Flora diversity survey and establishment of a plant DNA barcode database of Lomas ecosystems in Peru.

Lomas formations or "fog oases" are islands of vegetation in the desert belt of the west coast of South America, with a unique vegetation composition among the world's deserts. However, plant diversity and conservation studies have long been neglected, and there exists a severe gap in plant DNA sequence information. To address the lack of DNA information, we conducted field collections and laboratory DNA sequencing to establish a DNA barcode reference library of Lomas plants from Peru. This database provides 1,207 plant specimens and 3,129 DNA barcodes data corresponding with collections from 16 Lomas locations in Peru, during 2017 and 2018. This database will facilitate both rapid species identification and basic studies on plant diversity, thereby enhancing our understanding of Lomas flora's composition and temporal variation, and providing valuable resources for conserving plant diversity and maintaining the stability of the fragile Lomas ecosystems.

Reaffirming the Loma Mountains National Park in Sierra Leone as a critical site for the conservation of West African chimpanzee (Pan troglodytes verus).

The population of West African chimpanzees (Pan troglodytes verus) is declining rapidly mostly due to the impact of human activities and habitat loss. Sierra Leone harbors the third largest population of this subspecies, recently reclassified as Critically Endangered in the 2016 IUCN Red List. Population monitoring provides crucial data for planning and evaluating conservation and management policies. Therefore, to assess the status of the population size inhabiting the Loma Mountains National Park (LMNP) in Sierra Leone, we performed a nest count survey and estimated chimpanzee density and abundance using distance sampling. In total, 34 × 2-km-line transects were surveyed, with transects being systematically distributed across the LMNP area (288.5 km2 ). Concurrently, we compiled environmental data, which were used to model nest distribution and infer the most relevant environmental and anthropogenic drivers of the observed nest abundances. We encountered 10.03 nests/km and estimated a density of 3.47 ± standard error (SE) 0.92 individuals/km2 (i.e., 1002 ± SE 266 individuals in total). Compared to the figures obtained from a systematic literature review, our results suggest that the density and abundance of chimpanzees in the LMNP is among the highest across Africa. Contrary to expectation, no specific anthropogenic features predicted nest distribution and abundance. However, the nest distribution model indicated preference for elevated and steep areas covered by closed evergreen forest, which could be an indication of human avoidance. Based on these results, we highlight the value of LMNP for the conservation of the chimpanzees in Sierra Leone and the urgent necessity of guarantying long-term funding for this park's management to ensure the survival of this critically endangered subspecies in West Africa.

Cohort profile for the Loma Linda University Health BREATHE programme: a model to study continuously incentivised employee smoking cessation.

PURPOSE: The purpose of the Loma Linda University Health (LLUH) BREATHE cohort is to test the efficacy of a novel method of continuously incentivising participation in workplace smoking cessation on participation, long-term abstinence, health outcomes, healthcare costs and healthcare utilisation. PARTICIPANTS: In 2014, LLUH-a US academic medical centre and university-incentivised participation in a workplace smoking cessation programme (LLUH BREATHE) by lowering health plan costs. Specifically, LLUH introduced a Wholeness Health Plan (WHP) option that, for the smokers, continuously incentivises participation in nicotine screening and the LLUH BREATHE smoking cessation programme by offering an 'opt-in wellness discount' that consisted of 50%-53% lower out of pocket health plan costs (ie, monthly employee premiums, copayments). This novel 'continuously incentivised' model lowers annual health plan costs for smokers who, on an annual basis, attempt or maintain cessation from tobacco use. The annual WHP cost savings for smokers far exceed the value of short-term incentives that have been tested in workplace cessation trials to date. This ongoing health plan option offered to over 16 000 employees has created an open, dynamic LLUH BREATHE cohort of current and former smokers (n=1092). FINDINGS TO DATE: Our profile of the LLUH BREATHE cohort indicates that after 5 years of follow-up in a prospective cohort study (2014-2019), continuously incentivised smoking cessation produced a 74% participation (95% CI (71% to 77%)) in employer-sponsored smoking cessation attempts that were occurring less than a year after the incentive was offered. The cohort can be purposed to examine the effect of continuously incentivised cessation on cessation outcomes, health plan utilisation/costs, use of electronic nicotine delivery systems, and COVID-19 outcomes.

New isolate of Loma psittaca (Microsporidia: Glugeidae) infecting the stomach wall of cultured hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) in South China.

Loma psittaca, previously described as inhabiting the intestinal mucosa of an anadromous fish, Colomesus pisttacus, from the Amazon Basin, is reported as being found for the first time in a marine fish, the hybrid grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀), from Lingshui city, Hainan Province, China, expanding the geographical distribution and host range of this parasite. Numerous whitish xenomas (0.5-0.7 mm in diameter) of this new isolate of L. psittaca were found distinctly in the muscle layer of the host stomach wall. Electron microscopic observations showed a monokaryotic nucleus in all developmental stages. Round or elongated multinucleate merogonial plasmodia surrounded by numerous mitochondria were observed initially, subsequently transforming into uninucleate sporonts through multiple fissions. Sporonts, each with a large centrally positioned nucleus, further developed into sporoblasts. Each sporoblast mother cell gave rise to two uninucleate sporoblasts by binary fission. Mature spores were ellipsoidal, measuring 4.0 ± 0.3 (3.7-4.3) µm in length and 2.2 ± 0.2 (2.1-2.5) µm in width. Spores possessed a mushroom-like anchoring disk, a bipartite polarplast, isofilar polar filaments arranged in 12-14 turns in one row, and a trilaminar spore wall. The obtained partial SSU rRNA gene sequence of the new isolate was 1330 bp in length and showed 99.4% sequence similarity with an estuary isolate of L. psittaca previously reported in South America. SSU rRNA gene-based phylogenetic analyses demonstrated that the two L. psittaca isolates first clustered together and then formed a dichotomy that included the digestive-tract-infecting Loma species, L. acerinae, with high support values within group I.

Genetic diversity of Loma acerinae (Microsporidia: Glugeida) from different fish hosts and localities - Short communication.

Loma acerinae is a xenoma-forming fish microsporidium described from common ruffe Gymnocephalus cernua (Perciformes: Percidae) and also found in Ponto-Caspian gobies (Gobiiformes: Gobiidae). This casts doubt on the strict host specificity of this parasite. The largest subunit RNA polymerase II (rpb1) was used as a genetic marker of the parasite isolated from six host species of Perciformes (G. cernua from the Baltic Sea), Atheriniformes (Atherina boyeri from the Azov Sea) and Gobiiformes (Neogobius spp. and Zosterisessor ophiocephalus from the Black Sea and Ponticola kessleri from the Caspian Sea basin). Two major rpb1 haplogroups were found with 98.5% identity between the groups. Notably, Haplogroup I was associated with Neogobius spp. samples (n = 6) only, whereas Haplogroup II included the samples from other host species (n = 7). These findings confirm the broad distribution and host range of L. acerinae, but also indicate that certain patterns of host-driven intraspecific polymorphism may exist. Furthermore, the study revealed low similarity between the ribosomal RNA gene sequences of L. acerinae and the type species, Loma morhua (as well as other species of the genus). This suggests loose genetic association within the genus, and may raise the need for the taxonomic revision of L. acerinae.

Development of a continuous cell line from larval Atlantic cod (Gadus morhua) and its use in the study of the microsporidian, Loma morhua.

In vitro cell culture methods are crucial for the isolation, purification and mass propagation of intracellular pathogens of aquatic organisms. Cell culture infection models can yield insights into infection mechanisms, aid in developing methods for disease mitigation and prevention, and inform commercial-scale cultivation approaches. This study details the establishment of a larval cell line (GML-5) from the Atlantic cod (Gadus morhua) and its use in the study of microsporidia. GML-5 has survived over 100 passages in 8 years of culture. The line remains active and viable between 8 and 21°C in Leibovitz-15 (L-15) media with 10% foetal bovine serum and exhibits a myofibroblast phenotype as indicated by immuno-positive results for vimentin, α-smooth muscle actin, collagen I and S-100 proteins, while being desmin-negative. GML-5 supports the infection and development of two microsporidian parasites, an opportunistic generalist (Anncaliia algerae) and cod-specific Loma morhua. Using GML-5, spore germination and proliferation of L. morhua was found to require exposure to basic pH and cool incubation temperatures (8°C), in contrast to A. algerae, which required no cultural modifications. Loma morhua-associated xenoma-like structures were observed 2 weeks postexposure. This in vitro infection model may serve as a valuable tool for cod parasitology and aquaculture research.

Study of Loma acerinae (Microsporidia) detected from three Ponto-Caspian gobies (Gobiidae) in Ukraine.

A previously recognized Glugea-belonging Microsporidia (G. melanostomi, G. markievichi, G. naidenovi) were found in the intestinal mucosa of Neogobius melanostomus, N. fluviatilis, and Proterorhinchus semilunaris (Teleostei, Gobiidae) from lower Dnieper River and Black Sea coastal inlets, Ukraine. Light microscope observations demonstrated small spherical xenomes about 0.35 mm in diameter filled by developmental stages of parasites. Mature spores were ellipsoidal, slightly narrowed at anterior pole, sometimes ovoid or elongate in shape approximately 3.1-4.5 × 2.0-2.9 µm in size. The stained spores were elongate ovoid that are measured about 2.3-4.3 × 1.6-3.4 µm. Ultrastructural study showed that xenoma wall consisted of finely granular layer up to 350 nm wide. Numerous host-derived collagenous fibers adhered to the xenoma wall were noticed. Microtubular inclusions about 40 nm in diameter occupied the episporontal space. Mature spores displayed the polar filament arranged in one row. One to two last turns of polar filament were sharply tapered to half of diameter. The number of polar filament turns varied depending of host infected from 10 (9 + 1) in N. melanostomus to 13 (11 + 2) in N. fluviatilis and 14 (13 + 1) in P. semilunaris. The endospore thickness varied depending of host infected from 30-35 nm in case of N. fluviatiis to 40-50 nm in P. semilunaris and 60-80 nm in N. melanostomus. SSU 16S rRNA sequence analysis, blast searches, and ultrastuctural data revealed similarity of all studied microsporidians with Loma acerinae (Jirovec, 1930).

Integrative Approach for the Reliable Detection and Specific Identification of the Microsporidium Loma morhua in Atlantic Cod (Gadus morhua).

Microsporidia are fungal parasites that infect diverse invertebrate and vertebrate hosts. Finfish aquaculture supports epizootics due to high host density and the high biotic potential of these parasites. Reliable methods for parasite detection and identification are a necessary precursor to empirical assessment of strategies to mitigate the effects of these pathogens during aquaculture. We developed an integrative approach to detect and identify Loma morhua infecting Atlantic cod. We show that the spleen is more reliable than the commonly presumed gills as best organ for parasite detection in spite of substantial morphological plasticity in xenoma complexes. We developed rDNA primers with 100% sensitivity in detecting L. morhua and with utility in distinguishing some congeneric Loma species. ITS sequencing is necessary to distinguish L. morhua from other congeneric microsporidia due to intraspecific nucleotide variation. 64% of L. morhua ITS variants from Atlantic cod have a 9-nucleotide motif that distinguishes it from Loma spp. infecting non-Gadus hosts. The remaining 36% of ITS variants from Atlantic cod are distinguished from currently represented Loma spp., particularly those infecting Gadus hosts, based on a 14-nucleotide motif. This research approach is amenable to developing templates in support of reliable detection and identification of other microsporidian parasites in fishes.

Effects of Loma morhua (Microsporidia) infection on the cardiorespiratory performance of Atlantic cod Gadus morhua (L).

The microsporidian Loma morhua infects Atlantic cod (Gadus morhua) in the wild and in culture and results in the formation of xenomas within the gill filaments, heart and spleen. Given the importance of the two former organs to metabolic capacity and thermal tolerance, the cardiorespiratory performance of cod with a naturally acquired infection of Loma was measured during an acute temperature increase (2 °C h(-1)) from 10 °C to the fish's critical thermal maximum (CT(Max)). In addition, oxygen consumption and swimming performance were measured during two successive critical swimming speed (U(crit)) tests at 10 °C. While Loma infection had a negative impact on cod cardiac function at warm temperatures, and on metabolic capacity in both the CT(Max) and U(crit) tests (i.e. a reduction of 30-40%), it appears that the Atlantic cod can largely compensate for these Loma-induced cardiorespiratory limitations. For example, (i) CT(Max) (21.0 ± 0.3 °C) and U(crit) (~1.75 BL s(-1)) were very comparable to those reported in previous studies using uninfected fish from the same founder population; and (ii) our data suggest that tissue oxygen extraction, and potentially the capacity for anaerobic metabolism, is enhanced in fish infected with this microsporidian.

Development of the microsporidian parasite, Loma salmonae, in a rainbow trout gill epithelial cell line (RTG-1): evidence of xenoma development in vitro.

Growth and propagation of fish-infecting microsporidians within cell culture has been more difficult to achieve than for insect- and human-infecting microsporidians. Fish microsporidia tend to elicit xenoma development rather than diffuse growth in vivo, and this process likely increases host specificity. We present evidence that the fish microsporidian, Loma salmonae, has the capacity to develop xenomas within a rainbow trout gill epithelial cell line (RTG-1). Spore numbers increased over a 4 weeks period within cell culture flasks. Xenoma-like structures were observed using phase contrast microscopy, and then confirmed using transmission electron microscopy. Optimization of the L. salmonae-RTG-1 cell model has important implications in elucidating the process of xenoma development induced by microsporidian parasites.

Immunohistochemical localization of inflammatory cells and cell cycle proteins in the gills of Loma salmonae infected rainbow trout (Oncorhynchus mykiss).

Microsporidial gill diseases particularly those caused by Loma salmonae incur significant economic losses to the salmonid aquaculture industry. The gill responses to infection include the formation of xenomas and the acute hyperplastic inflammatory responses once the xenomas rupture releasing infective spores. The aim of this work was to characterize the inflammatory responses of the gill to both the presence of the xenomas as well as the hyperplasia associated with L. salmonae infection in the rainbow trout gill following an experimental infection using immunohistochemistry. Hyperplastic lesions demonstrated numerous cells expressing PCNA as well as an apparent increased expression of caspase-3 and number of apoptotic cells (TUNEL positive cells). There was an expression of TNFα in individual cells within the gill and increased expression of a myeloid cell line antigen indicating the presence of granulocyte infiltration of both the hyperplastic lesions as well as the xenomas. Similar immune-reactivity was seen in gill EGCs. Hyperplastic gill lesions showed a marked infiltration of CD8+ cells and expression of MHC class I antigens. These findings suggest that L. salmonae xenomas may be subject to infiltration by the host immune cells as well as the mounting or a marked cellular cytotoxic immunoreaction in the resultant hyperplasia following xenoma rupture and spore release.

Description of five new Loma (Microsporidia) species in pacific fishes with redesignation of the type species Loma morhua Morrison & Sprague, 1981, based on morphological and molecular species-boundaries tests.

Five new species of Loma were described from five Pacific fishes using light-microscopic and ultrastructural features along with phylogenetic analysis of the gene sequences of ribosomal RNA (rRNA) and elongation factor 1-alpha. Morphological data revealed both qualitative and quantitative differences in developmental stages and timing, vesicles, xenoma features, and spore sizes with statistical support that differentiated Loma pacificodae n. sp. in Pacific cod, Loma wallae n. sp. in walleye pollock, Loma kenti n. sp. in Pacific tomcod, Loma lingcodae n. sp. in lingcod, and Loma richardi n. sp. in sablefish from each other and other species in the genus. Phylogenetic analyses combined with monophyly tests supported species designations, but with low resolution in two cases perhaps due to rRNA paralogs or recent speciation. Loma branchialis in haddock was shown to be separate from Loma morhua in Atlantic cod, thereby making L. morhua, and not L. branchialis, the type species. A species from brook trout was shown to be a separate species from Loma salmonae, not a variant strain selected in the laboratory. By comparison with gadid host phylogeny, these Loma species appear to have coevolved with their hosts, first colonizing the Pacific basin about 12 million years ago.

Morphological and genetical description of Loma psittaca sp. n. isolated from the Amazonian fish species Colomesus psittacus.

A previously unrecognised fish-infecting microsporidia (Loma psittaca n. sp.), found adherent to the intestinal mucosa of the freshwater puffer fish Colomesus psittacus (Teleostei, Tetraodontidae) from lower Amazon River, was described based on light and transmission electron microscope and phylogenetic analysis. The whitish xenoma was completely filled by numerous spores, including several developmental stages of the parasite. In all of these stages, the nuclei were monokaryotic. The merogonial plasmodium divided by binary fission and the sporont gave rise to disporoblastic ovoid spores measuring 4.2 +/- 0.4 x 2.8 +/- 0.4 microm. In mature spores, the polar filament was arranged in 10-11 (rarely 12) coils in one row in turn of posterior vacuole. The polaroplast had two distinct regions around the manubrium. The polyribosomes were organised in coiled tapes. The small subunit rRNA gene was sequenced and maximum parsimony analysis placed the microsporidian described here in the clade that includes the genera Ichthyosporidium, Loma and Pseudoloma. Based on differences from previously described microsporidians, such as ultrastructural characteristics of the xenoma, developmental stages including the spore and phylogenetic analysis supported the recognition of a new species, herein named L. psittaca n. sp.

Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss).

In the present study, the pattern of immuno-reactive ladderlectin and intelectin in healthy rainbow trout is compared to rainbow trout infected with a variety of infectious agents. In healthy rainbow trout, both proteins were localized to individual epithelial cells of the gill and intestine and both proteins were clearly demonstrated within cytoplasmic granules of polymorphonuclear leucocytes and macrophages/monocytes found in blood vessels, hepatic sinusoids, renal interstitium, mucosal epithelium and submucosa of normal intestine. In tissue from infected rainbow trout, there was an overall relative increase in both lectins compared to healthy fish and both proteins were detected in extra-cellular spaces surrounding bacteria, fungi and protozoa. Increased distribution and density of both RTLL and RTInt was demonstrated along mucosal surfaces and within inflammatory leucocytes in infected tissues and immune related organs. These findings represent one of the few examples of in vivo association of defence lectins and infectious agents.

Effects of dexamethasone on host innate and adaptive immune responses and parasite development in rainbow trout Oncorhynchus mykiss infected with Loma salmonae.

The effects of dexamethasone (dex) treatment on infections with the microsporidian parasite, Loma salmonae and the effects of dex on initiation of the adaptive immune response were investigated in rainbow trout, Oncorhynchus mykiss experimentally infected with the parasite. Dex treatment resulted in significantly higher infections with the parasite in the gills and other internal organs, suggesting that dex inhibits aspects of the innate immune response to L. salmonae; the heavier infections in the gills and organs of rainbow trout resembled infections seen in Chinook salmon. Mean xenoma counts per microscope field in the gills of fish infected with L. salmonae treated with dex or left untreated were 169 and 30, respectively. Although higher numbers of xenomas were observed in dex treated fish, the xenomas were generally smaller in size than in infected control fish. The xenomas in dex treated fish showed morphological signs of degeneration including loss and degeneration of early parasite stages, accumulation of amorphous material in xenomas, and infiltration with phagocytic cells containing degenerated parasites. The xenomas in infected untreated fish had larger xenomas with a more uniform size and contained identifiable parasite stages in the cytoplasm. According to this study, once fish have developed an adaptive immune response to the parasite by previous exposure, then fish have 100% protection to reinfection even when treated with heavy doses of dex. L. salmonae immune fish treated or untreated with dex during reinfection with the parasite developed no xenomas in the gills 6 weeks post reinfection. These results indicate that once the cellular response is primed to L. salmonae, then dex related immunosuppression does not reduce the effectiveness of the adaptive immune response.

Development of an effective whole-spore vaccine to protect against microsporidial gill disease in rainbow trout (Oncorhynchus mykiss) by using a low-virulence strain of Loma salmonae.

In determining the effective vaccine spore dose of a low-virulence strain of Loma salmonae to limit microsporidial gill disease in trout, we found that fish receiving 10(3) to 10(5) killed spores had the best protection against experimental infection, with 85% fewer xenomas in their gills than in the controls. Intraperitoneal delivery of the vaccine was effective, and the addition of adjuvant did not improve vaccine performance against this disease-causing microsporidian.

Ultrastructural examination of the host inflammatory response within gills of netpen reared chinook salmon (Oncorhynchus tshawytscha) with Microsporidial Gill Disease.

The sequence of host changes following the rupture of spore-laden xenomas of the microsporidian Loma salmonae during Microsporidial Gill Disease of Salmon was deduced from ultrastructural examination of the gills of naturally infected, moribund, chinook salmon from a commercial aquaculture site. The gills contained many stages of parasite development suggesting fish were chronically exposed to the parasite. Intact xenomas were generally found beneath the endothelium in arteries and arterioles and were encapsulated by a layer of collagen containing fibroblasts sometimes joined by desmosomes. Xenoma dissolution was characterized by neutrophil infiltration and loss of the xenoma plasma membrane and encapsulation. The inflammatory responses associated with ruptured xenomas ranged from acute lesions, denoted by a marked neutrophil infiltration and vascular thrombosis, to chronic lesions with a macrophage-rich infiltrate variously accompanied by neovascularization and vascular remodelling. Dendritic-like cells and plasma cells were characteristic throughout. Basement membrane damage of the primary filament epithelium and subsequent transepithelial expulsion of spores were associated with severe inflammation. An unusual previously undescribed multifocal change, in which epithelial cells invaded deeply beyond the normal boundaries of the basement membrane, affected areas of gill filament epithelium with basement membrane damage. Some neutrophils that contained L. salmonae spores, or spore polar tube, displayed morphological changes that included irregular cell shape, cytoplasmic darkening associated with an abundance of free ribosomes, lysis of neighbouring cells, and type II nuclear clefts. Fusion of apparently intact neutrophils occurred in other areas of the lesion, where close contacts between neighbouring cells were established and in some areas plasma membrane fusion occurred. Closely associated neutrophils with intact plasma membranes were observed to contain type II nuclear clefts, abundant granules and rough endoplasmic reticulum. Other neutrophils in the lesion displayed type I nuclear pockets, which is suspected to be an early stage of apoptosis.

Whole body net ion fluxes, plasma electrolyte concentrations and haematology during a Loma salmonae infection in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum).

Loma salmonae infections of salmonids culminate in the development of branchial xenomas and subsequent focal hyperplasia of the lamellar or filament epithelium following xenoma rupture and spore release. The effects of this acute branchial disruption upon net ionic flux rates and plasma electrolyte concentrations were determined in juvenile rainbow trout given an experimental oral exposure to L. salmonae. Mean numbers of branchial xenomas peaked at week 5 post-exposure (PE), which coincided with a reduction in the specific growth rate, although there were no significant differences in mass, length or condition of Loma-exposed fish compared with unexposed controls. Following exposure, negative net whole body Na(+) and K(+) fluxes decreased, whereas net Cl(-) fluxes remained unchanged compared with non-exposed control fish. At week 3 PE during the initial branchial xenoma formation stage, there was a significant negative whole body net K(+) flux in Loma-exposed trout compared with other points during the exposure and subsequent infection. Additionally, Loma-exposed fish had marginally elevated plasma Na(+) and Cl(-) concentrations, whilst K(+) levels remained unchanged, compared with control fish. Although there was a progressive decrease in leucocrit, haematocrit remained unchanged over the course of the Loma exposure and subsequent infection. These results suggest that ionic compensation can occur at the gills during the development of xenomas during exposure to L. salmonae and the resultant infection, therefore allowing defence of plasma electrolyte concentrations, unlike the acute ionic disturbances seen with some other parasitic diseases.