Effects of Chronic Stress from High Stocking Density in Mariculture: Evaluations of Growth Performance and Lipid Metabolism of Rainbow Trout (Oncorhychus mykiss).

(1) Background: In aquaculture, chronic stress due to high stocking density impairs animals' welfare and results in declined fishery production with low protein quality. However, most previous studies evaluated the effects of high stocking density on trout in freshwater rather than seawater. (2) Methods: Juvenile trout were reared for 84 days in circular tanks under three stocking densities, including low density ("LD", 9.15 kg/m3), moderate density ("MD", 13.65 kg/m3), and high density ("HD", 27.31 kg/m3) in seawater. The final densities of LD, MD, and HD were 22.00, 32.05 and 52.24 kg/m3, respectively. Growth performance and lipid metabolism were evaluated. (3) Results: Growth performance and feeding efficiency were significantly reduced due to chronic stress under high density in mariculture. The digestive activity of lipids was promoted in the gut of HD fish, while the concentration of triglycerides was decreased in the blood. Furthermore, decreased acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), increased hormone-sensitive lipase (HSL) concentrations, and activated hepatic ß-oxidation processes were observed in trout under HD. Redundancy analysis showed that glycerol and HSL can be used as potential markers to evaluate the growth performance of trout in mariculture. (4) Conclusions: We showed that chronic high stocking density led to negative effects on growth performance, reduced de novo synthesis of fatty acids, and enhanced lipolysis.

The Odorant Receptor Recognizing Camphor in a Camphor Tree Specialist Orthaga achatina (Lepidoptera: Pyralidae).

Camphor has been used as an effective repellent and pesticide to stored products for a long history, but Orthaga achatina (Lepidoptera: Pyralidae) has evolved to specifically feed on the camphor tree Cinnamomum camphora. However, the behavioral response of O. achatina to camphor and the molecular basis of camphor perception are totally unknown. Here, we demonstrated that both male and female adults were behaviorally attracted to camphor, suggesting the adaptation of O. achatina to and utilization of camphor as a signal of C. camphora. Second, in 40 O. achatina OR genes obtained by analyzing antenna transcriptomes, only OachOR16/Orco significantly responded to camphor in the Xenopus oocyte system. Finally, by molecular docking analysis and site-directed mutagenesis, the Ser209 residue is confirmed to be essential for binding of the oachOR16 with camphor. This study not only reveals the camphor-based host plant choice and olfactory mechanisms of O. achatina but also provides a molecular target for screening more potential insect repellents.

Effects of Dietary Zinc on Growth Performance, Digestive Enzyme Activities, Antioxidant Status, and Immune Responses of Sea Cucumber Apostichopus japonicus.

Zinc is an essential micronutrient for organisms involved in regulating various biological processes. This study evaluated the effects of dietary zinc on growth performance, digestive enzyme activities, antioxidant status, and immune responses of sea cucumber Apostichopus japonicus. Five experimental diets were formulated with graded levels of zinc (0, 20, 40, 60, and 80 mg/kg, respectively), and the actual dietary zinc values were 31.4, 51.0, 68.2, 91.9, and 110.8 mg/kg diet, respectively. Sea cucumbers were fed with diets for 2 months. The results showed the growth performance, amylase, and trypsin activities of sea cucumber increased significantly with zinc supplementation, and the best growth performance and enzyme activities were observed at 40 mg/kg zinc diet. Zinc supplementation significantly increased activities of superoxide dismutase, catalase, anti-superoxide anion, and inhibiting hydroxyl radical, while significantly reduced the malondialdehyde content. Furthermore, the higher zinc supplementation levels resulted in significantly upregulated immune-related genes of hsp90, p105, rel, and lsz, suggesting that excessive zinc caused oxidative stress. The broken-line regression analysis of specific growth rate indicated dietary zinc requirement in juvenile sea cucumber was ~ 66.3 mg/kg diet. Overall, dietary zinc contributes to the growth and immune resistance of juvenile sea cucumber, and our study will provide insights into the rational use of dietary zinc in aquaculture.

Molecular mechanism of sex pheromone perception in male Mythimna loreyi revealed by in vitro system.

BACKGROUND: Mythimna loreyi is an important agricultural pest with a sensitive sex pheromone communication system. To clarify the pheromone binding proteins (PBPs) and pheromone receptors (PRs) involved in sex pheromone perception is important for both understanding the molecular olfactory mechanism and developing a new pest control strategy in M. loreyi. RESULTS: First, the electroantennogram (EAG) assay showed that male M. loreyi displayed the highest response to the major sex pheromone component Z9-14:Ac, and higher responses to two minor components, Z7-12:Ac and Z11-16:Ac. Second, the fluorescence competition binding assay showed that PBP1 bound all three pheromones and other tested compounds with high or moderate affinity, while PBP2 and PBP3 each bound only one pheromone component and few other compounds. Third, functional study using the Xenopus oocyte system demonstrated that, of the six candidate PRs, PR2 was weakly sensitive to the major pheromone Z9-14:Ac, but was strongly sensitive to pheromone analog Z9-14:OH; PR3 was strongly and specifically sensitive to a minor component Z7-12:Ac; PR4 and OR33 were both weakly sensitive to another minor component, Z11-16:Ac. Finally, phylogenetic relationship and ligand profiles of PRs were compared among six species from two closely related genera Mythimna and Spodoptera, suggesting functional shifts of M. loreyi PRs toward Spodoptera PRs. CONCLUSION: Functional differentiations were revealed among three PBPs and six PRs in sex pheromone perception, laying an important basis for understanding the molecular mechanism of sex pheromone perception and for developing new control strategies in M. loreyi. © 2023 Society of Chemical Industry.

Co-exposure of nanoplastics and arsenic causes neurotoxicity in zebrafish (Danio rerio) through disrupting homeostasis of microbiota-intestine-brain axis.

Nanoplastics (NPs) and arsenic (As) are toxic pollutants prevalent on the earth and have gained considerable attention in recent decades. Although numerous studies reported NPs and As can cause neurotoxicity there are still significant knowledge gaps in illustrating their combined toxicity and its mechanism. In this study, the co-exposure of environmentally relevant concentrations of NPs and As caused neurobehavioral toxicity in zebrafish, as evidenced by reduced swimming ability, anxiety and impaired short-term learning memory. Potentially, its toxicity mechanism is through disrupting the homeostasis of microbiota-intestine-brain axis in zebrafish. Specifically, the co-exposure reduced the 5-hydroxytryptamine (5-HT) production in intestine, which led to lower levels of 5-HT transported by the blood circulation to the brain. Ultimately, neurobehavior was adversely affected by the reduced binding of 5-HT to its receptors. Intestine, the primary source of 5-HT, its impaired health (aggravation in oxidative stress, mitochondrial damage and histopathological alterations) induced the dysregulation in the 5-HT system, which may be induced by the increased accumulation of As in the intestine by the co-exposure. Besides, the reduced 5-HT levels were correlated with decreased Firmicutes and Protecbacteria and increased Actinobacteriota and Chloroflexi in intestines. Potentially, intestinal microbiota adversely regulates the intestine-brain axis by reducing SCFAs levels. Thus, the alteration of intestinal microbiota structure may be the other reason for the dysregulation of intestine-brain axis. In summary, co-exposure of NPs and As induced neurobehavior toxicity probably through disrupting the homeostasis of microbiota-intestine-brain axis. This study provides insights into assessing the environmental health risks of the pollution of NPs and As to aquatic organisms.

Temperature acclimation improves high temperature tolerance of rainbow trout (Oncorhynchus mykiss) by improving mitochondrial quality and inhibiting apoptosis in liver.

Global warming is threatening the survival and growth of cold-water fish, and the methods to improve the high-temperature adaptability of cold-water fish need to be explored urgently. This study aims to explore the mechanism of improving high temperature tolerance of rainbow trout by temperature acclimation (TA). Rainbow trout were acclimated by two modes at 22 °C, including fluctuating TA (FA) and constant TA (CA), and thereafter subjected to heat stress (HS) at 25 °C. Results showed that TA markedly increased the critical temperature maximum (CTmax) of rainbow trout. Secondly, the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum and malondialdehyde (MDA) in liver of CA + HS group significantly decreased compared to those in HS group without TA, indicating the reduction of liver injury by CA. Moreover, HS significantly induced ROS production and reduced mitochondrial membrane potential (MMP) in rainbow trout liver, but TA reduced the levels of ROS and increased the MMP in liver of rainbow trout after HS, indicating the reduced oxidative stress and mitochondrial damage. Furthermore, TA up-regulated the expression of genes related to mitochondrial autophagy, fusion, fission and biogenesis, as well as the expression of marker proteins of autophagy (LC3II) and mitophagy (Parkin) in the liver, so as to maintain mitochondrial homeostasis. Moreover, TA also inhibited the occurrence of apoptosis (decrease in bax/bcl-2), which may be owing to the reduced ROS and mitochondrial damage by TA. Interestingly, CA significantly up-regulated the genes expression of methyltransferase in the liver, which may inhibit the genes or transcription factors related to oxidative stress and apoptosis by DNA methylation. In conclusion, TA increased the upper limit of heat tolerance of rainbow trout by improving mitochondrial quality and inhibiting apoptosis in liver. This study will provide an effective solution to the risk of high temperature in cold-water fish culture.

Ionotropic Receptor IR75q.2 Mediates Avoidance Reaction to Nonanoic Acid in the Fall Armyworm Spodoptera frugiperda (Lepidoptera, Noctuidae).

Ionotropic receptors (IRs) play an important role in olfaction, but little is known in nondrosophila insects. Here, we report in vitro and in vivo functional characterization of IR75q.2 in the invasive moth pest Spodoptera frugiperda. First, 13 IRs (including four coreceptor IRs) were found specifically or highly expressed in adult antennae. Second, these IRs were tested for responding profiles to 59 odorants using the Xenopus oocyte expression system, showing that only SfruIR75q.2 responded to 8-10C fatty acids and their corresponding aldehydes, with SfruIR8a as the only coreceptor. Third, the three acids (especially nonanoic acid) showed repellent effects on moth's behavior and oviposition, but the repellence significantly reduced to the insects with IR75q.2 knockout by CRISPR/Cas9. Taken together, our study reveals the function of SfruIR75q.2 in perception of acid and aldehyde odorants and provides the first in vivo evidence for olfactory function of an odor-specific IR in Lepidoptera.

An odorant receptor tuned to an attractive plant volatile vanillin in Spodoptera litura.

The insect olfaction plays crucial roles in many important behaviors, in which ORs are key determinants for signal transduction and the olfactory specificity. Spodoptera litura is a typical polyphagous pest, possessing a large repertoire of ORs tuning to broad range of plant odorants. However, the specific functions of those ORs remain mostly unknown. In this study, we functionally characterized one S. litura OR (OR51) that was highly expressed in the adult antennae. First, by using Xenopus oocyte expression and two-electrode voltage clamp recording system (XOE-TEVC), OR51 was found to be strongly and specifically responsive to vanillin (a volatile of S. litura host plants) among 77 tested odorants. Second, electroantennogram (EAG) and Y-tube behavioral experiment showed that vanillin elicited significant EAG response and attraction behavior especially of female adults. This female attraction was further confirmed by the oviposition experiment, in which the soybean plants treated with vanillin were significantly preferred by females for egg-laying. Third, 3D structural modelling and molecular docking were conducted to explore the interaction between OR51 and vanillin, which showed a high affinity (-4.46 kcal/mol) and three residues (Gln163, Phe164 and Ala305) forming hydrogen bonds with vanillin, supporting the specific binding of OR51 to vanillin. In addition, OR51 and its homologs from other seven noctuid species shared high amino acid identities (78-97%) and the same three hydrogen bond forming residues, suggesting a conserved function of the OR in these insects. Taken together, our study provides some new insights into the olfactory mechanisms of host plant finding and suggests potential applications of vanillin in S. litura control.

Nitrogen removal capability and mechanism of a novel heterotrophic nitrifying-aerobic denitrifying strain H1 as a potential candidate in mariculture wastewater treatment.

The nitrogen removal performance and mechanisms of Bacillus subtilis H1 isolated from a mariculture environment were investigated. Strain H1 efficiently removed NH4+-N, NO2--N, and NO3--N in simulated wastewater with removal efficiencies of 85.61%, 90.58%, and 57.82%, respectively. Strain H1 also efficiently degraded mixed nitrogen (NH4+-N mixed with NO2--N and/or NO3--N) and had removal efficiencies ranging from 82.39 to 89.54%. Nitrogen balance analysis revealed that inorganic nitrogen was degraded by heterotrophic nitrification-aerobic denitrification (HN-AD) and assimilation. 15N isotope tracing indicated that N2O was the product of the HN-AD process, while N2 as the final product was only detected during the reduction of 15NO2--N. The nitrogen assimilation and dissimilation pathways by strain H1 were further clarified using complete genome sequencing, nitrification inhibitor addition, and enzymatic activity measurement, and the ammonium oxidation process was speculated as NH4+ → NH2OH → NO → N2O. These results showed the application prospect of B. subtilis H1 in treating mariculture wastewater.

Conserved Odorant Receptors Involved in Nonanal-Induced Female Attractive Behavior in Two Spodoptera Species.

Moths rely on plant volatiles to locate appropriate plants for feeding and laying eggs. While extensive research has been conducted on the global agricultural pests, Spodoptera frugiperda and Spodoptera litura, their molecular mechanisms for detecting plant volatiles remain mostly unknown. Here, we have demonstrated that nonanal, a common plant volatile, is attractive for both virgin and gravid females of the two species. Second, we have identified a conserved odorant receptor clade (SfruOR47 clade) that is primarily tuned to nonanal. Finally, by three-dimensional (3D) structure prediction, molecular docking, and site-directed mutagenesis, we have revealed that the His57 and Glu61 residues, also shared by other six orthologous ORs, are essential for nonanal binding in SfruOR47 and SlituOR9, indicating the conserved structure and function of ORs in the SfruOR47 clade. These findings offer novel insights into the molecular mechanisms and evolutionary aspects of moth behavior in response to plant volatiles.

Nitrogen removal capability and mechanism of a novel heterotrophic nitrification-aerobic denitrification bacterium Halomonas sp. DN3.

Recently, the functional microorganisms capable of eliminating nitrogenous waste have been applied in mariculture systems. As a potential candidate for treating mariculture wastewater, strain DN3 eliminated 100% of ammonia and nitrate and 96.61%-100% of nitrite within 72 h, when single nitrogen sources at concentrations of 0-50 mg/L. Strain DN3 also exhibited the efficient removal performance of mixed-form nitrogen (ammonia, nitrate, and nitrite) at salinity 30 ‰, C/N ratio 20, and 180 rpm. The nitrogen assimilation pathway dominated inorganic nitrogen metabolism, with less nitrogen (14.23%-25.02% of TN) lost into the air via nitrification and denitrification, based on nitrogen balance analysis. Moreover, the bacterial nitrification pathway was explored by enzymatic assays and inhibition assays. These complex nitrogen assimilation and dissimilation processes were further revealed by bacterial genome analysis. These results provide important insight into nitrogen metabolism of Halomonas sp. and theoretical support for treating mariculture wastewater with strain DN3.

Functional differentiation of two general-odorant binding proteins in Hyphantria cunea (Drury) (Lepidoptera: Erebidae).

BACKGROUND: General odor-binding proteins (GOBPs) play critical roles in insect olfactory recognition of sex pheromones and plant volatiles. Therefore, the identification of GOBPs in Hyphantria cunea (Drury) based on their characterization to pheromone components and plant volatiles is remain unknown. RESULTS: In this study, two H. cunea (HcunGOBPs) genes were cloned, and their expression profiles and odorant binding characteristics were systematically analyzed. Firstly, the tissue expression study showed that both HcunGOBP1 and HcunGOBP2 were highly expressed in the antennae of both sexes, indicating their potential involvement in the perception of sex pheromones. Secondly, these two HcunGOBPs genes were expressed in Escherichia coli and ligand binding assays were used to assess the binding affinities to its sex pheromone components including two aldehydes and two epoxides, and some plant volatiles. HcunGOBP2 showed high binding affinities to two aldehyde components (Z9, Z12, Z15-18Ald and Z9, Z12-18Ald), and showed low binding affinities to two epoxide components (1, Z3, Z6-9S, 10R-epoxy-21Hy and Z3, Z6-9S, 10R-epoxy-21Hy), whereas HcunGOBP1 showed weak but significant binding to all four sex pheromone components. Furthermore, both HcunGOBPs demonstrated variable binding affinities to the plant volatiles tested. Thirdly, in silico studies of HcunGOBPs utilized homology, structure modeling, and molecular docking revealed critical hydrophobic residues might be involved in the binding of HcunGOBPs to their sex pheromone components and plant volatiles. CONCLUSION: Our study suggests that these two HcunGOBPs may serve as potential targets for future studies of HcunGOBPs ligand binding, providing insight in the mechanism of olfaction in H. cunea. © 2023 Society of Chemical Industry.

Identification and Functional Characterization of General Odorant Binding Proteins in Orthaga achatina.

The olfactory system in insects are crucial for recognition of host plants and oviposition sites. General odorant binding proteins (GOBPs) are thought to be involved in detecting odorants released by host plants. Orthaga achatina (Lepidoptera: Pyralidae) is one of the most serious pests of camphor trees, Cinnamomum camphora (L.) Presl, an important urban tree species in southern China. In this study, we study the GOBPs of O. achatina. Firstly, two full-length GOBP genes (OachGOBP1 and OachGOBP2) were successfully cloned according to transcriptome sequencing results, and real-time quantitative PCR measurements showed that both GOBP genes were specifically expressed in the antennae of both sexes, proposing their important roles in olfaction. Then, both GOBP genes were heterologous expressed in Escherichia coli and fluorescence competitive binding assays were conducted. The results showed that OachGOBP1 could bind Farnesol (Ki = 9.49 µM) and Z11-16: OH (Ki = 1.57 µM). OachGOBP2 has a high binding affinity with two camphor plant volatiles (Farnesol, Ki = 7.33 µM; α-Phellandrene, Ki = 8.71 µM) and two sex pheromone components (Z11-16: OAc, Ki = 2.84 µM; Z11-16: OH, Ki = 3.30 µM). These results indicate that OachGOBP1 and OachGOBP2 differ in terms of odorants and other ligands. Furthermore, key amino acid residues that bind to plant volatiles were identified in GOBPs using 3-D structure modeling and ligand molecular docking, predicting the interactions between the GOBPs and the host plant volatiles.

Nanoplastics promote arsenic-induced ROS accumulation, mitochondrial damage and disturbances in neurotransmitter metabolism of zebrafish (Danio rerio).

As a carrier, nanoplastics (NPs) can adsorb other toxic substances and thus modify their biological toxicity. Numerous studies have investigated the neurotoxic of high concentrations of arsenic (As, 2.83 mg/L-5 mg/L). However, it is still unknown whether the relatively low environmentally relevant concentrations of As (200 µg/L) can damage the structure and function of fish brains with the presence of NPs. In this study, zebrafish were exposed to polystyrene NPs, As and their mixture for 30 days respectively. Firstly, we found that the presence of NPs promoted the accumulation of As in zebrafish brains. Thereby the co-exposure of NPs and As further promoted the production of reactive oxygen species (ROS) in zebrafish brains compared with the single exposure of NPs or As, resulting in severe oxidative stress. Moreover, accumulated ROS directly damaged the mitochondrial membrane and mtDNA in zebrafish brains. Moreover, the mitochondrial damage was further aggravated due to inhibited mitochondrial fusion and activated mitochondrial division and mitophagy. Ultimately, the co-exposure led to mitochondrial damage in the zebrafish brain. Damaged mitochondria may not meet the high energy metabolic requirement for neuronal function. As a result, the normal function of nerve cells was adversely affected and eventually cell apoptosis may occur. Besides, the co-exposure caused more significant structural alterations in zebrafish brain tissue. Finally, the co-exposure of NPs and As caused abnormal biosynthesis and degradation of dopamine and acetylcholine. These resulted in decreased dopamine levels and increased acetylcholine levels in zebrafish brains. In conclusion, the presence of NPs promoted the accumulation of As, thereby inducing severe oxidative stress, which caused structural alterations and mitochondrial damage in the zebrafish brain, thus disordering neuromodulation, which may ultimately cause neurological dysfunction in zebrafish. This study will provide a risk assessment for evaluating the biotoxicity of NPs and As to fish and even other animals.

Selenium improved mitochondrial quality and energy supply in the liver of high-fat diet-fed grass carp (Ctenopharyngodon idella) after heat stress.

This study aims to explore the effects of dietary selenium on hepatic mitochondrial quality and energy supply of grass carp (Ctenopharyngodon idella) fed with high-fat diet (HFD) after heat stress (HS). Grass carp were fed with HFD, and HFD contained 0.3 mg/kg nano-selenium for 10 weeks, thereafter exposed to HS from 26 to 34 °C, and named the HFD + HS (control) group and the HFD + Se + HS group, respectively. The results show that selenium significantly prompted the growth, increased glutathione peroxidase (GPX) activity, but reduced malondialdehyde (MDA) content in the liver and the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of grass carp fed with HFD after HS. Further, selenium alleviated mitochondrial damage and increased the number of mitochondrial DNA copies in the liver of the grass carp fed with HFD after HS. And selenium also maintained mitochondrial homeostasis by upregulating the expression of mitochondrial quality control-related genes (pgc-1α, nrf1/2, tfam, opa1, mfn1/2, and drp1), mitophagy-related genes (beclin1, atg5, atg12, pink1, and parkin), and the protein expression of parkin and LC3-II/I in the liver of grass carp. Finally, selenium reduced the triglyceride (TG) level and increased the free fatty acid (FFA) level and adenosine triphosphate (ATP) production in the liver of grass carp fed with HFD after HS. In conclusion, dietary selenium alleviated liver damage and improved liver mitochondrial quality and ATP production by increasing liver antioxidant capacity and promoting liver mitochondrial quality in grass carp fed with HFD after HS, which help grass carp to resist these two stressors.

Deep-sea cage culture altered microbial community composition in the sediments of the Yellow Sea Cold Water Mass.

Environmental impacts of the first submersible salmonid culture cage, which was launched in the deep-sea of the Yellow Sea Cold Water Mass, were investigated. The temporal and spatial variations of several physicochemical parameters were observed in both the bottom and sediment pore water. Aquaculture activities decreased bacterial richness in the sediment. The dominant phyla Proteobacteria, Desulfobacterota and Acidobacteriota, accounted for 30.42 % of the total bacteria community. Principal component analysis indicated that the bacterial composition in December was different from that in the other three months, and the aquaculture activity affected the distribution of the sediment microbial community in June, August and April. Planctomycetes was selected as biomarkers in the aquaculture area by linear discriminant analysis effect size. Redundancy analysis showed that the biomarkers were positively correlated with temperature and the concentration of nitrite in June, and negatively correlated with the dissolved oxygen in August and April.

Dynamic transcriptome and LC-MS/MS analysis revealed the important roles of taurine and glutamine metabolism in response to environmental salinity changes in gills of rainbow trout (Oncorhynchus mykiss).

Recently, the frequent salinity fluctuation has become a growing threat to fishes. However, the dynamic patterns of gene expression in response to salinity changes remain largely unexplored. In the present study, 18 RNA-Seq datasets were generated from gills of rainbow trout at different salinities, including 0 ‰, 6 ‰, 12 ‰, 18 ‰, 24 ‰ and 30 ‰. Based on the strict thresholds, we have identified 63, 1411, 2096, 1031 and 1041 differentially expressed genes in gills of rainbow trout through pairwise comparisons. Additionally, weighted gene co-expression network analysis was performed to construct 18 independent modules with distinct expression patterns. Of them, green and tan modules were found to be tightly related to salinity changes, several hub genes of which are known as the important regulators in taurine and glutamine metabolism. To further investigate their potential roles in response to salinity changes, taurine, glutamine, and their metabolism-related glutamic acid and α-ketoglutaric acid were accurately quantitated using liquid chromatography-tandem mass spectrometry analysis. Results clearly showed that their concentrations were closely associated with salinity changes. These findings suggested that taurine and glutamine play important roles in response to salinity changes in gills of rainbow trout, providing new insights into the molecular mechanism of fishes in salinity adaptation.

Functional differentiation of three pheromone binding proteins in Orthaga achatina using mixed-type sex pheromones.

Pheromone-binding proteins (PBPs) play important roles in perception of insect sex pheromones, functioning to recognize and transport pheromone components onto the olfactory receptors of the odorant sensing neurons. Orthaga achatina, a serious pest of camphor trees, uses a mixture of three Type I (Z11-16:OAc, Z11-16:OH and Z11-16:Ald) and one Type II (Z3,Z6,Z9,Z12,Z15-23:H) sex pheromone components in its sex communication, in which Z11-16:OAc is the major component and others are minor components. In this study, we for the first time demonstrated that the three PBPs differentiated in recognition among pheromone components in a moth using mixed-type sex pheromones. First, tissue expression study showed that all three PBPs of O. achatina were expressed only in antennae and highly male-biased, suggesting their involvement in perception of the sex pheromones. Second, the three PBPs were expressed in Escherichia coli and the binding affinities of PBPs to four sex pheromone components and some pheromone analogs were determined by the fluorescence competition binding assays. The results showed that OachPBP1 bound all four sex pheromone components with high binding affinity, while OachPBP2 had high or moderate binding affinity only to three Type I components, and OachPBP3 had high binding affinity only to three minor pheromone components. Furthermore, key amino acid residues that bind to sex pheromone components were identified in three PBPs by 3-D structure modeling and ligand molecular docking, predicting the interactions between PBPs and pheromone components. Our study provides a fundamental insight into the olfactory mechanism in moths that use mixed-type sex pheromones.

Release of moth pheromone compounds from Nicotiana benthamiana upon transient expression of heterologous biosynthetic genes.

BACKGROUND: Using genetically modified plants as natural dispensers of insect pheromones may eventually become part of a novel strategy for integrated pest management. RESULTS: In the present study, we first characterized essential functional genes for sex pheromone biosynthesis in the rice stem borer Chilo suppressalis (Walker) by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana, including two desaturase genes CsupYPAQ and CsupKPSE and a reductase gene CsupFAR2. Subsequently, we co-expressed CsupYPAQ and CsupFAR2 together with the previously characterized moth desaturase Atr∆11 in N. benthamiana. This resulted in the production of (Z)-11-hexadecenol together with (Z)-11-hexadecenal, the major pheromone component of C. suppressalis. Both compounds were collected from the transformed N. benthamiana headspace volatiles using solid-phase microextraction. We finally added the expression of a yeast acetyltransferase gene ATF1 and could then confirm also (Z)-11-hexadecenyl acetate release from the plant. CONCLUSIONS: Our results pave the way for stable transformation of plants to be used as biological pheromone sources in different pest control strategies.

A hydroponic plants and biofilm combined treatment system efficiently purified wastewater from cold flowing water aquaculture.

Recently, more and more cold flowing water aquaculture has been adopted, but its wastewater treatment is always ignored, which causes great pressure on the environment. In this study, a compound in-situ treatment system that applied hydroponic plants and biofilm was constructed to treat the wastewater produced by cold flowing water culture of sturgeon. The removal efficiency of the nutrients from culture and the microbial composition in water and biofilm were tested, the correlation between the water quality indexes and bacterium was analyzed, and the abundance of nitrogen and phosphorus cycling genes was quantified. The results show that the system respectively achieved 90%, 100%, 100%, 100% and 48% removal efficiency of NH4+-N, NO3--N, TN, TP and COD which were produced by experimental sturgeon culture. Chinese cabbage (Brassica rapa var. chinensis) and water dropwort (Oenanthe javanica) showed obvious growth in the four plants, which contributed to the removal of nutrients from wastewater. Besides, in the biofilm, Proteobacteria, Bacteroidetes and Verrucomicrobia became the top three dominant flora at the phylum level, and Flavobacterium, Rhodoferax, Sphaerotilus and Chitinimonas became the top four dominant flora at the genus level, which promoted the removal of nitrogen in the wastewater. The FAPROTAX analysis result shows that the highest functions within the carbon and nitrogen metabolisms were significantly identified in the biofilm, such as chemoheterotrophy, aerobic chemoheterotrophy and nitrate reduction. Further, the abundance of denitrifying genes (narG and napA) was higher than the nitrifying related genes (nxrB and amoA), indicating the more active denitrifying process. In summary, the compound in-situ treatment system efficiently removed nutrients from cold flowing water aquaculture. And the combined purification of hydroponic plants and biofilm which is rich in denitrifying bacterium plays an essential role in this process.