Gut Microvilli Research Articles

Composition and abundance of midgut plasma membrane proteins in two major hemipteran vectors of plant viruses, Bemisia tabaci and Myzus persicae.

Multiple species within the order Hemiptera cause severe agricultural losses on a global scale. Aphids and whiteflies are of particular importance due to their role as vectors for hundreds of plant viruses, many of which enter the insect via the gut. To facilitate the identification of novel targets for disruption of plant virus transmission, we compared the relative abundance and composition of the gut plasma membrane proteomes of adult Bemisia tabaci (Hemiptera: Aleyrodidae) and Myzus persicae (Hemiptera: Aphididae), representing the first study comparing the gut plasma membrane proteomes of two different insect species. Brush border membrane vesicles were prepared from dissected guts, and proteins extracted, identified and quantified from triplicate samples via timsTOF mass spectrometry. A total of 1699 B. tabaci and 1175 M. persicae proteins were identified. Following bioinformatics analysis and manual curation, 151 B. tabaci and 115 M. persicae proteins were predicted to localize to the plasma membrane of the gut microvilli. These proteins were further categorized based on molecular function and biological process according to Gene Ontology terms. The most abundant gut plasma membrane proteins were identified. The ten plasma membrane proteins that differed in abundance between the two insect species were associated with the terms "protein binding" and "viral processes." In addition to providing insight into the gut physiology of hemipteran insects, these gut plasma membrane proteomes provide context for appropriate identification of plant virus receptors based on a combination of bioinformatic prediction and protein localization on the surface of the insect gut.

The influence of biofloc system on Vibrio composition, the growth and the gut microvilli performance of the Pacific white shrimp Penaeus vannamei

Biofloc technology has been shown to have a positive impact on shrimp culture by controlling pathogenic Vibrio. This study aimed to analyze the effect of biofloc on the Vibrio composition in water and shrimp gut, as well as the growth performance and microvilli of Pacific white shrimp (Penaeus vannamei). Shrimp post-larvae measuring 0.66 ± 0.02 g were reared in a glass aquarium (working volume 22 L) of 6 units with a density of 150 shrimp m-3 for 28 days. The treatments included rearing in the biofloc system with C/N ratio 10, and without a biofloc system as a control. The results showed that Vibrio was highly prevalent in the control gut (4.13%) and biofloc water (3.77%), but only a few were found in the control water (0.16%) and biofloc-treated gut (0.11%). V. hepatarius (1.10%) and V. nereis (1.06%) were found to dominate the Vibrio bacterial community in the biofloc system maintenance media, while Vibrio sp. Hep-1b-8 (2.26%) and V. parahaemolyticus (0.80%) known as a pathogenic bacteria dominated the control shrimp gut. The biofloc system significantly increased the digestive enzyme activity, growth performance, and microvilli length in the shrimp gut. In conclusion, the application of a biofloc system in shrimp culture can affect the composition and abundance of bacterial communities in both the culture environment and the shrimp gut, and improve growth performance with higher digestive enzyme activity and longer microvilli in the gut.
 
 Keywords: biocontrol, biofloc, microbiota, shrimp, Vibrio
 
 ABSTRAK
 
 Teknologi bioflok menunjukkan dampak positif pada kegiatan budidaya udang vaname dengan mengendalikan bakteri patogen salah satunya Vibrio. Penelitian ini bertujuan untuk menganalisis efek bioflok terhadap komposisi Vibrio pada air dan usus udang, serta kinerja pertumbuhan dan mikrovili udang vaname (Penaeus vanamei). Udang post-larva berukuran 0.66 ± 0.02 g dipelihara pada akuarium kaca (volume air 22 L) sebanyak 6 unit dengan kepadatan 150 ekor m-3 selama 28 hari. Perlakuan meliputi pemeliharaan udang pada sistem bioflok dengan rasio C/N 10, dan tanpa sistem bioflok sebagai perlakuan kontrol. Hasil pengamatan menunjukkan kelimpahan Vibrio yang cukup tinggi pada usus perlakuan kontrol (4.13%) dan air perlakuan bioflok (3.77%), tetapi sangat sedikit ditemukan pada air perlakuan kontrol dan usus perlakuan bioflok. Selanjutnya, V. hepatarius (2.26%) dan V. nereis (1.06%) terdeteksi mendominasi komunitas bakteri Vibrio di media pemeliharaan pada sistem bioflok, sedangkan Vibrio sp. Hep-1b-8 (2.26%) dan V. parahaemolyticus (0.80%) mendominasi usus udang vaname pada perlakuan kontrol. Sistem bioflok juga mampu meningkatkan aktivitas enzim pencernaan, performa pertumbuhan, dan panjang mikrovili usus secara signifikan (P < 0.05). Kesimpulan dari penelitian ini adalah penerapan sistem bioflok pada budidaya udang vaname mampu memengaruhi komposisi dan kelimpahan komunitas bakteri Vibrio pada lingkungan budidaya maupun pada usus udang, serta meningkatkan performa pertumbuhan dengan aktivitas enzim pencernaan yang lebih baik dan mikrovilli usus yang lebih panjang.
 
 Kata kunci: bioflok, biokontrol, microbiota, udang vaname, Vibrio

Citrobacter freundii, a natural associate of the Colorado potato beetle, increases larval susceptibility to Bacillus thuringiensis.

We assume that certain representatives of gut microflora mediate immune changes during dysbiosis, accelerating septicemia caused by Bacillus thuringiensis. Co-introduction of Citrobacter freundii with Bacillus thuringiensis var. tenebrionis (morrisoni) (Bt) led to an increase in Colorado potato beetle (CPB) larval mortality to 69.0% (1.3-5×) and a synergistic effect was observed from day 1 to day 6. Ultrathin sections of the CPB midgut showed autophagosome formation and partial destruction of gut microvilli under the influence of Bt, which was accompanied by pronounced hypersecretion of the endoplasmic reticulum with apocrine vesicle formation and oncotic changes in cells under the action of C. freundii. The destruction of gut tissues was accompanied by suppression of detoxification processes under the action of the bacteria and a decrease (2.8-3.5×) in the concentration of lipid oxidation products during Bt infection. In the first hours post combined treatment, we registered a slight increase in the total hemocyte count (THC) especially a predomination (1.4×) of immune-competent plasmatocytes. Oral administration of symbiotic and entomopathogenic bacteria to the CPB larvae significantly decreased the THC (1.4×) after 24 h and increased (1.1-1.5×) the detoxifying enzymes level in the lymph. These changes are likely to be associated with the destruction of hemocytes and the need to remove the toxic products of reactive oxygen species. The obtained results indicate that feeding of C. freundii and B. thuringiensis to the CPB larvae is accompanied by tissue changes that significantly affect the cellular and humoral immunity of the insect, increasing its susceptibility to Bt. © 2022 Society of Chemical Industry.

Flotillin 2 Facilitates the Infection of a Plant Virus in the Gut of Insect Vector.

ABSTRACTMost plant viruses require insect vectors for transmission. One of the key steps for the transmission of persistent-circulative plant viruses is overcoming the gut barrier to enter epithelial cells. To date, little has been known about viral cofactors in gut epithelial cells of insect vectors. Here, we identified flotillin 2 as a plasma membrane protein that facilitates the infection of rice stripe virus (RSV) in its vector, the small brown planthopper. Flotillin 2 displayed a prominent plasma membrane location in midgut epithelial cells. The nucleocapsid protein of RSV and flotillin 2 colocalized on gut microvilli, and a nanomolar affinity existed between the two proteins. Knockout of flotillin 2 impeded the entry of virions into epithelial cells, resulting in a 57% reduction of RSV levels in planthoppers. The knockout of flotillin 2 decreased disease incidence in rice plants fed by viruliferous planthoppers from 40% to 11.7%. Furthermore, flotillin 2 mediated the infection of southern rice black-streaked dwarf virus in its vector, the white-backed planthopper. This work implies the potential of flotillin 2 as a target for controlling the transmission of rice stripe disease.IMPORTANCE Plant viral diseases are a major threat to world agriculture. The transmission of 80% of plant viruses requires vector insects, and 54% of vector-borne plant viruses are persistent-circulative viruses, which must overcome the barriers of gut cells with the help of proteins on the cell surface. Here, we identified flotillin 2 as a membrane protein that mediates the cell entry of rice stripe virus in its vector insect, small brown planthopper. Flotillin 2 displays a prominent cellular membrane location in midgut cells and can specifically bind to virions. The loss of flotillin 2 impedes the entry of virions into the midgut cells of vector insects and substantially suppresses viral transmission to rice. Therefore, flotillin 2 may be a promising target gene for manipulation in vector insects to control the transmission of rice stripe disease and perhaps that of other rice virus diseases in the future.

Reduced fitness of the mosquito Culex pipiens (Diptera: Culicidae) after feeding on a blood meal with hepatitis C virus

Hepatitis C virus (HCV) is a blood-borne virus. Given that mosquitoes can take blood meals from HCV patients, we aimed to test whether HCV in the blood meal can induce alterations in the biology of Culex pipiens. To address this aim, Cx. pipiens females were fed HCV-negative blood from healthy individuals or HCV-positive fresh blood samples harvested from viremic HCV patients. Replication of HCV in mosquitoes was confirmed by negative strand-specific RT-PCR and sequencing of RNA extracted from the mosquito bodies 7 days post-feeding. In addition, several parameters that determine the fitness of the mosquitoes were measured. Virus acquisition was associated with alterations in the architecture of the gut microvilli and the immune response, indicated by an increase in phenol oxidase activity. Interestingly, the mosquitoes that were fed the HCV-positive blood meal showed shorter median longevity (8 days) and laid fewer eggs than the control mosquitoes. Furthermore, the offspring of females fed the HCV-positive blood meal demonstrated a lower emergence rate than the controls. In sum, the results indicate that feeding on HCV by Cx. pipiens decreases fitness, which may, in turn, affect its potential as a vector.

Biological Effects of Activated Carbon on Benthic Macroinvertebrates are Determined by Particle Size and Ingestibility of Activated Carbon.

The application of activated carbon (AC) to the surface of contaminated sediments is a promising technology for sediment remediation in situ. Amendment with AC has proved to be effective in reducing bioavailability and sediment-to-water release of hydrophobic organic contaminants. However, AC may cause positive or negative biological responses in benthic organisms. The causes of these effects, which include changes in growth, reproduction, and mortality, are unclear but are thought to be related to the size of AC particles. The present study investigated biological response to AC ranging from ingestible powdered AC to noningestible granular AC in two benthic deposit feeders: the polychaete Marenzelleria spp. and the clam Limecola balthica (syn. Macoma balthica). In the polychaete, exposure to powdered AC (ingestible) reduced both dry weight and carbon assimilation, whereas exposure to granular AC (noningestible) increased both dry weight and carbon assimilation. Responses in the clam were similar but less pronounced, indicating that response levels are species-specific and may vary within a benthic community. In addition, worms exposed to the finest ingestible AC particles had reduced gut microvilli length and reduced gut lumen, indicating starvation. These results strongly suggest that biological responses to AC depend on particle ingestibility, whereby exposure to ingestible particles may cause starvation through reduced bioavailability of food coingested with AC or due to rejection of AC-treated sediment as a food source. Environ Toxicol Chem 2021;40:3465-3477. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effects of synthetic and natural microfibers on Daphnia magna-Are they dependent on microfiber type?

Microfibers, which are sourced from textiles and some products from the fishery industry, are the biggest contributors to microplastic pollution in aquatic ecosystems. In addition to these synthetic microfibers, naturally derived microfibers can also be found in aquatic environments. However, there are limited studies on the ecotoxicity of natural microfibers. To shed light on this topic, this study assessed and compared the toxicity of natural and synthetic microfibers on Daphnia magna, using lyocell, polyester (PET) and polypropylene (PP) microfibers. To evaluate the adverse effect of microfibers on D. magna, after effects including depuration, food intake, growth, mortality, and immobilization rate were continually observed for up to 96h after the initial 48h of exposure to the microfibers. Immobilization rate decreased in the following order: PP, PET, and lyocell. However, the depuration of microfibers in the lyocell and PET treatment groups was similar, with higher mortality rates than in the PP treatment group. Furthermore, despite the high rates of food intake following exposure, the lyocell and PET exposed groups exhibited growth inhibition during the same period. This growth inhibition corresponded with, and was likely due to, reductions in the length of gut microvilli, probably an expression of gut damage, which is believed to have reduced nutrient absorption in the affected individuals. Based on the results of this study, it was confirmed that even natural microfibers, and not just synthetic microfibers, can have adverse effects on aquatic organisms. This study confirmed not only the toxicity of microfibers, but also the consequences of their after effects. These results could be the basis for future research on the after effects of microplastics on aquatic organisms and provide directions for further microplastic ecotoxicity studies.

Chitin Synthases Are Critical for Reproduction, Molting, and Digestion in the Salmon Louse (Lepeophtheirus salmonis).

Chitin synthase (CHS) is a large transmembrane enzyme that polymerizes Uridine diphosphate N-acetylglucosamine into chitin. The genomes of insects often encode two chitin synthases, CHS1 and CHS2. Their functional roles have been investigated in several insects: CHS1 is mainly responsible for synthesizing chitin in the cuticle and CHS2 in the midgut. Lepeophtheirus salmonis is an ectoparasitic copepod on salmonid fish, which causes significant economic losses in aquaculture. In the present study, the tissue-specific localization, expression, and functional role of L. salmonis chitin synthases, LsCHS1 and LsCHS2, were investigated. The expressions of LsCHS1 and LsCHS2 were found in oocytes, ovaries, intestine, and integument. Wheat germ agglutinin (WGA) chitin staining signals were detected in ovaries, oocytes, intestine, cuticle, and intestine in adult female L. salmonis. The functional roles of the LsCHSs were investigated using RNA interference (RNAi) to silence the expression of LsCHS1 and LsCHS2. Knockdown of LsCHS1 in pre-adult I lice resulted in lethal phenotypes with cuticle deformation and deformation of ovaries and oocytes in adult lice. RNAi knockdown of LsCHS2 in adult female L. salmonis affected digestion, damaged the gut microvilli, reduced muscular tissues around the gut, and affected offspring. The results demonstrate that both LsCHS1 and LsCHS2 are important for the survival and reproduction in L. salmonis.

WITHDRAWN: Reduced fitness of the mosquito Culex pipiens (Diptera: Culicidae) after feeding on a blood meal with hepatitis C virus

Hepatitis C virus (HCV) is a blood-borne virus. Given that mosquitoes can take blood meals from healthy individuals as wells as HCV patients, we proved in a previous study that Culex pipiens can transmit HCV to human blood samples. In the present study, we aimed to test whether HCV in the blood meal can induce alterations in the biology of Cx. pipiens. To address this aim, Cx. pipiens females were fed HCV-positive fresh blood samples harvested from viremic HCV patients. The control mosquitoes were fed fresh HCV-free blood samples. Fragments of the HCV RNA negative strand were extracted from the mosquito bodies after 7 days of feeding, and RT-PCR sequenced to confirm the virus replication. In addition, several parameters that measure the fitness of the mosquitoes were observed. HCV acquired by the mosquitoes was able to replicate. Virus acquisition was associated with alterations in the architecture of the gut microvilli and the immune response, indicated by an increase in phenoloxidase activity. Interestingly, the mosquitoes that were fed the HCV-positive blood meal showed shorter median longevity (8 days) and laid fewer eggs than the control mosquitoes. Furthermore, the offspring of females fed the HCV-positive blood meal demonstrated a lower emergence rate than the controls. In sum, our results indicate that feeding on HCV by Cx. pipiens decreases fitness, which may, in turn, affect potential as a vector.

Effects of dietary probiotic supplementation on the growth, gut health and disease resistance of juvenile Nile tilapia (Oreochromis niloticus)

This study investigated the effects of the Streptococcus agalactiae antagonizing probiotics Bacillus cereus NY5 and Bacillus subtilis as feed additives for Nile tilapia in terms of growth performance, intestinal health and resistance to S. agalactiae. A total of 720 apparently healthy juvenile Nile tilapia (0.20 ± 0.05 g) were randomly divided into 4 equal groups with 3 replicates for each group. Fish were fed a basal diet (control check group, CK group) supplemented with B. subtilis (1 × 108 CFU/g feed, BS group), B. cereus NY5 (1 × 108 CFU/g feed, BC group), and B. subtilis + B. cereus NY5 (0.5 × 108 CFU/g feed of each probiotic, BS + BC group) for 6 wk, and the probiotic supplementation groups were then fed the basal diet for 1 wk to investigate the gut microbial community. The results of this study showed that BS + BC and BC treatments significantly increased weight gain (WG), feed conversion ratio (FCR) and S. agalactiae resistance in Nile tilapia (P < 0.05). Gut microvilli length and density and c-type lysozyme (lyzc) gene expression were significantly increased by probiotic supplementation (P < 0.05). The results of high-throughput sequencing showed that the B. cereus NY5 and B. subtilis + B. cereus NY5-supplemented feed resulted in a significant improvement in tilapia autochthonous gut bacterial communities and had a stimulation effect on a variety of potential probiotics after 6 wk of feeding. After cessation of probiotic administration for 1 wk, the gut bacteria of the fish in the BS + BC and BC groups had minor changes and maintained a stable state. Consequently, it was inferred that, as a feed supplement, B. cereus NY5 and the mixture of B. subtilis and B. cereus NY5 at 1 × 108 CFU/g feed were able to promote growth and disease resistance, which may be associated with the supplement's effects on gut immune status, intestinal morphology, and intestinal microbial community composition.

Effects of dietary Lactobacillus rhamnosus JCM1136 and Lactococcus lactis subsp. lactis JCM5805 on the growth, intestinal microbiota, morphology, immune response and disease resistance of juvenile Nile tilapia, Oreochromis niloticus

The present study aimed to evaluate the individual and combined effects of Lactobacillus rhamnosus (LR) JCM1136 and Lactococcus lactis subsp. lactis (LL) JCM5805 on the growth, intestinal microbiota, intestinal morphology, immune response and disease resistance of juvenile Nile tilapia (Oreochromis niloticus). A total of 720 apparently healthy juvenile Nile tilapia (0.20 ± 0.05 g) were randomly divided into four equal groups. Fish were fed with a basal diet (CK) supplemented with JCM1136 (LR), JCM5805 (LL), and JCM1136 + JCM5805 (LR+LL) at 1 × 108 CFU/g basal diet for 6 weeks, followed by a basal diet for 1 week. After 6 weeks of feeding, the LL treatment significantly increased the growth and feed utilization of Nile tilapia when compared with the CK. Light microscopy and transmission electron microscopy images of the midgut revealed that probiotic supplementation significantly increased gut microvilli length and microvilli density compared to CK. The transcript levels of several key immune-related genes in the mid-intestine and liver of fish were analyzed by means of quantitative polymerase chain reaction (qPCR) at the end of the sixth week. The results showed the following: when compared to CK group, fish in LR had significantly increased transcript levels of IFN-γ, lyzc, hsp70 and IL-1β in the intestine; LL fish showed significantly increased expressions of TNF-α, IFN-γ, lyzc, hsp70 and IL-1β in the intestine and liver; and intestine lyzc, hsp70 and IL-1β and liver TNF-α, IFN-γ, hsp70 and IL-1β were significantly increased in LR+LL fish. Following a 6-week period of being fed probiotics or a control diet, the tilapia were challenged with an intraperitoneal injection of 20 μl of the pathogenic Streptococcus agalactiae (WC1535) (1 × 105 CFU/ml). The survival rates of the probiotic-fed groups were significantly higher than that of the CK group, and the LL group had the highest survival rate. High-throughput sequencing revealed a significantly higher presence of JCM5805 in the guts of LL fish during the period of probiotic application, but this was no longer detected in all LL samples 1 week post cessation of probiotic administration. Cessation of probiotic administration led to disorders of individual gut microbes within the LR and LL groups. Statistical analysis (LEfSe) demonstrated that three phyla, namely, Bacteroidetes, Fusobacteria and Actinobacteria were enriched in the CK group, while the abundance of Proteobacteria was greater in the probiotic-fed fish. At the genus level, Plesiomonas, which includes potential pathogens of fish, were significantly decreased in the probiotic-fed groups. In contrast, a significant increase of Rhizobium and Achromobacter, which can produce a variety of enzymes with cellulolytic and pectolytic activity, were observed in fish fed with probiotics, indicating that dietary probiotics were helpful in the propagation of some probiotic bacteria. Our data revealed that JCM1136 and JCM5805, as a feed additive at 108 CFU/g feed, could improve intestinal morphology, enhance immune status and disease resistance, and affect the gut microbiota of tilapia; thus, these additives could be used as probiotics for juvenile Nile tilapia. JCM5805 was more effective than JCM1136 or the mixture of the two for promoting the growth, enhancing the immune status and disease resistance of tilapia.

Dietary supplementation with rutin has pro-/anti-inflammatory effects in the liver of juvenile GIFT tilapia, Oreochromis niloticus

Dietary supplementation with rutin may have some pharmacological qualities including anti-inflammatory effects. Kupffer cell activation resulted in increased transcription of pro- and anti-inflammatory cytokines. The main purpose of this study was to investigate the pro- and anti-inflammatory activities in juvenile freshwater tilapia, Oreochromis niloticus, in response to 0.1 or 0.3 g/kg dietary supplementation of rutin. Results showed that hepatic IgM, anti-inflammatory-cytokines, and pro-inflammatory cytokines were significantly decreased in groups treated with high doses of rutin. Hepatic IgM and anti-inflammatory cytokines (IL-10 and IFN-γ) transcripts were significantly decreased, whereas the transcripts of the pro-inflammatory cytokines, TNFα and IL-1β were significantly decreased, whereas IL-8 was significantly increased. The number of Kupffer cells in rutin-treated groups was significantly decreased, and scanning electron micrographs showed that rutin enriched the number of gut microvilli and secretion pits. With the phenomena of cell apoptosis occurred in the rutin groups, the present study demonstrated that optimum levels of rutin may be beneficial but excessive level may cause liver impairment, which may be absorbed by the gut and then transported to the liver.

Effects of dietary live and heat-inactive baker's yeast on growth, gut health, and disease resistance of Nile tilapia under high rearing density.

In this study, the effects of baker's yeast as probiotics was evaluated in Nile tilapia reared at high density. Juvenile tilapia were distributed to tanks at high density (436fish/m(3)) and fed with basal diet (CK) or diets supplemented with live (LY) or heat-inactivated yeast (HIY). Another group of fish reared at low density (218fish/m(3)) and fed with basal diet was also included (LowCK). After 8 weeks of feeding, growth, feed utilization, gut microvilli morphology, digestive enzymes, and expressions of hsp70 and inflammation-related cytokines in the intestine were assessed. Intestinal microbiota was investigated using 16S rRNA gene pyrosequencing. Fish were challenged with Aeromonas hydrophila to evaluate disease resistance. High rearing density significantly decreased the growth, feed utilization, microvilli length, and disease resistance of fish (CK versus LowCK). Moreover, the intestinal hsp70 expression was increased in fish reared at high density, supporting a stress condition. Compared to CK group, supplementation of live yeast significantly increased gut microvilli length and trypsin activity, decreased intestinal hsp70 expression, and enhanced resistance of fish against A.hydrophila (reflected by reduced intestinal alkaline phosphatase activity 24h post infection). The gut microbiota was not markedly influenced by either rearing density or yeast supplementation. Heat-inactivated yeast (HIY) didn't display the beneficial effects observed in LY except an increase in gut trypsin activity, suggesting the importance of yeast viability and thus secretory metabolites of yeast. In conclusion, live baker's yeast may alleviate the negative effects induced by crowding stress, and has the potential to be used as probiotics for tilapia reared at high density.

Dietary microbial phytase exerts mixed effects on the gut health of tilapia: a possible reason for the null effect on growth promotion.

The present study evaluated the effects of dietary microbial phytase on the growth and gut health of hybrid tilapia (Oreochromis niloticus ♀×Oreochromis aureus ♂), focusing on the effect on intestinal histology, adhesive microbiota and expression of immune-related cytokine genes. Tilapia were fed either control diet or diet supplemented with microbial phytase (1000 U/kg). Each diet was randomly assigned to four groups of fish reared in cages (3×3×2 m). After 12 weeks of feeding, weight gain and feed conversion ratio of tilapia were not significantly improved by dietary microbial phytase supplementation. However, significantly higher level of P content in the scales, tighter and more regular intestinal mucosa folds were observed in the microbial phytase group and the microvilli density was significantly increased. The adhesive gut bacterial communities were strikingly altered by microbial phytase supplementation (0·41<similarity coefficient<0·54). Stimulated intestinal inflammation and stress status were observed in the fish fed diet supplemented with microbial phytase, as indicated by the up-regulated intestinal expressions of the cytokine genes (tnf-α and tgf-β) and hsp70. In addition, the gut microvilli height was significantly decreased in the phytase group. These results indicate that dietary microbial phytase may exert mixed effects on hybrid tilapia, and can guide our future selection of phytases as aquafeed additives - that is, eliminating those that can stimulate intestinal inflammation.

Transmission Electron Microscopy and Scanning Transmission X-Ray Microscopy Studies on the Bioaccumulation and Tissue Level Absorption of TiO2 Nanoparticles in Daphnia magna.

In this study, bioaccumulation and tissue-level absorption of TiO2 nanoparticles (NPs) in freshwater invertebrates were investigated using transmission electron microscopy (TEM) and scanning transmission X-ray microscopy (STXM). The TiO2 NPs were used to test impacts of core sizes (i.e., 5 ± 2 nm and 23 ± 7 nm for TiO2(SYN) and TiO2(P25), respectively) and agglomerations (i.e., well dispersed vs. highly agglomerated) on the uptake of TiO2 NPs in Daphnia magna (D. magna). Highly agglomerated TiO2 NPs, regardless of their core sizes, were heavily taken up into the digestive tract of D. magna and no detectable penetration of both TiO2 NPs into the gut epithelial cells of D. magna was observed in TEM and STXM images. However, significant damages involving morphological changes in the microvilli and gut epithelial cells (e.g., irregular shaped microvilli, epithelial cell protrusion, and dilatation of cytoplasmic inclusion) were observed only with the commercial TiO2 NPs (TiO2(P25)) with larger core size and mixed crystalline phase, while the laboratory synthesized TiO2 NPs (TiO2(Syn)) with smaller core size and single crystalline phase showed slight morphological changes in the gut microvilli and epithelial cells. In the case of D. magna exposed to the well dispersed synthetic TiO2 NP ((Cit)TiO2(Syn)), only a negligible amount of TiO2 NPs were found within the digestive tract of the D. magna without any significant damages in the gut microvilli and epithelial cells and any detectable penetrations of TiO2 NPs into epithelial cells of D. magna gut. These TEM and STXM observations confirmed us that uptake of NP into D. magna are strongly dependent on their agglomeration (i.e., hydrodynamic sizes), rather than their core sizes, while direct penetration of NPs into tissues of digestive tract seems unlikely without significant morphological changes (e.g., collapse of the epithelial tissue) caused by high toxicity of NPs or released metal ions.

Cloning and characterization of a novel sigma-like glutathione S-transferase from the giant panda parasitic nematode, Baylisascaris schroederi.

BackgroundBaylisascaris schroederi, an intestinal nematode of the giant panda, is the cause of the often fatal disease, baylisascariasis. Glutathione S-transferases (GSTs) are versatile enzymes that can affect parasite survival and parasite-host interactions and, are therefore, potential targets for the development of diagnostic tests and vaccines.MethodsIn this study, we identified a full-length cDNA that encoded a novel, secretory sigma-like GST (Bsc-GSTσ) from a B. schroederi-omic dataset. Following cloning and sequencing, sequence and structural analyses and comparative modeling were performed using online-bioinformatics and proteomics tools. The recombinant Bsc-GSTσ (rBsc-GSTσ) protein was prokaryotically expressed and then used to detect antigenicity and reactivity using immunoblotting assays. In addition, the native protein in female adult B. schroederi was located via immunofluorescence techniques, while the preliminary ELISA-based serodiagnostic potential of rBsc-GSTσ was assessed in native and infected mouse sera.ResultsBsc-GSTσ contained a 621-bp open reading frame that encoded a polypeptide of 206 amino acids with two typical sigma GST domain profiles, including a GST_N_Sigma_like at the N-terminus and a GST_C_Sigma_like at the C-terminus. The presence of an N-terminal signal sequence indicated that Bsc-GSTσ was a secretory protein. Sequence alignment and phylogenetic analyses showed that Bsc-GSTσ was a nematode-specific member of the Sigma class GSTs and shared the closest genetic distance with its homologue in Ascaris suum. Further comparative structure analyses indicated that Bsc-GSTσ possessed the essential structural motifs (e.g., βαβαββα) and the consensus secondary or tertiary structure that is typical for other characterized GSTσs. Immunolocalization revealed strong distributions of native Bsc-GSTσ in the body hypodermis, lateral chords, gut epithelium, gut microvilli, oviduct epithelium, and ovaries of adult female worms, similar to its homologue in A. suum. Building on good immunogenic properties, rBsc-GSTσ-based ELISA exhibited a sensitivity of 79.1% and a specificity of 82.0% to detect anti-B. schroederi IgG antibodies in the sera of experimentally infected mice.ConclusionThis study presents a comprehensive demonstration of sequence and structural-based analysis of a new, secretory sigma-like GST from a nematode, and its good serodiagnostic performance suggests that rBsc-GSTσ has the potential to detect B. schroederi and, therefore, could be used to develop an ELISA-based serological test to diagnose baylisascariasis in giant pandas.

X-ray and electron microscopy studies on the biodistribution and biomodification of iron oxide nanoparticles in Daphnia magna

Biodistribution and biomodification of iron oxide (Fe3O4 and α-Fe2O3) nanoparticles (NPs) in a well-known toxicity test organism, Daphnia magna (D. magna), were investigated using transmission electron microscopy (TEM) and scanning transmission X-ray microscopy (STXM). In addition to the morphological changes in the gut tissues of D. magna, biodistribution and biomodification of iron oxide NPs in the digestive tract of D. magna were also monitored in this study. Upon exposures to both iron oxide NPs, unique morphological changes (e.g., irregular shaped microvilli, epithelial cell protrusion, and dilatation of cytoplasmic inclusion) in the gut tissues of D. magna were observed along with bacterial colonization of the gut lumen. However, despite their heavy accumulations in the digesitive tract, TEM and STXM images confirmed us that both Fe3O4 and α-Fe2O3 NPs were not penetrating into the gut tissues of D. magna. Moreover, for the Fe3O4 NPs in direct contact with the gut microvilli of D. magna, slight but significant spectral changes were observed in their Fe L-edge X-ray absorption near edge structure (XANES) spectra, which indicated that there were biomodifications of Fe3O4 NPs, probably involving oxidative dissolution of Fe3O4 NPs followed by rapid precipitation of ferric oxide or hydroxide. However, no significant changes were observed in the Fe L-edge XANES spectra of the α-Fe2O3 NPs present in the gut lumen of D. magna. These X-ray and electron microscopic observations confirmed us that, despite similarities in core sizes and chemical compositions, NPs with different crystalline phase and dissolution rates can interact quite differently with their local environment, may result in different biodistribution and cause completely dissimilar toxicities.

Immunofluorescence localization and ultrastructure of Stewart's wilt disease bacterium Pantoea stewartii in maize leaves and in its flea beetle vector Chaetocnema pulicaria (Coleoptera: Chrysomelidae)

Pantoea stewartii is the causal agent of Stewart's wilt of sweet corn, the most serious bacterial disease of sweet corn and maize in the North-Central and Eastern USA. P. stewartii is transmitted mainly by the corn flea beetle Chaetocnema pulicaria (Coleoptera: Chrysomelidae) and this bacterium is assumed to overwinter in its vector beetle. Using immunofluorescence confocal and transmission electron microscopy (TEM) we localized P. stewartii in its host plant and insect vector. In infected maize, P. stewartii was found mainly in xylem vessels of major and minor veins as well as in intercellular spaces of infected leaves. In the vector beetle, P. stewartii was found by immunofluorescence in the foregut, midgut and hindgut up to 12 days post-acquisition (post-feeding on infected plants for 2 days). TEM of thin sections in the beetle's gut four and eight days post-acquisition revealed bacterial cells similar to those of P. stewartii in the gut lumen, close to or associated with the gut microvilli, as well as inside epithelial cells of the midgut. This suggests a stronger biological relationship between this bacterium and its vector, and that the corn flea beetle may carry P. stewartii intracellularly as well as extracellularly within the gut, which has implications in the persistence and overwintering of this bacterium in the vector and on the epidemiology of Stewart's wilt disease of corn.

Proteome changes in the intestinal mucosa of broiler (Gallus gallus) activated by probiotic Enterococcus faecium

Probiotics are supplemented to animal diet to support a well-balanced gut microbiota, finally contributing to improved health. The molecular mechanism of probiotics in animal intestine improvement is yet unclear. We investigated the production parameters, gut morphology and microbiota, and mucosal proteome of Arbor Acres broilers (Gallus gallus) supplemented with Enterococcus faecium by performing denaturing gradient gel electrophoresis, quantitative real-time PCR, two-dimensional fluorescence difference gel electrophoresis, and mass spectrometry. E. faecium supplementation promoted the development of immune organs and gut microvilli and enlarged the gut microbial diversity and population. However, it had no effects on daily weight gain and feed intake, and slightly enhanced feed conversion ratio. A total of 42 intestinal mucosal proteins were found to be differentially abundant. Four of them are related to intestinal structure and may extend the absorptive surface area. Of 17 differential proteins related to immune and antioxidant systems, only six are abundant in the broilers fed E. faecium, indicating that these chickens employ less nutrients and energy to deal with immune and antioxidant stresses. These findings have important implications for understanding the probiotic mechanisms of E. faecium on broiler intestine. Probiotic supplementation to animal diet is closely related with improved health. The objective of this study is to determine the molecular mechanisms of probiotic E. faecium achieving its biological mission in the gut of Arbor Acres broilers (G. gallus). E. faecium supplementation did not improve daily weight gain and feed intake; however, it had effects on immune organ and gut microvillus development, and gut microbial diversity and population. Quantitative proteomic analysis of the intestinal mucosa of broilers treated with E. faecium identified 42 intestinal mucosal proteins related to substance metabolism, immune and antioxidant systems, and cell structure. This study identified the E. faecium derived probiotic mechanism on the proteome level.

TRANSLATIONAL IMPACT

Enterohemorrhagic Escherichia coli (EHEC) is an emerging zoonotic pathogen that causes acute gastroenteritis and hemorrhagic colitis in humans. It also causes disease in newborn calves and asymptomatically colonizes the gut mucosa of adult bovines, therefore constituting the main reservoir for food and environmental contamination. The hallmark gut lesion induced by EHEC involves bacterial attachment to the gut epithelium and effacing of gut microvilli, but innate immune responses and inflammation are conspicuously limited. Given that EHEC is strictly a human and calf pathogen that does not infect mice, it has been a challenge to develop simple models that permit detailed investigation of virulence mechanisms. Tissue tropism cannot be mimicked in vitro because EHEC is neither species- nor cell-type-specific in culture. Although human in vitro organ cultures have been used, this model has a short lifespan, does not accurately mimic normal gut architecture and lacks an intact immune system. Therefore, a better model is needed to study the molecular bases for the human and calf specificity of EHEC, its tissue tropism and the innate immune response induced upon infection.To address this need, the authors developed an in vivo model of EHEC infection by xenotransplantation of human or bovine fetal intestinal tissue under the skin of SCID mice (which do not reject the foreign tissue owing to lack of an adaptive immune system). Xenografts of human and bovine tissue originally isolated from different gut regions develop into mature tissues and faithfully mimic the tissue of human and calf intestine. Infection of xenografts with EHEC induces the formation of typical attaching and effacing lesions and tissue damage resembling hemorrhagic colitis. By contrast, xenografts infected with an EHEC mutant deficient in the type III secretion system (a major EHEC virulence mechanism) remained undamaged. Notably, EHEC does not attach to or damage the epithelium of small intestinal tissue.This xenograft model of EHEC infection faithfully recapitulates several aspects of the natural human and calf disease, including host and tissue tropism, typical histopathology, and attenuation of innate immune responses. Therefore, this relatively simple and economical xenograft model should be effective for resolving molecular mechanisms of host and tissue tropism, as well as the immunomodulatory effects of EHEC in intact tissue. A challenging step to further developing this model would be to connect the xenografted segments within the intestines of mice in order to establish a system that is fully reconstituted with normal microflora as well as the human or bovine immune systems.