The induced favorable environment for bio-cathode formation might be the main reason for this improvement since the content of total extracellular polymeric substances (TEPS) of the substrate in the cathode area almost doubled (from 44.59 μg/g wet sludge to 87.70 μg/g wet sludge) as the percentage of PAC increased to 10%. This work provides another potential usage of PAC in CW-MFCs with a higher wastewater treatment efficiency and energy recovery.MFC centered hybrid technologies have attracted attention during the last few years due to their compatibility and dual advantages of energy recovery and wastewater treatment. In this study, a MFC was integrated into a dewatered alum sludge (DAS)- based vertical upflow constructed wetland (CW). Powder activate carbon (PAC) was used in the anode area in varied percentage with DAS to explore its influences on the performance of the CW-MFC system. The trial has demonstrated that the inclusion of PAC improved the removal efficiencies of COD, TN and RP. More significantly, increasing the proportion of PAC from 2% to 10% can significantly enhance the maximum power densities from 36.58 mW/m Natural resources for freshwater production and energy generation are depleting at an unprecedented rate. It was estimated that two-thirds of the global population will face water quality problems by 2025 while the demand for water consumption will increase more than 40% by 2050Most recently, a new hybrid technology based on the principle of MFCs was developed by embedding MFCs into constructed wetlands (CWs), giving the name CW-MFCs3453 under certain circumstances3 when the pure MFC’s volume increases to over 2 L2) at presentSignificant improvements of pure MFCs were achieved during last several years, with the highest power density reaching as high as 2870 W/mPowder activate carbon (PAC), a well-known cost-effective material with high specific area, has been widely applied as an adsorbent in various wastewater treatment processes for different pollutants removal. It has also been used to provide sufficient adhesive surface for microorganism growth, while these attached bacterial can also utilize the adsorbed and/or surrounding organic pollutants to maintain their metabolism121416171820In this study, five CW-MFC systems (four for testing and one for control) were set up, which employed dewatered alum sludge (DAS) as the wetland substrate while PAC was adopted to modify the DAS in the anode area to explore the enhanced performance on electricity generation and wastewater treatment. Emphasis was placed on the role of PAC in reducing the internal resistance (activation and ohmic losses). The influences of the percentage of PAC versus DAS on the performance of the CW-MFC were examined under continuous operation of the system in vertical flow mode.3-N and NO2-N. However, it is interesting to find out that the integration slightly improved the NH4-N and TN removal, which means the improvements in both nitrification and denitrification process since NO3-N in the effluent didn’t show the corresponding changes. Many previous studies have showed the possibility by using MFC for nitrogen removal through SND (simultaneously nitrification and denitrification) processDiluted swine water with the designed concentration was continuously fed to five parallel CW-MFC systems. Effluent quality and the related parameters of each system were examined throughout the stable operation period (about 4 months) and the results are shown in More specifically to those operated under close circuit, COD was significantly removed in all systems. Based on our previous studies426With regard to phosphate removal, it has already been shown that DAS has excellent phosphate adsorption ability due to the strong adsorption affinity between Al in DAS and P in the influent2831et al.et al.35373936Nitrobacter can directly oxidize nitrite into nitrate owing to the sufficient DO provision in the upper layer of the CW-MFC system In terms of nitrogen removal, ammonia-N removal efficiency of the control system is lower than 40% (from 26.40 ± 4.2 mg/L to 15.45 ± 3.3 mg/L). This is mainly due to the insufficient oxygen provision for the nitrification process, since without an artificial aeration system, nitrification can only happen near the surface of the system where the oxygen dissolved form air can be utilized (with DO of around 2.0 mg/L). It is also noted in At the end of the trial, 9 substrate samples from the top, middle and bottom layers of each CW-MFC system were sampled and the LOI of each sample was measured. Differences in LOI caused by different PAC additions can be observed from In order to explore the impact of PAC additions to the colonization of microbes in the system, TEPS of the substrates taken from top to bottom of each system were measured. It should be clear that more microbes refer to more secretion of metabolites to extracellular space, resulting in a higher concentration of EPS, which mainly consists of polysaccharide and protein45472 to 36.58 mW/m2 was observed. In contrast, when the percentage of PAC increased to 2%, MPD almost doubled (from 36.582 mW/m2 to 73.8 mW/m2). With further increase of PAC percentage to 10%, MPD increased to 87.79 mW/m2. From the polarization curves, it is also obvious that the open circuit voltage was significantly improved by PAC additions when its content is higher than 2% (from about 500 mV to over 700 mV).The main purpose of this study lies in exploring the role of PAC addition on the electrical performance of the CW-MFC systems. To this end, polarization and power density curves were generated  at the eIn order to explore the reasons behind those differences, electrode potentials of each system were measured . From thRecalling the content of TEPS within each system, the addition of PAC in the anode chamber will suppress the growth of microbes which might be the main reason for the decrease of the anode potential. Some previous studies found that when granular activated carbon was used as exoelectrogenic bacteria support material, it can function as an electron capacitor. With intermittent contact of the anode electrode, the fluidized particles are more efficient than packed-bed styleet al.As indicated by the changing trends of the amount of TEPS in substrates taken from each reactor, PAC additions can favor the growth of microbes near the surface of the reactor, which may contribute to the formation of a bio-cathode. Unlike abiotic cathodes, bio-cathodes can use bacteria as biocatalysts to intrinsically decrease the activation losses of cathode reactions, which can facilitate the oxygen reduction reaction in air-cathode MFCsint) can be regarded as the sum of anodic resistance (Ra), cathodic resistance (Rc), membrane resistance (Rm), and electrolyte resistance (Re). Here, the relationship between external voltage (E) and current (I) can be derived from the equation proposed by Fan et al.Internal resistance can be used to indicate the amount of energy lost during electricity production. The information about the changes of the internal resistances brought by the addition of PAC will help to explore the role of PAC in CW-MFC systems. Therefore, the distribution of internal resistance in each system was evaluated. For near linear polarization, the internal resistance (Rb (V) is the linear extrapolation open circuit voltage (LE-OCV); r (Ω m2) is the area-specific resistance (ASR) of respective electrode; S (m2) is the projected area and Sr is cross section area of the reactor; A is decided by the electrodes distance and electrolyte concentration.where, EEb, ra, rc and A are determined using the SOLVER function in Microsoft excel with a best fit of the experimental date with et al.The values of 6H12O6) as an example, with 1 mol of glucose oxidation, 24 mol electrons will be released which can provides enough electrons for 6 mol of oxygen reduction. Considering the slow catalytic kinetics of oxygen reduction, the electrons produced is abundant for cathode reactions if only oxygen was considered as the electron acceptor. Thus future work should focus on the improvement of cathode performance, rather than the anode compartment. Methods such as utilizing materials to promote the catalytic property of the electrode or adopting other electron acceptors  warrant further investigation. Since most of the internal resistance comes from electrolyte resistance which strongly relates to the electrode spacing, reducing the spacing and keeping respective electrodes working under its favorable conditions is the most effective way to minimize the energy losses from ohmic resistance.Based on the influences of PAC additions on power output of CW-MFC described above, it seems that though the amount of microorganism around the anode area decreased, the performance of the anode electrode was not influenced too much. In comparison with the anode, the performance of the cathode was greatly enhanced due to the bio-cathode formation. This reveals the fact that the anode is not the primary factor in controlling how much electricity can be generated in the CW-MFC. Taking glucose , which reflects the ability of MFC to convert organics in wastewater into electricity. Based on previous studies, the theoretical highest NER of pure MFCs is about 3.86 KW/Kg COD, which is considerably higher than most of the studies of MFCs at present (less than 1.0 KW/Kg COD)et al.Another key parameter used to assess the performance of MFCs is the NER  respectively. Among each CW-MFC, cathode and anode were buried close to the surface and bottom of the system, respectively, which resulted in an electrode spacing of about 200 mm. The cathode compartment was filled with a layer of granular graphite (GG)  and located at the air–water interface. The anode compartment consisted of the PAC modified DAS (DAS/PAC) with different percentages of PAC ranging from 1% wt (PAC/DAS ratio) to 10% wt. This configuration of CW-MFCs set up remains the same for four systems while one system had no PAC, and served as a control. Cathode and anode were connected by insulated titanium wire through an external circuit with a load of 950 Ω, which was chosen based on our previous workFive lab-scale vertical flow CW-MFCs, with identical dimensions (Φ 0.15 m × 0.32 m), were set up with their configuration shown in 4+-N) and reactive phosphorous (RP) concentrations of 40.5 ± 5.7 mg/L, 26.4 ± 4.2 mg/L and 9.8 ± 1.3 mg/L, respectively. The prepared wastewater was then continuously pumped into each CW-MFC system (from the same influent tank) at the bottom, passing through the anode compartment, cathode compartment and finally left the system from the upper outlet. Influent flow rate was controlled through peristaltic pumps, giving an average hydraulic retention time (HRT) of about 1 day for all systems. All the experiments were conducted at room temperature (15 ± 5 °C) during the whole operation period of 4 months.Swine wastewater was collected weekly from a local agriculture research farm. The influent wastewater was diluted with tap water to obtain a COD concentration of about 500 mg/L, which resulted in average total nitrogen (TN), ammonium ; DO was determined through a microprocessor oximeter ; COD, NH4+-N, NO3−-N, NO2−-N and RP were analysed using Hach DR/2400 spectrophotometer according to its standard operating procedures. TN was determined with persulfate methodAfter a period of steady operation, the performance of CW-MFCs in wastewater treatment was investigated via pH, DO, COD, TN, NHet al.LOI is usually used to provide a rough approximation of the total organic matter (TOC) presented in the solid fraction of the sample2/RS, mW/m2) were determined through basic electrical calculations, where U is the voltage (V), R is the resistance (Ω) and S is the surface area of the cathode (m2). To obtain the polarization curves, external resistance was varied over a range from 580,000 Ω to 20 Ω and the steady-state voltage across the resistors was measured. The electrode potentials were determined against a saturated Ag/AgCl electrode (Mettler Toledo).The electricity generation was monitored through either a digital multimeter or a data logger  in terms of the voltage drop (V) across the external resistor. Power densities  (the fraction of electrons used for electricity generation versus the electrons in the starting organic matter) was calculate through the formula shown in 2 (g O2/mol O2), which is 32. I is current (A) and F is Faraday’s constant (C/mol), which is 94,685. q is flow rate (L/s) while b is number of electrons donated per mole O2 (mol e−/mol O2), which is 4. Finally, ΔCOD represents the change in COD between influent and effluent (g/L).where, M is molecular mass of OHow to cite this article: Xu, L. et al. Promoting the bio-cathode formation of a constructed wetland-microbial fuel cell by using powder activated carbon modified alum sludge in anode chamber. Sci. Rep. 6, 26514; doi: 10.1038/srep26514 (2016).


Culex flavivirus. The potential implications of this relationship and the possible uses of these and other arbovirus-related insect-specific flaviviruses are reviewed.Three novel insect-specific flaviviruses, isolated from mosquitoes collected in Peru, Malaysia (Sarawak), and the United States, are characterized. The new viruses, designated La Tina, Kampung Karu, and Long Pine Key, respectively, are antigenically and phylogenetically more similar to the mosquito-borne flavivirus pathogens, than to the classical insect-specific viruses like cell fusing agent and  Many of the new ISVs appear to be members of the family Flaviviridae, genus Flavivirus, and are common in insect populations in nature, with a worldwide geographic distribution.During the past two decades, there has been a dramatic increase in the discovery and characterization of novel insect-specific viruses (ISVs).Diptera and that replicate in mosquito cells in vitro, but do not replicate in vertebrate cells or infect humans or other vertebrates.1 This is in contrast to the classical arthropod-borne viruses (arboviruses) that are maintained principally, or to an important extent, through biological transmission between susceptible vertebrate hosts by hematophagous arthropods.4 The arboviruses are dual host (vertebrate and arthropod) viruses, whereas the ISVs appear to involve only hematophagous insects.The terms “insect-specific” or “insect-restricted” viruses in current usage generally refer to viruses that naturally infect hematophagous 12 The ISFs can be separated into two distinct groups, based on their phylogenetic and antigenic relationships (Culex flavivirus (CxFV), and Kamiti River (KRV) viruses. The cISFs constitute a separate clade distinct from the vertebrate pathogenic flaviviruses. The second ISF group consists of the arbovirus-related or dual host affiliated insect-specific flaviviruses (dISFs).3 The dISFs are phylogenetically more similar to the flavivirus vertebrate pathogens than to the cISFs. Furthermore, as shown in this report, the dISFs are also closely related antigenically to some of the flavivirus pathogens, like West Nile (WNV), Zika, and dengue viruses. These similarities raise the possibility that some of the dISFs might modulate arbovirus infection and transmission in a dually infected mosquito host or that they could be useful in developing potential flavivirus vaccines or reagents.3To date (December 2016), approximately 35 insect-specific flaviviruses (ISFs) have been described.ionships . The firA total of 31 ISFs were included in this study. Their names, strain designations, GenBank numbers, host source, and geographic origin are given in Aedes scapularis mosquitoes collected on August 22, 1996, in a horse-baited Shannon trap by M.R. Mendez. The collection site was surrounded by irrigated rice fields near the village of La Tina, Piura Province, Peru . After isolation at the NAMRU-6 facility, the virus was subsequently sent to the University of Texas Medical Branch (UTMB) for further study and characterization.strain 49 LT96, was isolated in C6/36 cells at the U.S. Naval Medical Research Unit Number 6 (NAMRU-6) in Lima, Peru, from a pool of female Anopheles crucians collected on July 24, 2013, was designated as the prototype.A total of eight isolates of LPKV were made in C6/36 cells at UTMB from mosquitoes collected in CDC-type light traps placed in various habitats at Long Pine Key  within Everglades National Park in southern Florida. Mosquito collections were made between June 13 and July 25, 2013, by a team from Yale University studying the distribution, abundance, and species composition of mosquitoes and mosquito-borne viruses occurring in the Florida Everglades. Mosquito collections were approved by the U.S. National Park Service under Collecting Permit EVER-2013-SCI-0032. Anopheles tesselatus mosquito collected in a gravid mosquito trap (Bioquip 2800) on October 16, 2013, in the village of Kampang Karu, Kuching District, Sarawak, Malaysia . KPKV was originally isolated in C6/36 cell cultures at the University Malaysia Sarawak and was sent to UTMB for further study and characterization.strain SWK P44, was isolated from a single female 2 were added. The C6/36 monolayers were washed in 0.1 M cacodylate buffer, cells were scraped off and processed further as a pellet. The pellets were postfixed in 1% OsO4 in 0.1 M cacodylate buffer pH 7.3 for 1 hour, washed with distilled water and en bloc stained with 2% aqueous uranyl acetate for 20 minutes at 60°C. The pellets were dehydrated in ethanol, processed through propylene oxide, and embedded in Poly/Bed 812 . Ultrathin sections were cut on Leica EM UC7 μLtramicrotome , stained with lead citrate and examined in Philips 201 transmission electron microscope  at 60 kV.For ultrastructural analysis in ultrathin sections infected cells were fixed for at least 1 hour in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder, and 0.1% glutaraldehyde in 0.5 M cacodylate buffer pH 7.3 to which 0.01% picric acid and 0.03% CaCl13 All animal work and preparation of murine antibodies was covered by an approved UTMB Institutional Animal Care and Use Committee protocol (number 9505045).Since the ISFs by definition do not infect vertebrates or vertebrate cells, we were unable to produce ISF-specific antibodies. Attempts to immunize mice with ISF antigens produced from infected C6/36 cells inevitably resulted in antibodies that reacted with uninfected C6/36 cell controls. Attempts to absorb out the mosquito cell antibodies were unsuccessful; consequently, we used heterologous mouse hyperimmune ascitic fluids (MIAFs), prepared with infected mouse brain antigens of selected flavivirus pathogens, such as WNV, dengue type-2, Zika, yellow fever, and Japanese encephalitis viruses, in serologic tests. These antibodies were obtained from the World Reference Center for Emerging Viruses and Arboviruses; their homologous titers, as determined by hemagglutination-inhibition (HI) tests, are given in 14 using the heterologous mouse hyperimmune polyclonal antibodies described above at a 1:20 dilution.The antigens used in immunofluorescent studies were ISF-infected C6/36 cells. Six or 7 days after virus inoculation, the infected cells were scraped from the surface of the culture flask and spotted onto Cel-Line 12-well glass slides  for examination by indirect fluorescent antibody test (IFAT),15 we found that it was possible to prepare serologic antigens for some ISFs, using infected C6/36 cells. Selected ISFs were inoculated into flask cultures of C6/36 cells maintained at 28°C, as noted above. When most of the cells showed viral cytopathic effect (CPE), the entire flask, containing medium and cells, was frozen at −80°C. For preparation of an acetone-extracted antigen, the flask contents were thawed and dropped through a 26 gauge needle into 20 volumes of chilled acetone. Within 5 minutes, this mixture was centrifuged at 1,000 rpm for 2 minutes and the supernatant fluid discarded. The sediment was resuspended in another 20 volumes of chilled acetone, shaken, and held for 1 hour at 4°C. The mixture was then centrifuged at 1,600 rpm for 5 minutes, the acetone decanted and the sediment dried for 1 hour by vacuum. The dried sediment was rehydrated in a volume of borate saline solution pH 9.0 equal to the original fluid used for extraction and was stored frozen at −80°C until used.In preliminary studies,16 Nonspecific inhibitors in the antisera were acetone extracted by the method of Clarke and Casals.17 The ISF antigens (infected C6/36 cells) were described above. Antibodies (MIAFs prepared to various flavivirus pathogens) were tested at serial 2-fold dilutions from 1:20 to 1:5120 at pH 6.0–6.2 with 4 units of antigen and a 1:200 dilution of goose erythrocytes, following established protocols.16A standard HI test was done in microtiter plates, according to methods described previously.Aedes albopictus, clone C6/36 (CRL-1660); baby hamster kidney, BHK-21 (CCL-10); and African green monkey kidney, Vero E6 (CRL-1586). The three cell lines were originally obtained from the American Type Culture Collection (ATCC), Manassas, VA. The mosquito cells were maintained at 28°C and the vertebrate cells at 37°C in 12.5 cm2 tissue culture flasks with 5 mL of medium recommended in the ATCC specification sheets. When a confluent monolayer of cells was present, 200 uL of a stock of each virus  were inoculated into flasks of C6/36, BHK-21, and Vero E6 cells. After incubation for 2 hours, each flask was washed three times with 5 mL of maintenance medium, with aspiration to remove all remaining medium between washes. The medium was replaced and a sample (500 uL) taken as a day 0 control. Each day thereafter, for 7 consecutive days, the medium was completely removed, sampled, and fresh medium added to each flask, as described above. The daily samples (day 0–7) were subsequently assayed by reverse transcription-polymerase chain reaction (RT-PCR). Primer sequences available upon request.To determine the host range of the three new ISFs, their replication was studied in the following cell lines: N = 10) were inoculated intracranially with approximately 15 uL of C6/36 culture fluid from stocks of each of the three ISVs . After inoculation, the pups were returned to their dams and were examined daily for 14 days for signs of illness or death. Mice were purchased from Harlan Sprague–Dawley ; this animal work at UTMB was carried out under IACUC-approval protocol number 9505045.One litter of 1- to 2-day-old Institute for Cancer Research (ICR) mouse pups  once CPE was advanced. One milliliter of clarified supernatant from each virus was treated with a cocktail of DNases  for 1 hour at 37°C. Viral RNA was then extracted using Trizol and resuspended in 50 μL RNase/DNase and protease-free water .For all ISVs, fluid supernatant from cultures of infected C6/36 cells were used for RNA extraction and sequencing. Supernatants were harvested and clarified by low speed centrifugation , ∼340,000 (3.37%), ∼350,000 (6.0%), ∼3,100,000 (36.6%), ∼3,300,000 (29.0%), ∼7,870,000 (82.6%), ∼460,000 (4.1%), ∼3,770,000 (22.8%), ∼2,780,000 (25.3%), ∼2,150,800 (10.8%), and ∼6,400,000 (30.5%), respectively.Viral RNA (∼0.9 µg) was fragmented by incubation at 94°C for 8 minutes in 19.5 μL of fragmentation buffer (Illumina 15016648). A sequencing library was prepared from the sample RNA using an Illumina TruSeq RNA v2 kit following the manufacturer’s protocol. The sample was sequenced on a HiSeq 1500 using the 2 × 50 paired-end protocol. Reads in fastq format were quality-filtered, and any adapter sequences were removed, using Trimmomatic software.22The evolutionary history was inferred by using the maximum likelihood method based on the General Time Reversible model. The tree with the highest log likelihood (−366425.2857) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach, and then selecting the topology with superior log likelihood value. A discrete gamma distribution was used to model evolutionary rate differences among sites . The rate variation model allowed for some sites to be evolutionarily invariable . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analyses involved 93 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 5,981 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.LTNV, LPKV, and KPKV were each initially isolated in cultures of C6/36 cells. In this cell line, the three viruses produced a similar CPE (rounding and detaching of cells from flask surface) 6 or 7 days after inoculation. Subsequent inoculation of the C6/36 culture fluid into BHK-21 or Vero E6 cells failed to produce detectable CPE. Likewise, intracranial inoculation of the infected C6/36 culture fluid into newborn ICR mice failed to produce illness or death in the animals.To determine if LTNV, LPKV, and KPKV replication occurred in vertebrate cells without producing CPE, additional experiments were carried out in C6/36, Vero, and BHK cell cultures to assay for virus replication by RT-PCR. Samples of medium from the three ISV-inoculated cell lines were collected from day 0 to day 7, as described in the Methods section. After RNA extraction, a partial region of the following genes of each virus was amplified and run on gels: LTNV, a partial region covering part of the NS1 and NS2B genes with expected band size between 350 and 400 nucleotides (nt) long; KPKV, a partial region between the NS1 and NS2A genes with expected band size 500–600 nt long; and LPKV, a partial region of the NS3 gene with expected band size between 450 and 500 nt long (primer sequences available upon request). RNA extracted from culture fluid from the C6/36 cells infected with LTNV, LPKV, and KPKV from day 0 to day 7 postinoculation (dpi) displayed strong bands on all days (data not shown). In contrast, extracted and amplified viral RNA from the Vero and BHK cells inoculated with the three viruses showed decreasing intensity of the RNA bands from day 0 to day 7, indicating that the three new flaviviruses did not replicate in the two vertebrate cell lines (data not shown).In ultrathin sections of infected C6/36 cells, the three viruses had typical flavivirus morphology with intracellular localization and formation of smooth membrane structures (SMS) . VirionsIn preliminary studies, we observed that polyclonal MIAFs prepared against selected flavivirus pathogens reacted in HI tests with some of the dISFs, using antigens prepared from infected C6/36 cell cultures. However, for most of the dISFs, we were unable to prepare reactive hemagglutinins. In the case of the three new ISFs , LTNV was the only virus that produced a reactive hemagglutinin.Flavivirus genus  sequences were aligned to a dataset of sequences representing 93 flavivirus species. A consensus tree was obtained based on maximum-likelihood with bootstrap resampling of 1,000 replicates used to obtain confidence limits on individual branches. As expected the cISFs, represented by cell fusing virus (CFAV), KRV, CxFV, and others, clustered in a clade basal to all other member species of the us genus . On the The size of the positive sense, near complete single-strand genomes of the three identified viruses are 10,859, 10,968, and 10,882 nucleotides (nt) long, for LPKV (KY290256), KPKV (KY320648), and LTNV (KY320649), respectively. A single ORF of 10,365 , 10,311 , and 10,356  nt for LPKV, KPKV, and LTNV, respectively, are flanked by untranslated regions at the 5′ and 3′ ends .3 The first and most numerous group is the cISVs  as well as other agents such as NHUV, Marisma mosquito, and Donggang viruses . The dISWhat are the possible implications of this relationship between the ISFs and important mosquito-borne flavivirus pathogens? One possible implication is that the ISFs, and the dISFs in particular, may alter the vector competence of their dipteran hosts. Since most of the ISFs described to date have been associated with mosquitoes, the following discussion will focus on the mosquito-specific flaviviruses.24 In addition, flavivirus-derived endogenous elements (EVEs) or nonretroviral integrated RNA viruses have been reported in the genomes of several mosquito species.28 ISFs and EVEs are part of the microbiome of many species and genera of mosquitoes, but they appear to have no obvious deleterious effect on their natural insect hosts.3 The available evidence suggests that the ISFs are maintained in mosquito populations by vertical transmission, and that they do not use vertebrate hosts as part of their life cycle, like most of the classical arthropod-borne viruses of vertebrates (arboviruses).29The available data on the ISFs indicate that they are relatively common in mosquito populations in nature and that some, like CFAV and CxFV, have a wide geographic distribution.35 Some studies have suggested that coinfection reduces vector competence35 whereas others have found that it had no effect.31There has been considerable speculation, but conflicting experimental evidence, that infection of a mosquito with an ISF can alter the insect’s vector competence for certain mosquito-borne flavivirus pathogens, due to heterologous interference. But most of the experimental studies of this phenomenon to date have used cISFs, such as CxFV or Palm Creek viruses; and the results have been mixed. 36 so in vitro results of dual infection may not be indicative of what actually occurs in a live mosquito with a functional RNAi response (in vivo). One study34 with Culex quinquefasciatus infected with Nhumirim virus and then challenged with WNV indicated that the dually infected mosquitoes were less competent vectors of WNV than control mosquitoes infected with WNV alone, but additional studies of dual infection with dISF and related flavivirus pathogens are needed in live mosquitoes to clarify this possibility. If a dISF reduced the vector competence of Aedes aegypti for Zika or dengue viruses, for example, potentially it might be used as a disease control agent, as some strains of Wolbachia have been used.37 If the candidate dISF was also vertically transmitted in mosquitoes, then theoretically it would be maintained in the vector population.A second problem is that most of the experimental studies have been done in vitro, using the C6/36 mosquito cell line. However, the C6/36 cell line has a dysfunctional antiviral RNA interference (RNAi) response,1 The insect-specific alphavirus, Eilat,38 is defective for vertebrate cell infection39 and has been exploited by using recombinant DNA technology to generate Eilat chimeras, where the structural polyprotein ORF was swapped with that of a vertebrate-pathogenic alphavirus to generate a chimera that is structurally indistinguishable from the pathogenic virus.1 Eilat-based alphavirus chimeras have been developed as vaccines for chikungunya and Venezuelan equine encephalitis viruses.1 Chimeras between Eliat and chikungunya viruses have also been shown to serve as high-quality antigens for enzyme-linked immunosorbent assays.40Another potential application for the dISVs could be to use them as platforms for development of vaccines or diagnostics.35 it appears that some of these ISFs have an effect on the vector competence of their mosquito hosts for related mosquito-transmitted flavivirus pathogens. Although the frequency of ISF infections in field populations of mosquitoes is currently unknown, it is probably relatively low; furthermore, the infection rate likely varies by locality and species. But the variety and number of ISFs (and EVEs) serves as yet another example that all Ae. aegypti or Culex quinquefasciatus do not have the same vector potential. Other recent studies have demonstrated that the gut microbiota  of mosquitoes can also change the insect’s vector potential for some arboviruses by altering the mosquito’s basal innate immunity or by directly inhibiting the virus through bacterial metabolites.42The discovery of three new mosquito-specific flaviviruses brings the total number of these agents to 38 . This ilAe. aegypti population may be of equal importance in determining the character of an anticipated dengue or Zika outbreak, as the herd immunity of the local human population. Given the complexity of factors affecting vector competence of mosquitoes and other hematophagous insects, further research in this area is needed if we want to understand and control the transmission of vector-borne viral diseases.Although the effects of an insect’s microbiome on its vector competence are now being recognized by microbiologists and vector biologists, they apparently are not by most public health officials, epidemiologists, and modelers of arboviral diseases. It appears that the latter groups assume that all mosquitoes of a given species have the same vector potential and that it is possible to predict the risk, intensity of transmission, and spread of a disease caused by a mosquito-borne pathogen, such as dengue or Zika virus, based solely on climatic data, estimates of vector distribution and density, and susceptibility (immune status) of the local human population. But this approach is simplistic and misleading. The microbiome and resulting vector competence of a local 44 have shown that flavivirus-like and other negative-sense RNA viruses are much more numerous and diverse in invertebrates than in vertebrates, suggesting that the flavivirus pathogens may have evolved from earlier arthropod viruses. Thus it seems possible that an ISF could emerge as a vertebrate pathogen, although at present it is impossible to know how or when this might occur. However, this is another reason to discover, characterize, and monitor novel ISFs.In view of the similarity of some of the ISFs with the mosquito-borne flavivirus pathogens of vertebrates, one final consideration is whether one or more of the ISFs could evolve or mutate and acquire the ability to infect vertebrates. In other words, could an ISF like Marisma mosquito virus emerge sometime in the future as a human or animal pathogen? This is not a frivolous question. Recent metagenomics studies


Macrobrachium rosenbergii) is one of the most farmed freshwater crustaceans in the world. Its global production has been stalling in the past decade due to the inconsistent quality of broodstock and hatchery-produced seeds. A better understanding of the role of nutrition in maturation diets will help overcome some of the production challenges. Arachidonic acid  is a fatty acid precursor of signaling molecules important for crustacean reproduction, prostaglandins E and F of the series II (PGE2 and PGF2α), and is often lacking in maturation diets of shrimp and prawns. We examined the effects of ARA in a combination of different fish oil (FO) and soybean oil (SO) blends on females’ reproductive performance and larval quality. Adult females (15.22 ± 0.13 g and 11.12 ± 0.09 cm) were fed six isonitrogenous and isolipidic diets containing one of two different base compositions (A or B), supplemented with one of three levels of Mortierella alpine-derived ARA (containing 40% active ARA): 0, 1 or 2% by ingredient weight. The two base diets differed in the percentages of  and docosahexaenoic acid ). After the eight-week experiment, prawns fed diet B with 1 and 2% ARA supplement (B1 and B2) exhibited the highest gonadosomatic index (GSI), hepatosomatic index (HSI), egg clutch weight, fecundity, hatching rate, number of larvae, and reproductive effort compared to those fed other diets (p ≤ 0.05). Larvae from these two dietary treatments also had higher tolerance to low salinity (2 ppt). The maturation period was not significantly different among most treatments (p ≥ 0.05). ARA supplementation, regardless of the base diet, significantly improved GSI, HSI, egg clutch weight and fecundity. However, the diets with an enhanced ARA and LOA (B1 and B2) resulted in the best reproductive performance, egg hatchability and larval tolerance to low salinity. These dietary treatments also allow for effective accumulation of ARA and an n-3 lcPUFA, DHA in eggs and larvae.The giant river prawn ( Macrobrachium rosenbergii, is an economically important species cultured in the Indo-Pacific region  and linoleic acid     and has -6, LOA) . In crus-6, LOA)  and infl-6, LOA) . In gravy acids) , althoug−1 diet ingredients) can enhance reproduction of tank-domesticated P. monodon (M. rosenbergii reproduction (−1 DW) enhanced fecundity of females compared to those containing a low n-6 fatty acid level (approximately 4 mg g−1)  improved hatching rates and larval ammonia tolerance. For this current study, we built on this maturation diet and a diet formulation recommended by the Thai Department of Fisheries based on locally available diet ingredients (Schizochytrium sp. yielded the best growth performance in juvenile M. rosenbergii (unpublished data). Some individuals in this dietary treatment reached earlier maturation compared to the control fish oil (FO) diet.ARA is often inadequate in pelleted maturation diets because of the naturally low base level of ARA derived from fish oil  . Moreoveredients . Our preM. rosenbergii female broodstock and larval quality, namely the tolerance to low salinity. We examined the effects of ARA supplementation in two base diets varying in percentages of FO and SO . We hypothesized that increased dietary ARA along with a higher LOA level would improve reproductive performance of female M. rosenbergii. Our results provided insights to the understanding of these fatty acids in facilitating the prawn maturation process, and in larval development. Our findings also help fine tune the formulation of optimal maturation diets based on locally available material that will improve larval quality.The present study investigated the effect of ARA and LOA given adequate n-3 lcPUFA  on the reproductive performance of We used a 6 × 3 completely randomized design (CRD) in this experiment, with six treatments performed in triplicate. Each experimental unit (a cage)consisted of a group of 15 females receiving each of the six diet formulations. Each cage was sub-divided into fifteen 18 × 13 × 12 cm compartments, each of which held one individual female. To have a manageable number of individuals per tank, to ensure optimal water quality for growth and reproduction, and to have an adequate number of individuals for all analyses, we created the entire experimental setting in two 3 × 3 × 1.2 m recirculating concrete tanks. Each tank contained 270 individual female prawns. Individuals raised in one tank were used for the analyses of females’ reproductive performance and fatty acids of eggs and larvae. Those raised in the second tank were used for determining fatty acid compositions in muscle and stage II–V ovaries, as well as for estimating Gonadosomatic index (GSI) and Hepatosomatic index (HSI). Because the hepatopancreas is a major lipid storage organ in crustaceans, HSI is an indication of the status of lipid and nutrient storage. The experiment lasted eight weeks.M. rosenbergii adults were obtained from a commercial prawn farm in Chachoengchao Province, Thailand. Individuals were acclimatized to 28 °C in a freshwater recirculation system at Rajamangala University of Technology Tawan-ok and fed a  diet for seven days.Approximately four-month-old At the start of the experiment, females and males were randomly selected and stocked into the experimental set-ups. The initial weights and total lengths of females and males were 15.22 ± 0.13 g and 11.12 ± 0.09 cm, and 17.68 ± 0.22 g and 11.44 ± 0.09 cm, respectively. Males and females were kept separately until mating. The males used in the experiments were raised together in another two 1 × 3 × 1 m communal concrete tanks, each containing 250 individuals. Only females received the experimental diets; the males were fed a commercially formulated diet . Experimental animals were fed twice daily (07.00 and 18.00) at approximately 5% of body weight.−1, pH from 8.19–8.38, alkalinity from 178.42–242.33 mg L−1, hardness from 129.42–146.33 mg L−1. Nitrogenous compounds, namely ammonia nitrogen, NO2-N and NO3-N were 0.01–0.06, 0.01–0.04 and 0.05–0.08 mg L−1, respectively. Temperature, pH  and dissolved oxygen were monitored daily. Alkalinity  contained 2% FO and 2% SO by ingredient weight . Compared to the HH diet, base diet A had a comparable DHA content while diet B had comparable LOA contents  using standard procedures . Moisturr lipids .For female reproductive performance, we determined gonadosomatic index (GSI), hepatosomatic index (HSI), incubation period , fecundity, reproductive effort, hatching rate, larval length and larval tolerance to low salinity. For fatty acid analysis, we determined fatty acid profiles of muscle tissue , stage II–V ovaries, two stages of eggs (OE and BE stages) and larvae. For all analyses, we collected the data in triplicate and nine individuals per treatment were analyzed. With the exception of fatty acid analysis, all other analyses obtained nine independent values per replication. For fatty acid analysis of all tissue types, we prepared a homogenate for each replication from a pool of three individuals; only three values per treatment were obtained. We started to observe stage II ovaries at approximately one month after beginning the experiment. Stages III and IV developed one to two weeks after the prior stage. By the end of the experiment, most experimental animals developed stage V ovaries. The tissue samples were kept at −40 °C for fatty acid analysis.−1. The temperature program used was an initial 150 °C for 0.5 min, increasing to 170 °C at a rate of 5 °C min−1, held at 170 °C for 10 min, then increasing to 190 °C at a rate of 3 °C min−1, and then held at 190 °C for 28 min. Temperatures at the injection and detection ports were 230 °C and 250 °C, respectively. The fatty acid analyses were performed at the Institute of Marine Science, Burapha University, Thailand.Total lipids from experimental diets and various tissues were extracted by homogenizing each sample in 20 ml ice-cold chloroform:methanol  containing 0.1% butylated hydroxytoluene for 20 min before the liquid fraction was transferred to a separating funnel. The residual matter was then subjected to a second round of extraction, after which the liquid portion was transferred to a separating funnel. To separate the non-lipid phase, 0.88% (w/w) KCl  was then added to the separating funnel, agitated to mix the contents, and then left until the solution separated into two layers. Total lipid was obtained by filtering the lower layer through anhydrous sodium sulfate before evaporating the collected fraction . Fatty aMolting and ovarian development of each female were monitored daily. Maturation stages of the ovary were classified into one of five stages, based on the ovarian size and color observed through the carapace, following the criteria detailed in We paired a mature female to a male at a 1:1 ratio; the entire experiment required a total of 270 mating pairs. Each gravid female (with a stage V ovary) was mated in a compartment to a mature male immediately after molting. We manually placed a mature male, kept in one of the ‘male’ tanks (see above), in the compartment. After the eggs were fertilized (about 6–8 h after mating), the male was removed from the compartment to another tank without returning to the original male tank; each male was used only once. Approximately 8 h after mating, freshly fertilized eggs migrated to the female’s brood chamber in the abdominal area and became visible.The female prawns in individual compartments were observed daily to visually determine embryonic development stage, based on the egg color . As embrA 10-day-old egg clutch (brownish) (after OE become apparent), scraped from the brood chamber of each of three females per treatment was weighed to the nearest 0.01 g after removing any excess water by repeated blotting. Three independent samples of equal weight (0.2 g) from each clutch were counted under a microscope to estimate the total number of eggs per clutch. The averaged egg numbers did not differ statistically among treatments. We therefore extrapolated the averaged egg number per 0.2 g of 1,450 ± 72.13 eggs to an actual egg clutch weight for fecundity estimates for all treatments.An egg clutch weight was the difference between a gravid female’s weight before and after hatching. Gravid females bearing brownish eggs were weighed and transferred into an aerated 20-L tank containing 14 ppt water. After spawning, the females were weighed and transferred back into the original compartment, while the spawned eggs remained in the hatching tank and then were used for the subsequent experimental steps. Fecundity was estimated for each individual female as the total number of eggs ) per body weight . The reproductive effort for each individual female was estimated as the percentage of egg weight to body weight. We estimated the number of newly hatched larvae to a female’s weight  by counting the number of individuals in a sample of 100 ml water from a well-mixed tank and then extrapolating to 20 L. Hatching rates were determined from the number of larvae per total number of brownish eggs.We tested the tolerance to low salinity of newly hatched larvae of the experimental females. The larvae from each treatment were exposed to a low salinity level of 2 ppt for 24 h. Each experimental unit contained 30 individual larvae. A group raised in 14 ppt water were treated as a control. At the end of the test, we recorded the survival for each treatment.p value < 0.05. All statistical analyses were executed with SPSS version 17 for Windows . For the data  that were not normally distributed, we performed either square root arcsine or arcsine transformation before ANOVA. However, only non-transformed means are presented in the table.We determined the differences in female reproductive performance, larval quality and fatty acid compositions in diets and various tissues among dietary treatments using one-way analysis of variance (ANOVA). The significance of the differences in means was determined by Duncan’s new multiple range test at a To examine the variation of fatty acid profiles among various tissue types and treatments, we analyzed principal components from seven quantitative variables: percentages of five major fatty acids and total values of two fatty acid classes detected in our study . Total n3-lcPUFA was treated as a supplementary variable on a principal component analysis (PCA) biplot for a better understanding of the ordination based on the seven active quantitative variables; it did not interfere with the ordination. This multivariate approach utilized all information available for each individual while at the same time reduced the dimensionalities of the data (from seven dimensions to two dimensions in our case). The first few principal components typically captured most of the variation in the data set. PCA was performed using algorithms implemented in the FactoMineR package  and the −1.The proximate compositions of the experimental diets were similar . Each exThe fatty acid composition of each diet generally reflects the level of ARA supplementation and different proportions of lipid sources in two base compositions (A and B) . The proFor n-6 PUFA, all diets contained LOA as a major component  with B2 containing the highest level of total n-6 PUFA and A0 containing the lowest level. For total n-3 PUFA, LNA, EPA and DHA were primary components. All diets contained approximately 6–7% LNA, 4–5% EPA and 6–10% DHA of total fatty acids. The total n-3 PUFA and n-3 lcPUFA were highest in diet A1. In diets A0-A3, LNA contents were slightly higher than EPA but lower than DHA contents. In contrast, LNA contents were higher than DHA in diets B0–B2. In all diets, dietary EPA levels were lower than DHA; EPA contents were similar among all diets . EPA to DHA ratios were similar among experimental diets, approximately 0.5 (0.43–0.72).p < 0.05; p < 0.05). Females fed diets B1 and B2 had GSI of 8.59 ± 0.53 and 8.73 ± 0.51% and HSI of 4.61 ± 0.57 and 4.85 ± 0.43%, respectively. The average egg clutch weights for these groups were 2.89 ± 0.38 and 2.91 ± 0.11 g. Their average fecundities were 1,399.82 ± 187.96 and 1,354.62 ± 129.93 eggs per female; reproductive effort was 16.61 ± 1.78 and 16.79 ± 1.03 g of eggs per g BW female. Hatching rates were higher than 90% for these groups. Female fed diets A1 and A2 had HSI of 3.81 ± 0.34 and 4.05 ± 0.42%. The average egg clutch weights for these groups were 2.50 ± 0.24 and 2.49 ± 0.22 g, average fecundities were 1,191.15 ± 73.29 and 1,268.33 ± 128.62 eggs per female, for A1 and A2, respectively. Larval lengths and egg incubation periods (OE to hatching) of all dietary treatments were not statistically different.Females fed diets containing 0.4% and 0.8% ARA supplementation  had higher reproductive performance than those fed on diets without ARA addition (A0 and B0) . Variation of muscle ARA percentages to total fatty acids corresponded to the percentages of ARA added to diets and source of fatty acid in each base diet. Groups fed diets A2 had the highest proportions of muscle ARA followed by B2, A1and B1 while those fed diet A0 and B0 had the lowest muscle ARA level (p < 0.05). For n-3 PUFA, muscle EPA was highest in the A0 group (p < 0.05) while the remaining treatments showed similar values (p > 0.05). The total n-3 PUFA in muscle was highest in prawns fed diets A0 and A1, while the remaining dietary treatments exhibited similar values.Among experimental treatments, muscle total saturates and total monoenes were not statistically different, but some PUFAs, namely LOA, ARA, EPA and DHA, showed some significant variation. The differences in LOA and DHA in muscle were consistent with the differences of these two fatty acids between the two base diets. Dietary treatment B had a higher muscle LOA level while dietary treatment A had a higher muscle DHA level  . OvarianFor each ovarian maturation stage, females fed different experimental diets varied in their ovarian fatty acid compositions , especiaThe variation of ovarian ARA within each ovarian maturation stage corresponded to the dietary ARA supplementation. Compared to dietary treatments without ARA supplementation, groups fed diets with ARA supplementation had higher ovarian ARA levels in all ovarian stages, especially those fed diets B1 and B2 (from 4.47–5.65% in stage II ovary to 6.17–7.05% in stage IV). In all ovarian stages, females fed B2 and B1 typically had the highest ovarian ARA proportional contents, followed by those fed A2 and A1; this relationship between the base diet and ARA retention was opposite to that observed in muscle and diet where treatments A2 and A1 had the highest ARA percentages.Egg and larval fatty acid profiles were more similar to the ovarian profiles, especially at stage V ovaries, than to those of muscle . Howeverp < 0.05). Egg ARA was highest in treatments A2, B1, and B2 followed by A1 (p < 0.05). For n-3 PUFA, EPA was highest in treatments A0, A1, A2 and B0, but LNA and DHA were highest in B1 and B2 (p < 0.05).At the OE stage, the differences in dietary fatty acid levels in the two base diets were consistent only with the differences in total n-3 PUFA and total n-3 lcPUFA . Groups p < 0.05). For n-6 PUFA, LOA contents were similar across treatments (18.05–19.84%) although treatment B2 showed the highest level at 19.84 ± 0.24%, followed by A0, B0 and B1 (18.92–19.03%). Similar to OE, ARA was highest in treatment B2 followed by A2 and A1/B1 (p < 0.05). The variation of total n-6 PUFA among treatments was therefore similar to that of ARA and LOA, with treatment B2 showing the highest values, followed by treatments A2 and B1 (p < 0.05). For n-3 PUFA, EPA and DHA percentages were similar across treatments, with A1 showing the highest values , and B0 showing the lowest values .At the BE stage, groups fed diet A had higher LNA, EPA, total n-3PUFA, and total n-3 lcPUFA than those fed diet B (p < 0.05). For n-6 PUFA, LOA was highest in treatment B2 and lowest in A1. Similar to egg tissues, larval ARA was highest in treatment B2 followed by A2/B1 (p < 0.05). For n-3 PUFA, the total n-3 PUFA and total n-3 lcPUFA were higher in treatments A0, B0, B1 and B2 than those in treatment A2; an opposite pattern was observed in egg tissue. This n-3 PUFA variation was due to high levels of EPA detected in B0 and B1 and high DHA levels detected in A0, B1 and B2.In larvae, groups fed the base diet B had higher total saturates in tissue than those fed diet A, with the percentage being highest in treatment B1  to 9.43 ± 0.28% (A2) in diets. The basal dietary ARA levels were approximately 3% in treatments without ARA supplement. The enhanced reproduction in M. rosenbergii was similar to that of tank domesticated P. monodon broodstock fed ARA-supplemented diet . These positive effects of dietary ARA supplementation on M. rosenbergii reproduction and embryonic development may link to an ARA role as a precursor to prostaglandins (PGs).Our research highlighted the importance of ARA in females’ reproduction, especially for traits relevant to ovarian maturation and oogenesis, in ted diet . The expα in the cyclooxygenase pathway. PGE2 and PGF2α are important signaling molecules in several reproductive functions in crustaceans. The roles of PGE2 have been more extensively examined and demonstrated in several shrimp species, including M. rosenbergii. PGE2 is associated with oocyte maturation  were comparable to those detected in mature ovaries and eggs of wild P. monodon  and our base diets (diets A0 and B0). Our findings have an important implication for maturation diets for domesticated broodstock of M. rosenbergii in that the broodstock of this species requires ARA levels as high as marine shrimp even though their natural diets are derived mainly from freshwater systems, which may be lacking ARA. Formulating maturation diets from observations in the wild may not optimize the prawn’s ARA requirements. We did not detect negative effects of supplementing ARA at 0.8% (9.43 and 8.33%) of total fatty acids in diets A2 and B2, respectively).We detected much higher levels of ovarian and egg ARA in the females fed diets supplementing ARA  than those detected in mature ovaries and eggs of wild y acids, ). This dM. rosenbergii than treatments A1 and A2, containing 6.17–9.43% ARA and 14.85–13.35% LOA. Positive effects of dietary LOA levels on reproductive performance of broodstock and larval quality observed in this study were consistent with those observed by M. rosenbergii and M. amazonicum. −1 DW LOA could improve fecundity of mature female M. amazonicum.In addition to ARA, we detected a positive synergistic effect of enhanced dietary ARA and LOA on females’ reproductive performance, egg hatchability and larval quality . The dietary treatments B1 and B2, containing 5.7–8.33% ARA and 21.78–22.49 % LOA of total fatty acid, led to higher reproductive performance of Macrobrachium species, M. borllii, Although some authors speculated that LOA may serve as a raw material for the synthesis of ARA , some emM. rosenbergii. When the dietary LOA was present at a higher percentage (as in diets B1 and B2), LOA was utilized during muscle tissue formation, ovarian maturation and early embryogenesis. On the other hand, dietary LOA available at a slightly lower level (as in diets A1 and A2) was mostly retained and accumulated in muscle and ovarian tissues. In addition, females fed diets with higher LOA tended to retain higher proportions of LOA in ovaries and larvae. LOA may also have played a role in hatching as proportional contents of LOA in larvae were slightly lower than that of mid-stage embryos.LOA contributed to a large percentage of total fatty acids in all tissue types, suggesting its importance as an energy source and as cellular structural components for Eggs of females fed diets with enhanced dietary ARA and LOA also had higher hatchability and resulted in higher numbers of larvae than those of other dietary treatments. Also, larvae from these treatments were more tolerant to low salinity stress. The results suggested the importance of both fatty acids in yolk deposition, embryogenesis and possibly larval osmoregulation. A combination of enriched ARA and LOA in diets may have facilitated the accumulations of other important fatty acids, especially DHA, in eggs and larvae despite the lower n-3 lcPUFA available in diets. Having adequate energy required by these energetically expensive life stages, especially from fertilized eggs (containing mainly yolk) to advanced embryo and larvae (involving organ formation)  may alloM. rosenbergii . The dominance of these fatty acid classes in muscle, ovarian and egg tissues were similar to those observed in wild and farmed r growth , ovarianr growth , embryogr growth  and earlr growth  as they r growth .M. rosenbergii. Compared to marine shrimp species, M. rosenbergii tends to accumulate higher LOA, comparable EPA, but less DHA in the muscle tissue while it accumulates comparable LOA, but less n-3 lcPUFA in reproductive tissues. Tissues of M. rosenbergii adults from natural environments tended to contain higher amounts of LOA  compared to marine shrimp species  .The pattern of LOA, EPA and DHA accumulation and utilization observed in our study may be reflective of fatty acid requirements specific to this diadromous  tissue) . Marine  tissue)  compared10.7717/peerj.2735/supp-1Data S1Click here for additional data file.10.7717/peerj.2735/supp-2Data S2Click here for additional data file.


Betula platyphylla) ectopic overexpressing a late embryogenesis abundant (LEA) gene and a basic leucine zipper (bZIP) gene from the salt-tolerant genus Tamarix  show increased tolerance to salt (NaCl) stress. Co-transfer of TaLEA and ThbZIP in birch under the control of two independent CaMV 35S promoters significantly enhanced salt stress. PCR and northern blot analyses indicated that the two genes were ectopically overexpressed in several dual-gene transgenic birch lines. We compared the effects of salt stress among three transgenic birch lines  and wild type (WT). In all lines, the net photosynthesis values were higher before salt stress treatment than afterwards. After the salt stress treatment, the transgenic lines L-4 and L-8 showed higher values for photosynthetic traits, chlorophyll fluorescence, peroxidase and superoxide dismutase activities, and lower malondialdehyde and Na+ contents, compared with those in WT and L-5. These different responses to salt stress suggested that the transcriptional level of the TaLEA and ThbZIP genes differed among the transgenic lines, resulting in a variety of genetic and phenotypic effects. The results of this research can provide a theoretical basis for the genetic engineering of salt-tolerant trees.The aim of this study was to determine whether transgenic birch ( Betula Platyphylla) is one of the most extensively distributed broadleaf species in the northern and southwestern forested areas of China [Birch  seeds [LEA genes were subsequently found to be one of the most important stress-associated gene families. Many studies have demonstrated that LEA genes are associated with tolerance against salt and other stresses [Late embryogenesis abundant (LEA) proteins were first discovered in germinating cotton (m) seeds , and LEAstresses –19. Basistresses . bZIP trstresses . One of stresses .TaLEA and ThbZIP genes from Tamarix were transformed into birch and then subjected to salt stress treatments. The aim of these experiments was to determine whether these genes affected salt tolerance, and to detect the variation in physiological characters among the different transgenic lines. This information will provide a theoretical basis for molecular breeding in birch.Although birch has a strong cold resistance, it is weak in salt tolerance, which limits the popularization and application of birch in saline soils. In order to gain transgenic birch with salt tolerance, both TaLEA and ThbZIP genes were firstly cloned from Tamarix as described by Wang [TaLEA-Tnos) amplified from a pROK2-TaLEA vector was inserted into the pROK2-ThbZIP vector, concrete steps of which were described in detail in our previous research  plantlets were obtained by Agrobacterium-mediated transformation. To prepare the infection liquid, an Agrobacterium culture was incubated until the OD600 was 0.6–0.8, then centrifuged at 3000 r min−1 for 5 min, finally, the collected pellets were diluted with sterile water to a final concentration of OD600 = 0.1. Leaves from 60-d-old clones were cut in half and the pieces were gently shaken in the infection liquid for 5 min. Then, the leaves were removed and excess liquid was absorbed with sterile filter paper. The infected birch leaves were then co-cultured on antibiotic-free differentiation medium  at 25 ± 2°C in the dark for 2 d. To eliminate bacteria, the co-cultured leaves were washed with 200 mg L−1 cephalosporin solution for 3–5 min. The excess liquid was absorbed with sterile filter paper, and then leaves were cultured in new differentiation medium . In the first week, bacteria elimination was performed every 2 d; subsequently, bacteria were eliminated every 7 d until small buds differentiated. The resistant buds grew into leaves, which were cut off and cultured on a differentiation medium containing antibiotics. Finally, adventitious shoots were transferred onto medium  to allow shoot growth for 2 weeks. To induce rooting from the shoots, 2-cm shoot cuttings were transferred to rooting medium .To study the functions of the NptII (Kanamycin resistance gene), TaLEA and ThbZIP genes  according to the manufacturer’s protocol. The cycling conditions were as follows: pre-denaturing for 3 min at 94°C followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The PCR products were detected on 1.0% agarose gels.Total DNA was extracted from all transgenic and WT birch lines using a modified CTAB method . Using tIP genes . The vecTaLEA and ThbZIP in birch, total RNA from WT and transgenic birch clones was isolated as described by Qu [+ nylon membrane, and fixed with UV cross-linking  for the northern blot analysis. The membrane was hybridized with full-length TaLEA and ThbZIP genes labeled with DIG-dUTP. Hybridization and detection were using a DIG Northern starter Kit,.To detect ed by Qu . Subsequ−1, 4 g L−1 or 6 g L−1 NaCl. Moreover, the wild-type and the transgenic plants exhibiting similar height (about 3 cm in length) were grown on 1/2 MS medium supplemented with 4 g L−1 or 6 g L−1 NaCl for rooting. The growth condition was controlled at 25°C in a 16 h light/8 h dark photoperiod at an intensity of ~2000 lux. The phenotypes of seedlings were photographed and measured after 20 d of growth.To test the salt stress tolerance of transgenic birchs, the shoot of tissue cultured seedlings was cut into 1- cm pieces and cultured on WPM differentiation medium containing 2 g L−2 s−1.For growth comparison of plants in soil, three transgenic birch lines  and one wild type line (WT) were used in this study. In April 2014, the four lines were propagated and grown in separate pots in a greenhouse. After 60 d, 200 healthy plants in each line were selected as the experimental materials (about 40 cm in height). The greenhouse was controlled at a relative humidity of 65–75% with an average temperature of 27 ± 2°C. Cool white fluorescent lights supplied photons at 200 μmol m−1). NaCl solutions were applied at 18:00–19:00 every 2 d for 16 d. The phenotypes of each group were observed and instantaneous net photosynthesis rate (Pn) and chlorophyll fluorescence parameters (Fv/Fm) were measured at 0, 2, 4, 6, 8, 10, and 12 d during the stress treatment. Pn values were measured from 8:30 a.m. to 11:30 a.m. with a Lico-6400 portable photosynthesis measuring system  on the third to fifth fully expanded leaves of each plant. The conditions during photosynthetic trait measurements were as follows: leaf temperature, 28°C; PPFD, 1400 μmol m-2 s-1; relative humidity, 60%; ambient CO2 concentration, 400 μmol mol-1. Chlorophyll fluorescence parameters were measured with the same leaves using a pulse amplitude modulation chlorophyll fluorometer MINI-PAM2500 . Minimal fluorescence, F0, was measured in 30-min dark-adapted leaves using weak modulated light of < 0.15 μmol m-2 s-1. Maximal fluorescence, Fm, was measured after an 0.8-s saturating white light pulse (6000 μmol m-2 s-1) in the same leaf with 2.9. Maximal variable fluorescence (Fv = Fm–F0) and the photochemical efficiency of PSII (Fv/Fm) for dark-adapted leaves were calculated.Thirty uniform WT seedlings were selected and divided into five group as the preliminary material, One group was designated as the control group and the remaining four groups were treated with NaCl at various concentrations  were selected as the experimental materials. All plants were watered with a 4 g L−1 NaCl solution every 2 d, and the Pn-photosynthetic photon flux density (PPFD) curves and Pn-CO2 concentration in air (Ca) curves were measured and analysesed by the method of Zhao [2 saturation point (CSP) and CO2 compensation point (CCP) were evaluated by fitting the data to the model function as follows:Y is the Pn value, X is the PPFD (or Ca), b0 is a constant, and b1 and b2 are coefficients.We selected 4 g L of Zhao  on 8 d o2 saturation point (CSP) and CO2 compensation point (CCP) were evaluated by fitting the data to the model function.The CO+ contents were conducted on 0, 4, 8, 12, and 16 d of the salt stress treatment, using 10 plants from each line. Also the third to fifth fully expanded leaves of each plant were used for photosynthetic parameters, chlorophyll fluorescence parameters, antioxidant enzyme activity and Na+ contents assays. Photosynthetic parameters , stomatal conductance (Gs), and transpiration rate (Tr)) were measured by lico-6400, chlorophyll fluorescence parameters (Fv/Fm) were measured by MINI-PAM2500, the methods were the same with as ahead. The total superoxide dismutase (SOD) activity was assayed as described by Giannopolitis [+ concentration was determined using atomic absorption spectroscopy as described by Chen [Measurements of photosynthetic parameters, antioxidant enzyme activity, malondialdehyde (MDA) and Naopolitis , total popolitis , Malondiopolitis  and Na+  by Chen .F-tests. Variation among lines in different time was analyzed by ANOVA according to Hansen and Roulund [ij is the performance of an individual of line i within time j, μ is the overall mean, Li is the line effect , Tj is the time effect  and εij is the random error.Statistical analyses were carried out using the Statistical Product and Service Solutions (SPSS 19.0) software. All the parameters were compared using analysis of variance; the significance of fixed effects was tested with  Roulund .yij=μ+LTaLEA gene (GenBank accession NO.: DQ663481) was isolated from T. androssowii, which belongs to late embryogenesis abundant 3 superfamily protein. The ThbZIP gene (NO.: FJ752700) was isolated from T. hispida, a member of basic leucine zipper superfamily of plant G-box binding factor 1 (GBF1)-like transcription factors, which are involved in developmental and physiological processes in response to stimuli such as light, hormones or stress. To investigate the physiological functions of TaLEA and ThbZIP genes in birch, we produced transgenic birch lines ectopically overexpressing these two genes  and five contained both genes, as confirmed by PCR with specific TaLEA and ThbZIP primers  and co-transfer (TaLEA and ThbZIP) both can enhance the salt and osmosis tolerance of transgenic tobacco plants [Abiotic stresses in plants involve a series of physiological and biochemical responses. A great many genes are associated with the abiotic stress-tolerance trait of plants, and multiple transcription factors were activated in different signal transduction pathways to respond single stress. As a result of the limited contribution of single gene to stresses, transfer of multiple transcription factors have been reported to produce additive or synergistic effects on stress tolerance in plants . LEA proo plants –25.Crossbreeding has been one of the most important traditional approaches to obtain new materials for tree breeding. However, this method is very time-consuming, because forest trees have long life cycles with extended vegetative phases ranging from one to many decades . Genetic−1 was determined to be appropriate; this is the same concentration that was used in a study on poplar [Determining the appropriate concentration of salt was the most important factor in the design of these experiments. Salt concentrations that were too high led to a rapid decrease in the photosynthetic index, which was not conducive to observation, and those that were too low did not affect the photosynthetic index sufficiently, or required a very long experimental period to detect any effects. In this research, the NaCl concentration of 4 g Ln poplar .−2 s−1, indicating that birch can adapt to strong illumination intensity. The LSPs of transgenic lines were higher than that of WT, suggesting that the plants harboring TaLEA-ThbZIP showed greater resistance to high illumination intensity. After 8 d of salt stress, the Pn of the different lines increased slowly with increasing PPFD, and the LSPs and maximum Pns at the LSP were lower than those before the stress treatment. The results also showed that salt stress affected antioxidant enzyme activity and the Na+ content in plant cells, which ultimately affected photosynthesis, consistent with the results reported by Deng [Measurement of the Pn–PPFD curve is an important method to analyze the ability of plants to adapt to high or low light conditions . In this by Deng .2 concentrations are projected to double from the current concentration of 350 μmol mol−1 to 700 μmol mol−1. This increase will further stimulate plant growth and result in ecosystem changes [2 is the most important substrate for photosynthesis. Within a certain concentration range, enhanced CO2 concentrations can promote the instantaneous photosynthetic rate [2 concentration [−1. Below the CSP, the Pn of the four lines ranged from 11.23 to 14.52 μmol m−2 s−1, similar to the Pn values reported for poplar seedlings [2. The instantaneous Pn under CSP was lower at 8 d of the salt stress treatment than before the stress treatment, indicating that salt stress affected photosynthesis in birch. The instantaneous Pn values were higher in L-4 and L-8 than in WT and L-5, indicating that the new genes had affected the physiology of the transgenic lines.In the next 80 years, atmospheric COtic rate . There intration . In thiseedlings . These rv/Fm values differed significantly (P < 0.01) among the four lines and the five time points. The Fv/Fm values of L-4 and L-8 were higher than those in L-5 and WT after 8 d of salt stress, indicating that the new genes had altered the efficiency of energy transfer from photosystem II, and increased the salt resistance of these lines.Chlorophyll fluorescence measurements are easy and rapid to conduct, do not damage plants, and sensitively reflect the relationship between the physiological status of the plant and the environment . In receGmbZIP78 leads to not only reducing salt resistance, will also affecting plant growth [bZIP genes in Arabidopsis thaliana  leads to slow growth, dwarf, and abnormal phenotype [bZIP may exist the most appropriate concentration range in vivo, too high may lead to the imbalance of transcriptional regulation.During plant growth and development, many metabolic pathways produce reactive oxygen species, which can damage cell membranes and lead to cell death . During t growth . Overexphenotype . That is+ content in leaves indirectly reflects the amount of Na+ absorbed by the roots, and is an indicator of the degree of stress. In this study, as the duration of the salt stress treatment extended, the Na+ content in leaves of all lines increased to different degrees, indicating that salt stress affected the growth of the lines differently. The average Na+ content showed little change after 4 d of salt stress, but had increased markedly after 8 d of salt stress. The Na+ contents were lower in Lines L-4 and L-8 than in WT and L-5, indicating that the former two lines were more salt-resistant than the latter two lines.The ability of a plant to tolerate salinity is related to its ability to maintain ion homeostasis in cells. Many different soluble salts can reduce the osmotic potential of plant rhizosphere, making it more difficult for the plant to absorb water, leading to physiological drought . The Na+TaLEA and ThbZIP genes were transformed into birch. Because the transcriptional level of these exogenous genes differed among the lines, the various transgenic lines showed different physiological properties under salt stress. Lines L-4 and L-8 were selected as excellent lines because of their strong salt resistance. Further research should characterize their growth and stem traits, and the regulation mechanism of the exogenous genes. Also, we should fully consider the safety of releasing and cultivating transgenic plants in the field.With the development of science and technology, increasing numbers of transgenic varieties of various crop species  have been produced . In foreS1 Table(DOCX)Click here for additional data file.


Pinna nobilis was detected across a wide geographical area of the Spanish Mediterranean Sea and linked to a haplosporidian parasite. In 2017–2018, mass mortality events affecting the pen shell Pinna nobilis were recorded in two different regions of Italy, Campania and Sicily, in the Tyrrhenian Sea (Mediterranean Sea). Histopathological and molecular examinations of specimens showed the presence of Haplosporidium sp. in only one specimen in one area. Conversely, in all of the surveyed moribund animals, strong inflammatory lesions at the level of connective tissue surrounding the digestive system and gonads and linked to the presence of intracellular Zhiel-Neelsen-positive bacteria were observed. Molecular analysis of all of the diseased specimens (13) confirmed the presence of a Mycobacterium. Blast analysis of the sequences from all of the areas revealed that they were grouped together with the human mycobacterium M. sherrisii close to the group including M. shigaense, M. lentiflavum and M. simiae. Based on pathological and molecular findings, it is proposed that a mycobacterial disease is associated with the mortality episodes of Pinna nobilis, indicating that, at this time, Haplosporidium sp. is not responsible for these events in Campanian and Sicilian waters.Disease is an increasing threat for marine bivalves worldwide. Recently, a mass mortality event (MME) impacting the bivalve  Pinna nobilis is an endemic Mediterranean species and among the largest bivalves worldwide, playing an important ecological role for soft bottom communities and contributing to the increase in local biodiversity. This species can reach up to 120 cm in length and a maximum reported age of 27 years2. The species commonly lives in seagrass fields of Posidonia oceanica or Cymodocea nodosa2 but also in non-vegetated estuarine areas and the soft bottoms of marine lakes3. The family of Pinnidae includes two genera (Pinna and Atrina) with 61 species described worldwide4. Different anthropogenic factors have adverse effects on the pen shell lifecycle and distribution, bringing structural and functional alterations in habitats and species physiology. Currently, P. nobilis has become a threatened and vulnerable species and is legally protected under Annex II of the Barcelona Convention (SPA/BD Protocol 1995), Annex IV of the EU Habitats Directive (EU Habitats Directive 2007), and the Spanish Catalogue of Threatened Species .The bivalve pen shell 7 and have also been described in the Mediterranean Sea10. Evidence suggests that a combination of predisposing and necessary biotic factors is involved in the disease causation, resulting in a scenario of new emerging complex multifactorial diseases11. In fact, these diseases have been often linked to changes in host/pathogen interactions and have been associated with increased water temperature, pathogen distribution and virulence, host reduced immune competence and growth13. The spread of infectious diseases of molluscs due to pathogens of different types has been intensively described in the last years, especially in farmed species15, with new outbreaks continuing to be recorded globally, representing a large limitation for the aquaculture industry. Conversely, in wild animal populations, only a few descriptions are present, and these outbreaks can involve keystone species, with consequences for the whole ecosystem.To date, disease conditions and mortality outbreaks have been reported in marine benthic population worldwide, such as corals, sea urchins, molluscs, sea turtles and marine mammalsPinna nobilis were reported over hundreds of kilometres of the western Mediterranean coast of Spain, except for Catalonia, due to a haplosporidian parasite17.Recently, mass mortality events of the pen shell P. nobilis populations were detected in Campania and Sicily, affecting animals of all sizes and affecting 85–100% in prevalence. The aims of this paper are to unravel the bivalve health status and to define the possible causes of the mortality events involving the pen shell P. nobilis in these two geographical areas in late 2017 through May 2018.In Italy, since 2016, mortality episodes of the pen shell have been described by local SCUBA divers and scientists along the Tyrrhenian coastline and encompassing different regions. In early 2017, mass mortality episodes of Following mortality episodes from different areas of the regions of Campania and Sicily, samples were collected and provided to our laboratory by divers in the Area Marina Protetta (AMP) Punta Campanella from the north-southeast part of the Campania region from December 2017 to May 2018. At the same time, samples were also collected by local fisherman in Sicily (Messina) in May 2018  were negative for all of the diagnostic analyses.Details about animal dimensions, macroscopic lesions and disease diagnosis are reported in Table P. rudis was healthy and negative for all of the diagnostic analyses performed for the different pathogens.The pen shells did not show any sign of illness, and the only collected individual of Regarding diseased animals, during collection, anomalous behavioural signs were demonstrated in debilitated specimens, showing difficulty in closing the valves (gaping) or slow responses to touching. The animals also showed retracted mantles or mantles with watery cysts.Pontonia pinnophylax; in 7 cases (53%), there were two per bivalve.On laboratory examination, on the external valves, the animals presented with attached epibionts of different types; among them, polychaetes, bryozoans, red and brown algae, ascidians, sponges and small bivalves were present. All of the specimens contained in the valves the Palaemonidae shrimps In 9 of the exanimated specimens (69%), gross examination of the bivalves revealed diffuse tissue oedema, mainly visible at the level of the gill and mantle Table , Fig. 3A18 as both hyalinocytes and granulocytes. The inflammatory condition was characterized by large nodular aggregates of the above immune cells, which were filled with long, slightly shaped, acid-fast positive bacteria , both cytology and histopathology showed the presence of different phases of development of a haplosporidian parasite in the digestive tissue. Large numbers of multinucleate stages were disseminated in the digestive tubule epithelia Fig. . SporogoElectron microscopy (EM) was used to further characterize these bacteria in the absence of a cultured strain. Transmission electron microscopy (TEM) performed on three infected individuals was used to assess the features of the bacteria Fig. , allowinMycobacterium sp., three different PCR sets were run for different pathogens: the Herpes virus OsHv-119; haplosporidian parasites20; and Mycobacterium sp21 detection.Considering the pathogens responsible for mortality episodes reported in Italian bivalves and considering the observed P. nobilis examined apart from the non-moribund specimens from south of Campania ; moreover, it showed 92% similarity to the 18S small subunit ribosomal RNA of uncultured Haplosporidium sp. from environmental samples but was not grouped with any definite haplosporidian species (sequence accession number: MH572222)  of sessile benthic invertebrates, including ascidians, sponges, anthozoans and bivalves, have been reported in the aquatic environment and many indeed in the Mediterranean Sea. Several factors have contributed to mass-mortality episodes. Many papers have agreed that global warming might be linked to the occurrence of such catastrophic events in the Mediterranean Sea, which could alter the host/pathogen range, due to alteration in host immunocompetence/pathogen virulence and thus modify pathogen transmission rates. In this scenario, opportunistic pathogens are suspected to play an important role, with a modified condition of virulence and pathogenicity25. With the exception of Mycobacterium leprae, the species responsible for tuberculosis in human and animals consists of a group of highly related mycobacterial lineages collectively known as the mycobacterium tuberculosis complex (MTBC)25. Finally, MTBC is distinguished from nontuberculous mycobacteria (NTM): free-living organisms that are ubiquitous in the environment and can cause a wide range of mycobacterial infections in humans and animals (more frequently pulmonary infections)26.Mycobacteriosis is a serious and generally lethal infectious disease, affecting a wide range of species from human to animals, both in farmed and wild conditions. The Mycobacteriaceae family includes 128 validly published species30. In particular, in marine environments, three species have been frequently cited as the main causative agents of infections in fish M. marinum, M. fortuitum and M. chelonae but also several other mycobacterial species, including a number of novel species, have been reported33. Thus far, mycobacteria in molluscs have only been detected in different gastropod species from the freshwater environment, while in bivalves, frequently oysters of the genus Crassostrea have been reported to be vector of infection involved in human disease, and one case was instead reported in the pecten Placopecten magellanicus with no human infection24  system. In the case of M. ulcerans, Mycobacteria are primary intracellular parasites of phagocytes. Phagosomes containing mycobacteria are believed to resist the normal processes of acidification and phago-lysosomal fusion42, thus promoting bacterial survival45. However, according to electron microscopy results, the Mycobacterium detected in our cases were located in the cytoplasm of immune cells without evidence of phagosome membranes around them. This finding seems to be in accordance with early evidence, suggesting that mycobacteria might eventually escape from phagosomes by translocating to the cytosol, as in the cases of M. tuberculosis and M. marinum46. It has been suggested that cytosolic translocation might reflect a strain-dependent virulence mechanism of pathogenic mycobacteria, conferring to cytosol-preferring strains a gain in virulence function by acquiring resistance to autophagy48. In this context, Mycobacterium sherrisii has been so far associated with several human diseases49 and only a few cases have been reported in animals, all of them in mammals50. To our knowledge, this report is the first concerning evidence for the involvement of M. sherrisii in disease outbreaks of aquatic organisms and in a bivalve mollusc. Over thousands of years, mycobacteria have undergone extensive specialization, particularly with vertebrate hosts or specific environmental ecosystems retaining the flexibility to occupy new niches by continually infecting different animals as primary pathogens or opportunists. In our cases, the zoonotic potential of the observed Mycobacterium should be considered and clarified.With the exception of 28, surviving and replicating within various hosts51, so vectors are potentially present throughout the food web. In many cases, the modality of transmission is not clear. Water and associated biofilms are natural habitats for Mycobacterium spp. including M. marinum, M. fortuitum, and M. chelonae, so waterborne transmission seems likely. In our case, the origin and transmission of the Mycobacterium remain unknown, but aquatic environments constitute the natural habitat of M. simiae complex.Mycobacteria are known to infect a number of aquatic organisms other than fish11. In this context, it is important to emphasize that the boundaries between pathogen and symbiont can be, in some cases, unfixed, with an alteration in the association, even in evolutionary short periods of time52. About haplosporidians, the group includes more than 50 described species, many of them responsible for important diseases in aquatic invertebrates53. An haplosporidian parasite was detected across a wide geographic area of the Spanish Mediterranean Sea (western Mediterranean Sea) in early autumn 2016 and was recognized as possibly responsible for the MME of P. nobilis population in the area17. A previous hypothesis reported that the observed Haplosporidium was instead a symbiont that modified its relationship with the host following environmental changes, finally leading to the mortality outbreak17.Disease is the outcome of complex interactions among the host, causative agent(s) and environment. Many causes frequently cooperate to induce diseases (complex of causes), and some of them are necessary (their absence prevents the onset of the effect), while others are predisposing (preparing the ground for the action of the necessary cause)P. nobilis, in which two possibly opportunistic pathogens are involved in two different areas of the Mediterranean Sea, suggesting that both are activated by further possible common unidentified causes. Based on our pathological and molecular findings, we are more likely to consider that a mycobacterial disease is associated with the observed mortality episodes, indicating that, at this time, Haplosporidium sp. is not responsible for the events in Campanian and Sicilian waters. Further knowledge of the modality of transmission, distribution and source of this pathogens is key to devising methods for the identification and understanding of disease pathogenesis. In our cases, the lack of genomic and proteomic data does not allow us to go beyond the consideration that further studies are needed to clarify the pathogenicity and potential virulence for the reported mycobacteriosis other than its origin and evolution. The present study is especially relevant since it uncovers additional information about the complexity in understanding the multifactorial diseases that we have experienced in recent years in the aquatic environment. Future collaboration among wildlife ecologists, environmental biologists, animal pathologists and related disciplines should be conducted to deeply explore emerging diseases in marine wildlife.In such a scenario, we consider that data reported in our study drive a hypothesis of a more complex disease pathogenesis involved in the disease outbreak of the bivalve P. nobilis from 5 sites: in December 2017 in Massa Lubrense 1  and Massa Lubrense 2 (40°34′57.5″N 14°21′22.6″E); Ischia island, AMP Regno di Nettuno  and Positano in April 2018 ; and in May 2018 Cilento 1  and Cilento 2 (40°10′23.9″N 15°05′08.4″E). In Sicily, there were two places: Torre Faro  and Paradiso  . During the macroscopic evaluation of the individuals, a description of each specimen’s condition was conducted, and the presence of epibionts and valve length were recorded. Animal valves were opened with the help of a blade, and the flesh observed was examined macroscopically, checking for eventual external signs. Impression smears of the digestive glands, gills, mantle and ovaries were obtained, air dried, fixed in absolute ethanol, and stained with May–Grunwald–Giemsa quick stain  for cytological examination. Samples from the digestive glands, mantle, labial palps, gills, gonads and adductor and retractor muscles were fixed in Davidson’s solution for 48 h at room temperature. Pieces of digestive gland and mantle were also preserved in 2.5% glutaraldehyde for TEM examination. Fragments of bivalve tissues and shrimp P. pinnophylax were fixed in absolute ethanol for DNA isolation.The study area in Campania covers a large geographical scale including the main habitat for P. nobilis and P. pinnophylax tissue samples were embedded in paraffin blocks and cut to 5 μm with a rotary microtome . Tissue sections were deparaffinized, stained with Carazzi haematoxylin and eosin and examined by light microscopy (Zeiss Axioscope A1). Additional staining techniques were also performed: Gram, Mallory Trichrome, Ziehl-Neelsen and PAS-BA (periodic acid Shiff)54.Five animals from different areas were processed for TEM observation, searching for pathogens of a different nature. From each animal, pieces of digestive gland tissue were placed in 2.5% glutaraldehyde, post-fixed in 2% OsO4, and embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate and were examined in a JEOL JEM 1010 transmission electron microscope at 80 kV.DNA was isolated from pieces of different tissues using the Qiagen Blood and Tissue Kit (Qiagen). DNA quality and quantity were checked with a Nanodrop ND-1000 spectrophotometer .19, generic haplosporidian primers (HAPF1- HAPR3)20 and primers derived from the 16S rRNA sequence of mycobacteria, mycgen-f (5′-AGAGTTTGATCCTGGCTCAG-3′) mycgen-r (5′-TGCACACAGGCCACAAGGGA-3′), as described by21. A positive control was used for OsHv-1 PCR reaction. The PCR was performed in 25 μl of reaction volume containing 1 μl of genomic DNA, 12.5 μl of GoTaq MasterMIX (Promega) at 1x concentration, 6.5 μl of water and 2.5 μl of each primer (10 μM). PCR products were electrophoresed on 2% agarose gels in 1x TAE buffer. The amplified fragments were gel eluted and directly sequenced. Negative controls were included in the PCR reaction.DNA was amplified by PCR with OsHv-1 primers (OsHVDPFor/OsHVDPRev) byBLASTN analysis was conducted using the nucleotide sequences obtained in the present study. The sequences were then submitted to GenBank (the accession numbers are listed in Figs Mycobacterium sp. obtained in the present study and those of different Mycobacterium species present in GenBank, selected on the basis of the highest BLASTN score. The analysis also included the 16S sequence of Mycobacterium sp. KR822678 isolated from sea scallops24 and of M. marinum and M. ulcerans, which are able to infect marine molluscs23. The nucleotide alignment was constructed using ClustalW and the neighbour-joining tree was obtained using the Maximum Composite Likelihood model implemented in MEGA X61 with 1000 bootstrap replicates.The pairwise p-distances were calculated among the 16S nucleotide sequences of