function of binned pre-stimulus alpha activity are illustrated in p = 0.01; R2 = .04) and 57%  = 15.59, p = 9.22×−510; R2 = .06) contrast levels, but no effect for the 2% contrast condition  = 1.84, p = 0.175; R2 = .004). These data are illustrated in We further tested whether the relationship between pre-stimulus alpha and P1 amplitude was due to interactions with post-stimulus alpha activity during the P1 timeframe. To test this, the ERP data were bandstop filtered from 8–14 Hz on every trial and the same binning procedure as above was performed. This analysis still revealed a significant relationship between pre-stimulus alpha and P1 amplitudes for the 8%  = 6.62, p = .044; 8%: r = −.49, p = .04; 57%: r = −.53, p = .024).Pearson correlation analysis did not show a significant relationship between any alpha-band measures and CPZ equivalents in patients (p>.2). The Brief Psychiatric Rating Scale (BPRS) total scores were negatively correlated with right-hemisphere ERD across each contrast condition  since patients with schizophrenia exhibit severe deficits in both these rhythms see , but alsSeveral caveats are important to note. First, pre-stimulus alpha and P1 amplitude are both quite low in patients, particularly at the 2% contrast level. Therefore, the lack of correlation between these two indices in patients may reflect reductions in SNR, which in turn may have hindered the likelihood of observing a correlation with our methods. Second, our task required little effortful attention. Thus, further work is necessary to determine whether the background alpha deficits as well as the pre-stimulus alpha/P1 relationship are similarly affected in paradigms that involve more effortful and demanding cognitive states. Third, all patients were receiving medication at the time of testing.


Although previous studies using non- elderly groups have assessed the factorial invariance of the Center for Epidemiological Studies Depression Scale (CES-D) across different groups with the same social-cultural backgrounds, few studies have tested the factorial invariance of the CES-D across two elderly groups from countries with different social cultures. The purposes of this study were to examine the factorial structure of the CES-D, and test its measurement invariance across two different national elderly populations.A total of 6806 elderly adults from China (n = 4903) and the Netherlands (n = 1903) were included in the final sample. The CES-D was assessed in both samples. Three strategies were used in the data analysis procedure. First, a confirmatory factor analysis (CFA) was carried out to determine the factor structures of the CES-D that best fitted the two samples. Second, the best fitting model was incorporated into a multi-group CFA model to test measurement invariance of the CES-D across the two population groups. Third, latent mean differences between the two groups were tested.The results of confirmatory factor analysis (CFA) showed: 1) in both samples, Radloff's four-factor model resulted in a significantly better fit and the four dimensions  of the CES-D seem to be the most informative in assessing depressive symptoms compared to the single-, three-, and the second-order factor models; and 2) the factorial structure was invariant across the populations under study. However, only partial scalar and uniqueness invariance of the CES-D items was supported. Latent means in the partial invariant model were lower for the Dutch sample, compared to the Chinese sample.Our findings provide evidence of a valid factorial structure of the CES-D that could be applied to elderly populations from both China and the Netherlands, producing a meaningful comparison of total scores between the two elderly groups. However, for some specific factors and items, caution is required when comparing the depressive symptoms between Chinese and Dutch elderly groups. Depression is considered an important public health problem because of its relatively high prevalence in the general population  and its In the initial report, Radloff 1977) examined the factor structure of the CES-D using principal components analysis with varimax rotation and identified four factors, including Depressed Affect, Positive Affect, Somatic Symptoms/Retarded Activity and Interpersonal problems . Followi77 examinHowever, there are some inconsistencies concerning the factor structure of the CES-D in the research literature. A three-factor solution is another widely accepted model. Using the data of Hispanic Health and Nutrition Examination Survey (Hispanic HANES), Guarnaccia et al. (1989) identified a three-factor model , with somatic symptoms and depressive affect combined as one factor rather than two distinct factors . Other sThe elderly population represents a fast growing segment in most societies. Although there is no direct causal relationship between age and depression , depresIn addition, it is well known that social and cultural differences between countries may result in disagreement about the definitions of depressive symptoms. For example, in the Eastern culture, especially for the Chinese, strongly positive affects run counter to and emotional controls are highly valued by the social culture. Consistent with this notion, previous studies have demonstrated that the Chinese are more likely to value low arousal positive affect  than the Western participants, whereas Western participants value high arousal affect  more than the Chinese participants . In addiVandenberg & Lance (2000) suggested that when using the same measure in two different  groups, measurement invariance should be established to ascertain whether a given set of measures taps a particular latent construct (such as depression) similarly across groups, so that meaningful comparisons between groups can be made . Put simAlthough previous studies using non-elderly groups have assessed the factorial invariance of the CES-D across different immigrant ,34, ethnBy using two elderly groups recruited from China and the Netherlands, the current study attempted to test the measurement invariance of the CES-D to ascertain whether these two socially and culturally contrasting groups interpret the constructs underlying the CES-D items in a conceptually similar manner. First, various hypothesized factorial structures of the CES-D are tested . Second, the equality across the two samples of the parameters characterizing the relationship between the items of the CES-D and the underlying latent constructs are tested. Third, when the measurement invariance was established, the latent mean differences between the two groups were assessed.The Chinese sample was from the National Survey of Mental Health among Chinese Elderly Adults, conducted by the Institute of Psychology, Chinese Academy of Science. The target population consisted of elderly adults aged 55 and over, residing in the major districts of the Chinese mainland. Data was collected in 2007-2008 through a multistage area national probability sample. A total of 4,903 elderly Chinese adults were included in our final analysis. Of all the participants in the Chinese sample, 2,415 were male (mean age = 67.35 ± 8.21 years) and 2,464 were female (mean age = 66.36 ± 7.97); 24 did not report their gender.The Dutch sample was from the NESTOR (The Netherlands Program for Research on Aging) Study on Living Arrangements and Social Networks (LSN) which was continued in the Longitudinal Aging Study Amsterdam (LASA), an ongoing longitudinal study with secondary studies on various topics. The target population consisted of elderly adults aged 55 to 85 years of age, residing in urban and rural areas in the West, North-East and South of the Netherlands. Data was used from the fifth wave of the LASA study, which was collected in 2005-2006. A total of 1,903 elderly adults were included in the Dutch sample. Of all participants in the Dutch sample, 853 were male (mean age = 70.43 ± 8.76 years) and 1,050 were female (mean age = 71.79 ± 9.41). A detailed discussion of the LASA sample was provided in the paper of Deeg et al. published in 2002 .In both samples, less than 1% of itemscores were missing. This amount of missing data can be deemed inconsequential . As a reBoth surveys were performed with the approval of two appropriate ethics committees. For the Chinese sample, the survey was approved by the ethics committee of the Institute of Psychology. Written informed consent was obtained from each participant. For the Dutch sample, informed consent was obtained at the beginning of the study, in accordance with legal requirements in the Netherlands. Ethical aspects of the research procedures were approved in 1992 by the committee on ethics of research in humans of the Faculty of Medicine of the Vrije Universiteit.The Center for Epidemiological Studies Depression Scale (CES-D) was used to measure levels of depressive symptoms among elderly participants. The CES-D consists of 16 negative affect and 4 positive affect items, such as "I felt depressed", "I felt lonely", and "I was happy". Participants were asked about the number of days on which they experienced depressive symptoms during the previous week. Each item was accompanied by a standard four-point Likert scale of potential responses: 1 = none, 2 = one or two days a week, 3 = three or four days per week, and 4 = five days or more per week. Higher scores on the CES-D indicate more depressive symptoms  and exteRadloff 1977) identified four factors in the CES-D in the general population, including somatic complaints, depressed affect, positive affect and interpersonal problems. Items associated with the four factors are listed in table 977 identFour competing models were tested in the present study: a one, three and four-factor model, and an additional second-order factor model. In the second-order model, the four factors suggested by Radloff (1977) were considered to be dependent on a single second-order factor. The three-factor  model combines somatic complaints and depressive affect factors and was examined in a number of earlier studies . A one-fConfirmatory factor analysis (CFA) with maximum likelihood estimation, using LISREL 8.70 , was empSecond, after the best fitting model was determined for each sample, it was incorporated into a multi-group CFA model to test measurement invariance of the CES-D across the two population groups. Measurement invariance can be established by running a multi-group analysis of the factor structure underlying the data of these two groups . TraditiThird, partial invariance of each model was allowed to refine the structural models , as invat-statistic of the estimated latent mean in the Dutch group [d values, were calculated according to the guidelines of Hancock (2001) [Fourth, following the assessment of measurement invariance, latent mean differences for each latent construct were tested,. In the analysis, latent mean values were fixed to zero in the Chinese group, and freely estimated for the Dutch group. Based on the difference from zero of the latent mean in the Dutch group, latent means can be compared. Statistical significance of the difference can be based on the ch group . Howeverk (2001) .To evaluate model fit in the current study, Minimum Fit Function Chi-Square χ2, df, RMSEA , NNFI , CFI , and AIC  values are reported. Among these indices, differences of χ2 and df statistics between two invariant models are frequently used to determine whether models' invariance constraints are likely to hold or not. However, a number of problems result from using the χ2 value to evaluate model fit: the χ2 (or Δχ2) is sensitive to minor departures from multivariate normality and is nearly always large and statistically significant with complex models and/or large samples, which have been well documented in previous research ,49. ObviTable Based on the above CFA results, reliability estimates for the 4 factors  were computed. Although internal consistency coefficient alpha is widely used as a reliability estimate, a number of problems arise from its use . The omIn subsequent analyses, the four-factor structure of the CES-D was used as a baseline model for testing measurement invariance across the Chinese and Dutch sample.The first test of configural invariance assessed whether the CES-D was best described by a four-factor structure for the two samples. The results showed that the configural invariance model fitted the data reasonably well, RMSEA = 0.059 , CFI = 0.976 , CFI = 0.972 , CFI = 0.958  on the Depressive Affect and Positive Affect factor. Results showed that the fit indices for the partial scalar invariance model were adequate: RMSEA = 0.066 , CFI = 0.967  were eventually lifted. The fit indices of the partial uniqueness invariance model showed better model fit: RMSEA = 0.070 , CFI = 0.959 . Results showed lower latent mean values for the Dutch group on all four dimensions of the CES-D. Means, standard deviations and effect sizes are presented in table The purpose of this study was to test the measurement invariance of the CES-D using confirmatory factor analysis in two large elderly populations from China and the Netherlands. The results reveal that in both samples, Radloff's four-factor model  resultedResults obtained from the test of configural invariance confirmed the four factor structure across both samples. That is, both populations demonstrate equivalence in the pattern of factor loadings of the CES-D, suggesting that the CES-D measures the same concept across the Chinese and Dutch elderly. Our analysis also supported metric invariance across the two samples. This finding seems to imply that the twenty items of CES-D measure depressive symptoms (or depressed affects) in the same way across the two national samples. According to the interpretation of factor loadings suggested by Oort (2005) , reporteAt the intercept level, full invariance was not supported. Two intercepts in the Depressed Affect factor (failure) and Positive Affect factor (good) differed across the Chinese and Dutch elderly. Specifically, the intercepts for failure and good were larger in the Chinese sample, which indicates a difference in internal standards across the Chinese and Dutch elderly . Chinesedepressed and fearful on the Depressed Affect factor and dislike in the Interpersonal Problems factor did not hold across the two samples. Uniquenesses of depressed, fearful and dislike were larger in the Chinese sample, suggesting that the items' measurement errors were larger for Chinese elderly adults than for Dutch elderly adults.Our analysis also did not support full uniqueness invariance across the two samples. The partial invariance analysis revealed that the Depressed Affect and Interpersonal Problems domain of the CES-D is less invariant than the other two domains. Specifically, the invariance of The differences in intercepts and uniquenesses of these items may result from the cultural differences and differing social norms, which could influence the way one experiences and expresses feelings of depression. For example, Nikelly (1998) suggested that the expression of affective distress causes the individual to appear self-centered, which may be threatening to close relationships and therefore discouraged in collective cultures . In addiAlthough only partial metric and scalar invariance were supported, the meaningful comparison of factor means of the CES-D across the Chinese and Dutch elderly seems possible. Cheung and Rensvold  suggested that if the proportion of non-invariant items of a scale is small, the comparison of factor means can still be meaningful even if full measurement invariance does not hold, as the non-invariant items will not heavily affect the comparison ,56. TherLatent mean differences between the Chinese and Dutch sample were found on all four CES-D factors, with the Dutch scoring about half a standard deviation lower than the Chinese elderly. This indicates that, on average, the Chinese elderly reported more feelings of depression than the Dutch elderly.The current study has two implications. First, based on the number of previous studies on the psychometric properties of the CES-D ,12, the There are several limitations to the current study. First, only the equivalence of factor validity was studied. This is insufficient to demonstrate that it is an effective measurement both for populations from two countries. A goal for future research is to examine whether the other types of validity, such as predictive concurrent and content validity of the CES-D are also equivalent across the two population groups. Second, although China and the Netherlands serve as examples of countries with different social and cultural backgrounds in the current study, future studies should be conducted using samples from other typically Western and Eastern countries to see whether the results can be replicated, in order to demonstrate the generalization of the CES-D across different cultural backgrounds. Third, when interpreting the loading or intercept differences of non-invariant items, caution should be applied because of chance capitalization. Releasing parameter restrictions based on modification indices and expected change is a data driven procedure, and susceptible to capitalization on chance characteristics of the data . The mod01. I was bothered by things that usually don't bother me.02. I did not feel like eating; my appetite was poor.03. I felt that I could not shake off the blues even with the help of my family or friends.04. I felt I was just as good as other people.05. I had trouble keeping my mind on what I was doing.06. I felt depressed.07. I felt that everything I did was an effort.08. I felt hopeful about the future.09. I thought my life had been a failure.10. I felt fearful.11. My sleep was restless.12. I was happy.13. I talked less than usual.14. I felt lonely.15. People were unfriendly.16. I enjoyed life.17. I had crying spells.18. I felt sad.19. I felt that people disliked me.20. I could not get "going."01. 我最近烦一些原来不烦心的事02. 我不想吃东西，胃口不好03. 我觉得沮丧，就算有家人和朋友帮助也不管用04. 我觉得自己和别人一样好05. 我不能集中精力做事06. 我感到消沉07. 我觉得做每件事都费力08. 我感到未来有希望09. 我觉得一直以来都很失败10. 我感到害怕11. 我睡不安稳12. 我感到快乐13. 我讲话比平时少14. 我觉得孤独15. 我觉得人们对我不友好16. 我生活愉快17. 我哭过或想哭18. 我感到悲伤难19. 我觉得别人不喜欢我20. 我提不起劲儿来做事01. De afgelopen week maakte ik me zorgen om dingen waar ik me anders geen zorgen over maak.02. De afgelopen week had ik geen zin in eten, was mijn eetlust slecht.03. De afgelopen week kon ik een neerslachtige stemming niet van me afschudden, zelfs niet met behulp van mijn familie en vrienden.04. De afgelopen week voelde ik me evenveel waard als andere mensen.05. De afgelopen week had ik moeite mijn gedachten te houden bij wat ik aan het doen was.06. De afgelopen week voelde ik me depressief.07. De afgelopen week had ik het gevoel dat alles wat ik deed me moeite kostte.08. De afgelopen week was ik hoopvol gestemd over de toekomst.09. De afgelopen week vond ik mijn leven een mislukking.10. De afgelopen week voelde ik me angstig.11. De afgelopen week had ik een onrustige slaap.12. De afgelopen week was ik gelukkig.13. De afgelopen week praatte ik minder dan gewoonlijk.14. De afgelopen week voelde ik me eenzaam.15. De afgelopen week waren de mensen onvriendelijk.16. De afgelopen week had ik plezier in het leven.17. De afgelopen week moest ik soms huilen.18. De afgelopen week voelde ik me bedroefd.19. De afgelopen week had ik het gevoel dat de mensen me niet aardig vonden.20. De afgelopen week kon ik maar niet goed op gang komen.The authors declare that they have no competing interests.BZ designed the protocol, analyzed the data and prepared the manuscript. MF conceived the study, participated in the study design and gave significant comments on the manuscript. PC helped to conceive the study and gave his significant comments for improving the manuscript. JL had full access to the Chinese data in the study, helped to design the protocol and draft the manuscript, and took responsibility for the integrity of the data. NS helped to conceive the study and gave significant comments for improving the manuscript. AB had full access to the LASA data and gave significant comments on the manuscript.All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/11/74/prepub


The process is characterized by formation of black crusts and metal pitting. The mechanism is usually explained by the corrosiveness of formed H2S, and scavenge of ‘cathodic’ H2 from chemical reaction of Fe0 with H2O. Here we studied peculiar marine SRB that grew lithotrophically with metallic iron as the only electron donor. They degraded up to 72% of iron coupons (10 mm × 10 mm × 1 mm) within five months, which is a technologically highly relevant corrosion rate (0.7 mm Fe0 year−1), while conventional H2-scavenging control strains were not corrosive. The black, hard mineral crust  deposited on the corroding metal exhibited electrical conductivity (50 S m−1). This was sufficient to explain the corrosion rate by electron flow from the metal (4Fe0 → 4Fe2+ + 8e−) through semiconductive sulfides to the crust-colonizing cells reducing sulfate (8e− + SO42− + 9H+ → HS− + 4H2O). Hence, anaerobic microbial iron corrosion obviously bypasses H2 rather than depends on it. SRB with such corrosive potential were revealed at naturally high numbers at a coastal marine sediment site. Iron coupons buried there were corroded and covered by the characteristic mineral crust. It is speculated that anaerobic biocorrosion is due to the promiscuous use of an ecophysiologically relevant catabolic trait for uptake of external electrons from abiotic or biotic sources in sediments.Iron (Fe In technology, the process is often referred to as microbially influenced corrosion (MIC).Iron, the fourth most abundant element in the earth's crust, is the principal redox-active metal in metabolic processes of essentially all living organisms. It is either involved in catalytic quantities as a component of a vast number of proteins, or in much higher, substrate quantities as the external electron donor or acceptor for specially adapted environmental microorganisms referred to as ‘iron bacteria’  or ‘iron-respiring bacteria’ respectively. In most biological functions, iron has the +II (ferrous) or +III (ferric) oxidation state. From a physiological point of view it appears astounding that also the native, metallic element (Fe8 t year−1) than the second most widely employed metal, aluminum  and finally leads to the formation of brittle ferric oxides/hydroxides. Another ubiquitous electron acceptor is protons yielding hydrogen . However, this is technologically only serious in rare instances of acidic surroundings. Proton reduction in circumneutral H2O and thus the net reaction with organic nutrients react more rapidly with iron-derived electrons than do protons from or in  H2O, according toFirst, undissociated protons in H2O’, viz. the abundant building structure 〈H-C-OH〉) asIn such way, SRB act indirectly through an excreted chemical agent. We here refer to this process as ‘chemical microbially influenced corrosion’ (CMIC). The net reaction  is thought to ‘pull’ the primary oxidation . For the present biocorrosion experiments intended to examine iron destruction under conditions comparable to those in situ during much longer incubation, the ratio of the culture (and gas phase) volume to metal mass had to be increased. Because macroscopic corrosion phenomena were of central interest, the iron specimens (10 mm × 10 mm × 1 mm) could not be miniaturized to any extent, thus necessitating much bigger culture volumes. An appropriate medium volume was 1.4 l, which was still small enough for precise monitoring of sulfate consumption. Indeed, corrosion rates did not significantly decrease over months. Corrosive cultures reached values as high as 0.7 mm Fe0 year−1 and deposited steadily growing black crusts , showed essentially no signs of iron corrosion within the incubation period. Iron in these control cultures was not more affected than in sterile incubations  crust. Sulfur-free patches were never colonized.Indeed, virtually no planktonic (free-living) cells could be observed, and scanning electron microscopy revealed a densely colonized crust surface in the corrosive cultures of strains IS4 or IS5. Colonized areas of the structurally heterogeneous crust contained the element S in addition to Fe, C and O  and amorphous ferrous sulfide at the expected ratio (3).The bulk composition of the crust formed by strain IS4 was analysed quantitatively by combining EDX, X-ray diffraction (XRD), inductively coupled plasma optical emission spectroscopy (ICP-OES), and infrared spectroscopy. This revealed siderite  part of a silty marine mud flat  at g device . The coug device . Their eg device  as well 2 (known from sterile control incubations) indicated corrosive SRB at numbers of more than 107 cells per gram wet sediment  by Fe0-derived electrons on the metal surface is a par excellence example of a kinetically ‘impeded’, slow electrochemical reaction. Availability of H+ ions at the metal surface and combination of the primarily formed atomic hydrogen [e− + H+ → H(adsorbed)] to H2 are commonly understood as kinetic ‘bottle neck’ that also explains the high negative electrochemical overpotential  of electrochemical H2-formation on iron ; this is even higher than that of many typical semiconductors + + H+; apK = 8.8] and precipitation of Fe2+ as soon as it enters the high-pH carbonate-containing medium . These may be tiny interstices or fissures. An apparent, striking ion bridge, also strongly supporting the model of transcrustal electron flow, emerged at ructures . Their fg medium .In conclusion, anaerobic corrosion caused by the direct, lithotrophic mode of iron utilization according to E°′av = −0.218 V, average) coupled to iron dissolution  + CH4 (g) + 5HO−, does not lead to a conductive precipitate. One may speculate that in this case cell-metal contact must be sustained so that hindrance by crust coverage may become obvious during long-term incubations (which have not been carried out so far). Nevertheless, the process may play a role in MIC because methanogenic archaea may take advantage of electroconductive FeS precipitated by co-occurring sulfate reduction. In axenic laboratory cultures, there is some FeS precipitation by sulfide added as reductant.There is first evidence that also special methanogenic archaea obtained through enrichment with metallic iron as the only source of reducing equivalents can bypass the slow abiotic Hlectrons  accordinnFeEMIC, is the sum of the amount oxidized catabolically by sulfate reduction, nFeCatab, and the amount oxidized due to the anabolic need of electrons, nFeAnab, i.e.An understanding of the properties of the precipitate formed during corrosion also requires knowledge of the proportion of formed cell mass that may be embedded. Because there is presently no convenient method for determining cell mass in the solid corrosion crust, its organic content was estimated. According to the principle of bifurcate substrate flow in every chemotrophic organism, the amount (e.g. in mol or mmol) of total iron oxidized in cultures of SRB performing EMIC, nFeEMIC is identical with the loss of metallic iron, nΔFe(0), and nFeCatab is fourfold higher than the amount of sulfate being reduced  or contribution of anabolic iron oxidation to total iron oxidation during EMIC can be formulated asnΔFe(0) = nFe(II). The resulting nFeAnab (mBio (e.g. in g or mg), is formed per amount of iron used for the anabolism, viz. they allow to formulate an anabolic yield coefficient,Iron loss is equivalent with ferrous iron formation, i.e.  nFeAnab  can betr4H8O2N , or require in addition an organic substrate such as acetate . The resulting assimilation equations areHere we used the simplified bulk formula C4H8O2N  with ‘M’andc. ⅔ of cell carbon in SRB is derived from acetate and c. ⅓ from bicarbonate  precipitated during lithotrophic corrosion, mMin, can be calculated from the same measurable parameters as nFeAnab (nΔFe(0)[or nFe(II)] and nSR  = nFe(II)). They are applicable if EMIC is the only process of corrosion, as in our cultures, and if one assumes complete precipitation of ferrous iron (and variable precipitation of Ca/MgCO3).can be derived  for auton▵Fe(0) = 4.31 and 9.86 mmol Fe0, respectively, during reduction of nSR = 0.95 (± 0.06) and 2.14 (± 0.08) mmol SO42− respectively. Hence, the amounts accounting for biosynthesis , as common in the strictly anaerobic SRB, and that the corrosion crust is essentially inorganic, in agreement with its hard, mineral-like appearance. The corrosion crust thus profoundly differs from typical biofilms that are largely constituted of cells and an organic matrix.in situ will remain a relevant issue.Protection of iron against corrosion by alloying or coating is technically and economically not possible at any extent. For instance, the most important alloying metal, chromium, is produced at a 48-fold lesser extent than iron  and chemical, electrochemical and microbial reactions in MIC. The most promising research strategy is, in the opinion of the authors, an experimental ‘dissection’ into individual and elementary processes for later synopsis. In the previous .in situ, recommendation of control measures is also limited. As long as MIC in a particular situation is largely CMIC, prevention would be possible if the organic nutrient of the anaerobic bacteria can be identified and eliminated. If MIC is essentially EMIC, avoidance of sulfate-containing water would be a causative solution. Extended in vitro and pilot studies may clarify as to which extent an addition of nitrate, which is applied to control biogenic H2S (souring) in petroleum reservoirs , so that qEMIC = nFeEMIC/nFe(II). Further calculation so as to substitute the unknown nFeEMIC and introduce the measurable amount of precipitated sulfide, nFeS, leads to the formula  is needed. For approximate calculation, qAnab may be omitted (because qAnab << 3). Still, such formal treatment is only applicable for anoxic conditions and absence of processes other than sulfate reduction, for instance methanogenesis (3 to FeS (further remarks in nFeS/nFe(II) = 0.20–0.24. By assuming, for convenience, qAnab ≍ 0.1, we obtain qEMIC = 0.98–1.03 . This suggests that corrosion under the conditions prevailing at the studied marine sediment site was indeed only EMIC, viz. due to SRB capable of direct electron uptake. Such crust analysis  of their abundance at various natural sites and physiological studies are needed to unravel their real significance in anaerobic mineralization.The specific ability to utilize metallic iron as an electron donor is a physiologically striking capability, the ecological significance of which is presently unknown. Apart from rare cases  and rearrangement of bound atoms. With many simple electron donors and acceptors, electron transfer takes place without specific catalysis simply according to redox potentials. This is a classical principle in biochemistry, for instance if electron accepting or donating dyes such as viologens or hexacyano-ferrates are used to react unspecifically with various redox proteins. In the cell, cofactors that can transfer electrons as such would be even critical in their free, dissolved form because of unspecific redox reactions. Their reducing or oxidizing power must be controlled for instance by embedding in a protein  or by restriction to the lipophilic cytoplasmic membrane (quinones). Substances that merely accept or donate electrons are much less critical outside of the cell, and the ability to use them via transmembrane electron transport components is obviously a typical domain of environmental prokaryotes.2 production from H2O  provided at growth-limiting rate through a silicon rubber membrane inside an anoxic cultivation device; some acetate (1 mM) was provided for cell synthesis. After two subcultures, the Desulfovibrio strain HS3 was isolated via agar dilution  headspace at 28°C. SO42− was usuallyprovided at 28 mM; exceptions were cultures with 5 mM SO42− (not resulting in a noticeable decrease of the growth rate) for precise determination of its consumption. The reductant was usually Fe0 alone. However, some big cultures (1400 ml) received also 75 µM Na2S, which shortened the lag phase. All strains except strain IS4 were supplemented with 1 mM acetate for heterotrophic cell synthesis. H2-grown inocula were flushed with N2-CO2 to prevent transfer of H2 substrate and sulfide to Fe0 cultures. Iron specimens  were degreased with acetone (2 min), freed from oxide layers with 2 M HCl (2 min) and washed in sterile anoxic water (2 × 20 s). Coupons were then rapidly dried under N2, weighed (in sterile Petri dishes) and added to cultures. The ratio of medium volume to metal surface was between 15 ml cm−2  and 600 ml cm−2 (for corrosion rate determination).Cultures were grown in COr medium  in butyl2 or lactate (+ yeast extract), and sequencing of 16S rRNA genes.Strain purity was routinely checked microscopically upon growth with H0 as electron donor and 1 mM acetate as a carbon source. SO42− consumption was quantified after 6 months of incubation at 20°C. Sterile controls were included to calculate sulfate reduction solely based on the measured abiotic H2 formation. Marginal background sulfate reduction was measured in iron-free controls.Cells of corrosive SRB in sediment were quantified via triplicate serial 1:10 dilutions with Fe2 chamber) scraped off the water-rinsed and dried coupons, finely ground in a mortar, and kept under anoxic N2 until analysis to prevent secondary oxidation. Corrosion coupons for weight loss determination were freed from the crust in aqueous 2 M HCl and 0.7 M hexamethylenetetramine (hexamine). The freed iron was washed in anoxic water and dried under N2 before weighing.Corrosion crust for analysis was (inside an Nσ = I d/(V a); I, current; d, distance between coupons; V, voltage; a, split area (split height × coupon thickness). For photographic documentation, the mounted coupons were transferred to anoxic water.Shaped and polished mild steel coupons were fixed at exactly defined distance on a polycarbonate support . The fou8 Pa) to smooth pills in a type 15.011 hydraulic press (Specac Ltd). Impedance was measured via a ‘piston electrode’  was determined with specimens compressed  at 5–15 kV was performed with a Leo 1550 FE instrument (Zeiss).Crust-covered iron specimens were fixed with 2% (v/v) glutaraldehyde in anoxic artificial seawater at 4°C (12 h), washed with anoxic seawater, dehydrated with anoxic ethanol at increasing concentration , pre-dried chemically with hexamethyldisilazane  to smooth pills in a type 15.011 hydraulic press (Specac Ltd) and surface-mapped (using triplicate samples) by energy-dispersive X-ray microanalysis (EDX) at 10 kV in combination with SEM. Data were analysed with the INCA software (Oxford Instruments). C and S were subsequently quantified after combustion (to CO2 and SO2 respectively) using a CS-444 infrared spectrometer (Leco). Na, Mg, Al, Si, P, K, Ca and Fe were quantified by inductively coupled plasma optical emission spectroscopy (ICP-OES) with an IRIS Intrepid HR Duo instrument (Thermo Fisher Scientific). Si was quantified after alkaline pulping. The O-content was calculated as the remaining fraction. H (not detectable with the used instruments) is assumed to constitute a marginal fraction of the dried mineral crust.For element identification, finely ground crust homogenate (see above) was compressed (9 × 10α radiation from 1 µm aperture in a D8 Advance diffractometer (Bruker). The 2θ angle was increased in 0.08° steps with 15 s counting time from 20 to 74.08°; the total scan time per diffractogram was 169 min. Data were analysed with the EVA software (Bruker).Crystalline mineral phases in finely ground corrosion crust were identified by means of X-ray diffraction (XRD) analysis in Bragg-Brentano geometry with CuK2 was quantified in samples withdrawn (with syringes through hypodermic needles) from the culture headspace via a GC-8A gas chromatograph (Shimadzu) equipped with a Porapak Q N80/100 (Machery-Nagel) column  and a thermal conductivity detector.H42− in filtered (0.45 µm pores) FeS-free samples was quantified via a 761 Compact Ion Chromatograph (Metrohm) by conductivity detection. Ions were separated via a Metrosep A Supp 5–100 column with an eluent of 3.2 mM Na2CO3 and 1 mM NaHCO3 at 0.7 ml min−1. Fe2+ dissolved in such filtrates was quantified by ICP-OES (see above).SO