Feeding behavior and hormoligosis associated with imidacloprid resistance in Asian citrus psyllid, Diaphorina citri.

Imidacloprid is a neonicotinoid insecticide used for managing the Asian citrus psyllid, Diaphorina citri Kuwayama, which serves as vector of phytopathogens causing citrus greening. However, development of resistance to neonicotinoids among populations of D. citri has coincided with occasional control failures in the field. The objectives of this research were to (1) survey current levels of imidacloprid resistance in Florida citrus; (2) compare feeding behavior between imidacloprid-resistant and susceptible D. citri using electrical penetration graph recordings, and (3) investigate the possible amplification of insecticide hormoligosis associated with resistance. Field surveys confirmed that the susceptibility of D. citri populations to imidacloprid has decreased in commercial Florida citrus groves compared with a laboratory-susceptible population. Following 12 generations of selection, resistance to imidacloprid increased by 438 fold compared with the susceptible strain. Imidacloprid-susceptible D. citri feeding on citrus exhibited significantly more bouts associated with intercellular pathway (C), phloem penetration (D), phloem salivation (E1), and nonprobing (Np) activities than imidacloprid-resistant counterparts. However, there were no differences observed in the frequency or duration of phloem ingestion or xylem feeding between susceptible and resistant D. citri. There was no statistical difference in fecundity between resistant and susceptible strains. However, the fecundity of imidacloprid-susceptible female D. citri treated with a sublethal concentration of imidacloprid (LC25 ) increased significantly compared with controls, while such hormoligosis was less pronounced among imidacloprid-resistant psyllids. Our results suggest that imidacloprid-resistant psyllids may cease feeding sooner than susceptible counterparts following sublethal exposure to this insecticide, indicative of a behavioral resistance mechanism.

Thiomicrorhabdus heinhorstiae sp. nov. and Thiomicrorhabdus cannonii sp. nov.: novel sulphur-oxidizing chemolithoautotrophs isolated from the chemocline of Hospital Hole, an anchialine sinkhole in Spring Hill, Florida, USA.

Two sulphur-oxidizing, chemolithoautotrophic aerobes were isolated from the chemocline of an anchialine sinkhole located within the Weeki Wachee River of Florida. Gram-stain-negative cells of both strains were motile, chemotactic rods. Phylogenetic analysis of the 16S rRNA gene and predicted amino acid sequences of ribosomal proteins, average nucleotide identities, and alignment fractions suggest the strains HH1T and HH3T represent novel species belonging to the genus Thiomicrorhabdus. The genome G+C fraction of HH1T is 47.8 mol% with a genome length of 2.61 Mb, whereas HH3T has a G+C fraction of 52.4 mol% and 2.49 Mb genome length. Major fatty acids of the two strains included C16 : 1, C18 : 1 and C16 : 0, with the addition of C10:0 3-OH in HH1T and C12 : 0 in HH3T. Chemolithoautotrophic growth of both strains was supported by elemental sulphur, sulphide, tetrathionate, and thiosulphate, and HH1T was also able to use molecular hydrogen. Neither strain was capable of heterotrophic growth or use of nitrate as a terminal electron acceptor. Strain HH1T grew from pH 6.5 to 8.5, with an optimum of pH 7.4, whereas strain HH3T grew from pH 6 to 8 with an optimum of pH 7.5. Growth was observed between 15-35 °C with optima of 32.8 °C for HH1T and 32 °C for HH3T. HH1T grew in media with [NaCl] 80-689 mM, with an optimum of 400 mM, while HH3T grew at 80-517 mM, with an optimum of 80 mM. The name Thiomicrorhabdus heinhorstiae sp. nov. is proposed, and the type strain is HH1T (=DSM 111584T=ATCC TSD-240T). The name Thiomicrorhabdus cannonii sp. nov is proposed, and the type strain is HH3T (=DSM 111593T=ATCC TSD-241T).

Dissolved Inorganic Carbon-Accumulating Complexes from Autotrophic Bacteria from Extreme Environments.

In nature, concentrations of dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO2 fixation. Such mechanisms have been well studied in Cyanobacteria, but much remains to be learned about their activity in other phyla. Novel multisubunit membrane-spanning complexes capable of elevating intracellular DIC were recently described in three species of bacteria. Homologs of these complexes are distributed among 17 phyla in Bacteria and Archaea and are predicted to consist of one, two, or three subunits. To determine whether DIC accumulation is a shared feature of these diverse complexes, seven of them, representative of organisms from four phyla, from a variety of habitats, and with three different subunit configurations, were chosen for study. A high-CO2-requiring, carbonic anhydrase-deficient (ΔyadF ΔcynT) strain of Escherichia coli Lemo21(DE3), which could be rescued via elevated intracellular DIC concentrations, was created for heterologous expression and characterization of the complexes. Expression of all seven complexes rescued the ability of E. coli Lemo21(DE3) ΔyadF ΔcynT to grow under low-CO2 conditions, and six of the seven generated measurably elevated intracellular DIC concentrations when their expression was induced. For complexes consisting of two or three subunits, all subunits were necessary for DIC accumulation. Isotopic disequilibrium experiments clarified that CO2 was the substrate for these complexes. In addition, the presence of an ionophore prevented the accumulation of intracellular DIC, suggesting that these complexes may couple proton potential to DIC accumulation. IMPORTANCE To facilitate the synthesis of biomass from CO2, autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multisubunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, raising the question of whether these complexes share this capability across the 17 phyla of Bacteria and Archaea where they are found. This study shows that DIC accumulation is a trait shared by complexes with various subunit structures, from organisms with diverse physiologies and taxonomies, suggesting that this trait is universal among them. Successful expression in E. coli suggests the possibility of their expression in engineered organisms synthesizing compounds of industrial importance from CO2.

Comparative transcriptome analysis of thiamethoxam susceptible and resistant Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), using RNA-sequencing.

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits the causal pathogen of huanglongbing and is a global pest of citrus. D. citri populations exhibit resistance to multiple insecticide modes of action in areas where these chemicals have been overused. We performed genome-wide transcriptional analysis for two field populations of D. citri (Wauchula and Lake Alfred, Florida, USA) that exhibit 1300-fold resistance to the neonicotinoid insecticide, thiamethoxam, and compared it to that of susceptible psyllids collected from the same area and without imposed selection. The Lake Alfred population responded to insecticide resistance by up-regulation of 240 genes and down-regulation of 148 others. The Wauchula population exhibited similar patterns to the Lake Alfred population with up-regulation of 253 genes and down-regulation of 115 others. Gene Ontology annotation associated with cellular processes, cell, and catalytic activity were assigned to differentially expressed genes (DEGs). The DEGs from Lake Alfred and Wauchula populations were mapped to Kyoto Encyclopedia of Gene and Genomes pathways and implicated enrichment of metabolic pathways, oxidative phosphorylation, extracellular matrix-receptor interaction, terpenoid backbone biosynthesis, and insect hormone biosynthesis in the resistant populations. Up-regulation of 60s ribosomal proteins, UDP-gluscoyltransferases, cytochrome c oxidases, and CYP and ABC transporters among thiamethoxam-resistant D. citri implicates a broad array of novel and conventionally understood resistance mechanisms.

Insecticide rotation scheme restores insecticide susceptibility in thiamethoxam-resistant field populations of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), in Florida.

BACKGROUND: We investigated rotation using insecticides with multiple modes of action as a resistance management strategy for Asian citrus psyllid, Diaphorina citri, Kuwayama (Hemiptera: Liviidae), in Florida. The stability of thiamethoxam resistance was investigated in the laboratory by establishing populations of field-collected, resistant D. citri and rearing them under no insecticide exposure. Furthermore, recovery of susceptibility was investigated in the field by initiating rotation to insecticides in plots that previously were treated with consecutive thiamethoxam applications. RESULTS: The resistance ratio (RR) for thiamethoxam reached between 1266.29- and 1395.00-fold after three and four consecutive applications of thiamethoxam, respectively. However, the RR for thiamethoxam remained low (1.71-5.28-fold) under both rotations at both Lake Alfred and Wauchula. Thiamethoxam was cross-resistant with imidacloprid (RR site 1 = 1059.65-fold, RR site 2 = 1595.43-fold) and clothianidin (RR site 1 = 1798.78-fold, RR site 2 = 1270.57-fold) in the nonrotated treatment at both sites. There was very low cross-resistance to other insecticides with different modes of action. Both laboratory and field investigations indicated that susceptibility to thiamethoxam fully recovered after five D. citri generations. Expression of CYP4C67 was significantly increased in resistant populations. CONCLUSION: Our results revealed that D. citri populations develop a high level of resistance following only three or four consecutive neonicotinoid sprays; this was associated with subsequent product failure. Our data suggest that metabolic detoxification by cytochrome P450s contributes to thiamethoxam resistance in D. citri. Overall, the investigation demonstrated that resistance to thiamethoxam can be managed readily in populations of D. citri by rotating modes of action.