Occurrence, source tracking and removal of antibiotics in recirculating aquaculture systems (RAS) in southern China.

The recirculating aquaculture system (RAS) has attracted much attention in China as a way to rapidly transform and upgrade aquaculture ponds to realize zero-emissions of pollutants in aquaculture tail water. Tail water purification ponds (TWPPs) play an important role in the treatment of aquaculture wastewater. However, until now, there have been few reports on the occurrence of antibiotics in RAS and the removal of antibiotics from the TWPPs of RAS. Therefore, this study focused on the occurrence of antibiotics in a typical ecological RAS. For comparison, the same measurements were simultaneously carried out in nearby open aquaculture ponds and rivers. The pollution level and spatial distribution of antibiotics in the RAS and the removal of antibiotics in the TWPPs were explored. The results showed that (1) eleven and twelve antibiotics were detected in water and sediment samples in the RAS, respectively, but no antibiotics were found in fish muscles and feed. Erythromycin (ERY), lincomycin (LIN), and ciprofloxacin (CFX) were the three main types of antibiotics found in water and sediment samples. (2) The TWPPs of the RAS can effectively remove antibiotics in aquaculture water. The antibiotic concentration in recirculating aquaculture ponds of the RAS was as high as 180 ng/L. After treatments in the TWPPs, the antibiotic concentration of aquaculture water decreased to 81.6 ng/L (3) The antibiotic concentrations in recirculating aquaculture ponds (25.2-180 ng/L) were lower than those in the nearby open aquaculture ponds (126-267.3 ng/L), and the concentration of antibiotics in the sediments of recirculating aquaculture ponds was up to 22.9 ng/g, while that in TWPPs was as high as 56.1 ng/g. In conclusion, the antibiotic residues in the RAS were low after antibiotics were banned in feed in China, and the removal of antibiotics in the TWPPs was more pronounced. Furthermore, cross-contamination was found between the RAS, surrounding open aquaculture ponds and the river, and the water supply of the RAS was likely to be the main contributor of antibiotics in the aquaculture environments. This study can help the government formulate discharge standards for antibiotics in aquaculture and also provide a reference for the transformation and upgrading of aquaculture ponds to achieve a zero-emission aquaculture mode.

Genomic and transcriptomic analyses reveal metabolic complementarity between whiteflies and their symbionts.

Nutritional mutualism between insects and symbiotic bacteria is widespread. The various sap-feeding whitefly species within the Bemisia tabaci complex associate with the same obligate symbiont (Portiera) and multiple secondary symbionts. It is often assumed that some of the symbionts residing in the whiteflies play crucial roles in the nutritional physiology of their insect hosts. Although effort has been made to understand the functions of the whitefly symbionts, the metabolic complementarity offered by these symbionts to the hosts is not yet well understood. We examined two secondary symbionts, Arsenophonus and Wolbachia, in two species of the B. tabaci whitefly complex, provisionally named as Asia II 3 and China 1. Genomic sequence analyses revealed that Arsenophonus and Wolbachia retained genes responsible for the biosynthesis of B vitamins. We then conducted transcriptomic surveys of the bacteriomes in these two species of whiteflies together with that in another species named MED of this whitefly complex previously reported. The analyses indicated that several key genes in B vitamin syntheses from the three whitefly species were identical. Our findings suggest that, similar to another secondary symbiont Hamiltonella, Arsenophonus and Wolbachia function in the nutrient provision of host whiteflies. Although phylogenetically distant species of symbionts are associated with their respective hosts, they have evolved and retained similar functions in biosynthesis of some B vitamins. Such metabolic complementarity between whiteflies and symbionts represents an important feature of their coevolution.

B-vitamin nutrition in the pea aphid-Buchnera symbiosis.

Various insects that utilize vitamin-deficient diets derive a supplementary supply of these micronutrients from their symbiotic microorganisms. Here, we tested the inference from genome annotation that the symbiotic bacterium Buchnera aphidicola in the pea aphid Acyrthosiphon pisum provides the insect with vitamins B2 and B5 but no other B-vitamins. Contrary to expectation, aphid survival over five days of larval development on artificial diets individually lacking each B-vitamin not synthesized by Buchnera was not significantly reduced, despite significantly lower carcass B1, B3, B6 and B7 concentrations in the aphids on diets lacking each of these B-vitamins than on the vitamin-complete diet. Aphid survival was, however, significantly reduced on diet containing low concentrations (≤0.2 mM) or no pantothenate (B5). Complementary transcriptome analysis revealed low abundance of the sense-transcript, but high abundance of the antisense transcript, of the Buchnera gene panC encoding the enzyme mediating the terminal reaction in pantothenate synthesis. We hypothesize that metabolic constraints or antisense transcripts may reduce Buchnera-mediated production of pantothenate, resulting in poor aphid performance on pantothenate-free diets. The discrepancy between predictions from genome data and empirical data illustrates the need for physiological study to test functional inferences made from genome annotations.

A newly recorded Rickettsia of the Torix group is a recent intruder and an endosymbiont in the whitefly Bemisia tabaci.

The bacterium Rickettsia is found widely in phytophagous insects and often exerts profound effects on the phenotype and fitness of its hosts. Here, we decrypt a new, independent, phylogenetically ancient Torix Rickettsia endosymbiont found constantly in a laboratory line of an economically important insect Asia II 7, a putative species of the Bemisia tabaci whitefly complex (Hemiptera: Aleyrodidae), and occasionally in field whitefly populations. This new Rickettsia distributes throughout the body of its whitefly host. Genetically, compared to Rickettsia_bellii_MEAM1 found earlier in whiteflies, the new Rickettsia species has more gene families and pathways, which may be important factors in shaping specific symbiotic relationships. We propose the name 'Candidatus Rickettsia_Torix_Bemisia_tabaci (RiTBt)' for this new endosymbiont associated with whiteflies. Comparative genomic analyses indicate that RiTBi may be a relatively recent intruder in whiteflies given its low abundance in the field and relatively larger genome compared to Rickettsia_bellii_MEAM1.

Conservation of transcriptional elements in the obligate symbiont of the whitefly Bemisia tabaci.

BACKGROUND: Bacterial symbiosis is widespread in arthropods, especially in insects. Some of the symbionts undergo a long-term co-evolution with the host, resulting in massive genome decay. One particular consequence of genome decay is thought to be the elimination of transcriptional elements within both the coding region and intergenic sequences. In the whitefly Bemisia tabaci species complex, the obligate symbiont Candidatus Portiera aleyrodidarum is of vital importance in nutrient provision, and yet little is known about the regulatory capacities of it. METHODS: Portiera genomes of two whitefly species in China were sequenced and assembled. Gene content of these two Portiera genomes was predicted, and then subjected to Kyoto Encyclopedia of Genes and Genomes pathway analysis. Together with two other Portiera genomes from whitefly species available previously, four Portiera genomes were utilized to investigate regulatory capacities of Portiera, focusing on transcriptional elements, including genes related with transcription and functional elements within the intergenic spacers. RESULTS: Comparative analyses of the four Portiera genomes of whitefly B. tabaci indicate that the obligate symbionts Portiera is similar in different species of whiteflies, in terms of general genome features and possible functions in the biosynthesis of essential amino acids. The screening of transcriptional factors suggests compromised ability of Portiera to regulate the essential amino acid biosynthesis pathways. Meanwhile, thermal tolerance ability of Portiera is indicated with the detection of a σ32 factor, as well as two predicted σ32 binding sites. Within intergenic spacers, functional elements are predicted, including 37 Shine-Dalgarno sequences and 34 putative small RNAs.

Genome-wide identification and characterization of HSP gene superfamily in whitefly (Bemisia tabaci) and expression profiling analysis under temperature stress.

Heat shock proteins (HSP) are essential molecular chaperones that play important roles in the stress stimulation of insects. Bemisia tabaci, a phloem feeder and invasive species, can cause extensive crop damage through direct feeding and transmission of plant viruses. Here we employed comprehensive genomics approaches to identity HSP superfamily members in the Middle East Asia Minor 1 whitefly genome. In total, we identified 26 Hsp genes, including three Hsp90, 17 Hsp70, one Hsp60 and five sHSP (small heat shock protein) genes. The HSP gene superfamily of whitefly is expanded compared with the other five insects surveyed here. The gene structures among the same families are relatively conserved. Meanwhile, the motif compositions and secondary structures of BtHsp proteins were predicted. In addition, quantitative polymerase chain reaction analysis showed that the expression patterns of BtHsp gene superfamily were diverse across different tissues of whiteflies. Most Hsp genes were induced or repressed by thermal stress (40°C) and cold treatment (4°C) in whitefly. Silencing the expression of BtHsp70-6 significantly decreased the survival rate of whitefly under 45°C. All the results showed the Hsps conferred thermo-tolerance or cold-tolerance to whiteflies that protect them from being affected by detrimental temperature conditions. Our observations highlighted the molecular evolutionary properties and the response mechanism to temperature assaults of Hsp genes in whitefly.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci.

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Sequencing and comparison of the Rickettsia genomes from the whitefly Bemisia tabaci Middle East Asia Minor I.

The whitefly, Bemisia tabaci, harbors the primary symbiont 'Candidatus Portiera aleyrodidarum' and a variety of secondary symbionts. Among these secondary symbionts, Rickettsia is the only one that can be detected both inside and outside the bacteriomes. Infection with Rickettsia has been reported to influence several aspects of the whitefly biology, such as fitness, sex ratio, virus transmission and resistance to pesticides. However, mechanisms underlying these differences remain unclear, largely due to the lack of genomic information of Rickettsia. In this study, we sequenced the genome of two Rickettsia strains isolated from the Middle East Asia Minor 1 (MEAM1) species of the B. tabaci complex in China and Israel. Both Rickettsia genomes were of high coding density and AT-rich, containing more than 1000 coding sequences, much larger than that of the coexisted primary symbiont, Portiera. Moreover, the two Rickettsia strains isolated from China and Israel shared most of the genes with 100% identity and only nine genes showed sequence differences. The phylogenetic analysis using orthologs shared in the genus, inferred the proximity of Rickettsia in MEAM1 and Rickettsia bellii. Functional analysis revealed that Rickettsia was unable to synthesize amino acids required for complementing the whitefly nutrition. Besides, a type IV secretion system and a number of virulence-related genes were detected in the Rickettsia genome. The presence of virulence-related genes might benefit the symbiotic life of the bacteria, and hint on potential effects of Rickettsia on whiteflies. The genome sequences of Rickettsia provided a basis for further understanding the function of Rickettsia in whiteflies.

Equine schlafen 11 restricts the production of equine infectious anemia virus via a codon usage-dependent mechanism.

Human schlafen11 is a novel restriction factor for HIV-1 based on bias regarding relative synonymous codon usage (RSCU). Here, we report the cloning of equine schlafen11 (eSLFN11) and the characteristics of its role in restricting the production of equine infectious anemia virus (EIAV), a retrovirus similar to HIV-1. Overexpression of eSLFN11 inhibited EIAV replication, whereas knockdown of endogenous eSLFN11 by siRNA enhanced the release of EIAV from its principal target cell. Notably, although eSLFN11 significantly suppressed expression of viral Gag protein and EIAV release into the culture medium, the levels of intracellular viral early gene proteins Tat and Rev and viral genomic RNA were unaffected. Coincidently, similar altered patterns of codon usage bias were observed for both the early and late genes of EIAV. Therefore, our data suggest that eSLFN11 restricts EIAV production by impairing viral mRNA translation via a mechanism that is similar to that employed by hSLFN11 for HIV-1.

Genome reduction and potential metabolic complementation of the dual endosymbionts in the whitefly Bemisia tabaci.

BACKGROUND: The whitefly Bemisia tabaci is an important agricultural pest with global distribution. This phloem-sap feeder harbors a primary symbiont, "Candidatus Portiera aleyrodidarum", which compensates for the deficient nutritional composition of its food sources, and a variety of secondary symbionts. Interestingly, all of these secondary symbionts are found in co-localization with the primary symbiont within the same bacteriocytes, which should favor the evolution of strong interactions between symbionts. RESULTS: In this paper, we analyzed the genome sequences of the primary symbiont Portiera and of the secondary symbiont Hamiltonella in the B. tabaci Mediterranean (MED) species in order to gain insight into the metabolic role of each symbiont in the biology of their host. The genome sequences of the uncultured symbionts Portiera and Hamiltonella were obtained from one single bacteriocyte of MED B. tabaci. As already reported, the genome of Portiera is highly reduced (357 kb), but has kept a number of genes encoding most essential amino-acids and carotenoids. On the other hand, Portiera lacks almost all the genes involved in the synthesis of vitamins and cofactors. Moreover, some pathways are incomplete, notably those involved in the synthesis of some essential amino-acids. Interestingly, the genome of Hamiltonella revealed that this secondary symbiont can not only provide vitamins and cofactors, but also complete the missing steps of some of the pathways of Portiera. In addition, some critical amino-acid biosynthetic genes are missing in the two symbiotic genomes, but analysis of whitefly transcriptome suggests that the missing steps may be performed by the whitefly itself or its microbiota. CONCLUSIONS: These data suggest that Portiera and Hamiltonella are not only complementary but could also be mutually dependent to provide a full complement of nutrients to their host. Altogether, these results illustrate how functional redundancies can lead to gene losses in the genomes of the different symbiotic partners, reinforcing their inter-dependency.

Draft genome sequence of Rickettsia sp. strain MEAM1, isolated from the whitefly Bemisia tabaci.

We report the draft genome sequence of the Rickettsia sp. strain MEAM1, which is a facultative symbiont from an invasive species of the whitefly Bemisia tabaci. The total length of the assembled genome is approximately 1.24 Mb, with 335 scaffolds and 1,247 coding sequences predicted within the genome.

[Mossy fiber sprouting and synapse formation in the dentate gyrus of temporal lobe epilepsy rats induced by pilocarpine].

OBJECTIVE: To investigate the relationship between mossy fiber axon sprouting (MFS), synaptic reorganization in the dentate gyrus, and the mechanism of chronic temporal lobe epilepsy induced by pilocarpine. METHODS: The mossy fiber sprouting and synapse formation were observed in rats after the acute status epileticus and chronic spontaneous temporal lobe epilepsy induced by pilocarpine using in situ hybridization of GAP-43 mRNA (a marker of MFS), an immunohistochemical staining of synaptophysin (P38, a marker of synaptogenes), and Neo-Timm staining. RESULTS: In hippocampus granule cell, there was a significant increase in the GAP-43mRNA levels measured 6 - 12 h after the end of status epileticus induced by pilocarpine. In contrast, synaptophysin immunoreactivity progressively increased in the molecular layer of the dentate gyrus from 7 d, which was in line with the Neo-Timm stain results that revealed a time-dependent growth of aberrant MFS into the dentate inner molecular layer. The GAP-43 mRNA levels in pilocarpine treated animals were significantly decreased after status epileticus. CONCLUSION: These results provide evidence for newly formed excitatory recurrent circuits, which might constitute a structural basis for chronic enhanced excitation and epileptogenesis in the hippocampus of pilocarpine-treated rats.