Transmission competence of a new mesonivirus, Yichang virus, in mosquitoes and its interference with representative flaviviruses.

Advances in technology have greatly stimulated the understanding of insect-specific viruses (ISVs). Unfortunately, most of these findings are based on sequencing technology, and laboratory data are scarce on the transmission dynamics of ISVs in nature and the potential effects of these viruses on arboviruses. Mesonivirus is a class of ISVs with a wide geographical distribution. Recently, our laboratory reported the isolation of a novel strain of mesonivirus, Yichang virus (YCV), from Culex mosquitoes, China. In this study, the experimental infection of YCV by the oral route for adult and larvae mosquitoes, and the vertical transmission has been conducted, which suggests that YCV could adopt a mixed-mode transmission. Controlled experiments showed that the infectivity of YCV depends on the mosquito species, virus dose, and infection route. The proliferation curve and tissue distribution of YCV in Cx. quinquefasciatus and Ae. albopictus showed that YCV is more susceptible to Ae. albopictus and is located in the midgut. Furthermore, we also assessed the interference of YCV with flaviviruses both in vitro and in vivo. YCV significantly inhibited the proliferation of DENV-2 and ZIKV, in cell culture, and reduced transmission rate of DENV-2 in Ae. albopictus. Our work provides insights into the transmission of ISVs in different mosquito species during ontogeny and their potential ability to interact with mosquito-borne viruses.

Effects of Propoxur Exposure on Insecticidal Susceptibility and Developmental Traits in Culex pipiens quinquefasciatus.

Propoxur-sel strains of Culex pipiens quinquefasciatus were derived from a lab-bred strain following 16 generations of propoxur exposure under sublethal concentrations of LC25 (lethal concentration of 25%) and LC50 (lethal concentration of 50%), respectively. This resulted in resistance development in F16 with ratios of 8.8× and 6.3×, respectively, compared with F0. The fecundity, longevity, sex ratio (F/M), and hatchability of the propoxur-exposed Cx. quinquefasciatus adult survivors and their offspring were decreased, with no effect on the emergence ratio and pupa survival rate. In addition, the intrinsic rates of increase (r), the net reproduction (R0), and the finite rate of increase (λ) of the Cx. quinquefasciatus offspring generations were also decreased significantly compared to F0. Correspondingly, the mean generation time (T) and the population double time (DT) in propoxur-sels were increased. Enhanced activities of cytochrome P450 monooxygenase and esterase were also observed in propoxur-sels, indicating that a detoxification mechanism might be responsible for resistance development in Cx. quinquefasciatus. Except for the three genes cyp4d42v1, cyp4c52v1, and cyp6aa9 which displayed a coincidence in some degree in different treatments, induction by different doses of propoxur and constitutive expression in different generations of propoxur-sel strains resulted in an inconsistent identification of the P450 genes probably related with resistance.

Regulator DegU is required for multicellular behavior in Lysinibacillus sphaericus.

DegS and DegU make up a two component system belonging to a class of signal transduction systems that play important roles in a broad range of bacterial responses to the environment. However, little study has been done to explore the physiological functions of DegS-DegU in mosquitocidal Lysinibacillus sphaericus. In this study, it was found that deletion of degU or degS-degU inhibited the swarming motility, biofilm formation, sporulation and binary toxin production through regulating the related genes, and phosphorylation was necessary for the functions of DegU. Based on the findings, a regulation network mediated by DegU was delineated. Both DegU-pi and Spo0A-pi positively regulates genes which are linked with the transition from stage Ⅱ to the end of the sporulation process and also influences the production of binary toxins via regulation on sigE. Both DegU-pi and Spo0A-pi negatively regulate abrB/sinR and influence the biofilm formation. DegU-pi can positively regulate the motility via the regulation on sigD. Whether the regulations are directly or indirectly need to be explored. Moreover, Spo0A-pi may indirectly regulate the swarming motility through negatively regulating DegU. It was concluded that DegU is a global transcriptional regulator on cell swarming motility, biofilm formation, sporulation and virulence in L. sphaericus.

A novel transcriptional activator, tubX, is required for the stability of Bacillus sphaericus mosquitocidal plasmid pBsph.

Stable maintenance of the low-copy-number plasmid pBsph in Bacillus sphaericus requires a partitioning (par) system that consists of a filament-forming protein, B. sphaericus TubZ (TubZ-Bs); a centromere-binding protein, TubR-Bs; and a centromere-like DNA site, tubC, composed of three blocks (I, II, and III) of 12-bp degenerate repeats. Previous studies have shown that mini-pBsph replicons encoding the TubZ system are segregationally highly unstable, whereas the native pBsph is stably maintained. However, the mechanism underlying the stability discrepancy between pBsph and its minireplicon is poorly understood. Here orf187 (encoding TubX), a gene downstream of tubZ-Bs, was found to play a role in plasmid stabilization. Null mutation or overexpression of tubX resulted in a defect in pBsph stability and a significant decrease in the level of tubRZ-Bs expression, and the TubX-null phenotype was suppressed by ectopic expression of a wild-type copy of tubX and additional tubRZ-Bs. An electrophoresis mobility shift assay (EMSA) and a DNase I footprinting assay revealed that the TubX protein bound directly to five 8-bp degenerate repeats located in the par promoter region and that TubX competed with TubR-Bs for binding to the par promoter. Further studies demonstrated that TubX significantly stimulated the transcription of the par operon in the absence of tubR-Bs, and a higher level of gene activation was observed when tubR-Bs was present. These results suggested that TubX positively regulates tubRZ-Bs transcription by interfering with TubR-Bs-mediated repression and binding directly to the tubRZ-Bs promoter region.

A new tubRZ operon involved in the maintenance of the Bacillus sphaericus mosquitocidal plasmid pBsph.

pBsph is a mosquitocidal plasmid first identified from Bacillus sphaericus, encoding binary toxins (Bin toxins) that are highly toxic to mosquito larvae. This plasmid plays an important role in the maintenance and evolution of the bin genes in B. sphaericus. However, little is known about its replication and partitioning. Here, we identified a 2.4 kb minimal replicon of pBsph plasmid that contained an operon encoding TubR-Bs and TubZ-Bs, each of which was shown to be required for plasmid replication. TubR-Bs was shown to be a transcriptional repressor of tubRZ-Bs genes and could bind cooperatively to the replication origin of eleven 12 bp degenerate repeats in three blocks, and this binding was essential for plasmid replication. TubZ-Bs exhibited GTPase activities and interacted with TubR-Bs : DNA complex to form a ternary nucleoprotein apparatus. Electron and fluorescence microscopy revealed that TubZ-Bs assembled filaments both in vitro and in vivo, and two point mutations in TubZ-Bs (T114A and Y260A) that severely impaired the GTPase and polymerization activities were found to be defective for plasmid maintenance. Further investigation demonstrated that overproduction of TubZ-Bs-GFP or its mutant forms significantly reduced the stability of pBsph. Taken together, these results suggested that TubR-Bs and TubZ-Bs are involved in the replication and probably in the partitioning of pBsph plasmid, increasing our understanding of the genetic particularity of TubZ systems.

Single nucleotide deletion of cqm1 gene results in the development of resistance to Bacillus sphaericus in Culex quinquefasciatus.

The entomopathogen Bacillus sphaericus is one of the most effective biolarvicides used to control the Culex species of mosquito. The appearance of resistance in mosquitoes to this bacterium, however, remains a threat to its continuous use in integrated mosquito control programs. Previous work showed that the resistance to B. sphaericus in Culex colonies was associated with the absence of the 60-kDa binary toxin receptor (Cpm1/Cqm1), an alpha-glucosidase present in the larval midgut microvilli. In this work, we studied the molecular basis of the resistance developed by Culex quinquefasciatus to B. sphaericus C3-41. The cqm1 genes were cloned from susceptible (CqSL) and resistant (CqRL/C3-41) colonies, respectively. The sequence of the cDNA and genomic DNA derived from CqRL/C3-41 colony differed from that of CqSL one by a one-nucleotide deletion which resulted in a premature stop codon, leading to production of a truncated protein. Recombinant Cqm1S from the CqSL colony expressed in Escherichia coli specifically bound to the Bin toxin and had α-glucosidase activity, whereas the Cqm1R from the CqRL/C3-41 colony, with a deletion of three quarters of the receptor's C-terminal lost its α-glucosidase activity and could not bind to the binary toxin. Immunoblotting experiments showed that Cqm1 was undetectable in CqRL/C3-41 larvae, although the gene was correctly transcribed. Thus, the cqm1R represents a new allele in C. quinquefasciatus that confers resistance to B. sphaericus.

Occurrence of psychrotolerant Bacillus cereus group strains in ice creams.

The occurrences of Bacillus cereus group strains in 40 ice cream samples were investigated. Among 109 isolated B. cereus group strains confirmed by 16S rDNA sequence analysis only 50 were identified as B. cereus and one as B. thuringiensis by using FDA (U.S. Food and Drug Administration) standard, indicating the two identification standards were highly inconsistent. Furthermore, the psychrotolerant growth properties and the occurrence of specific psychrotolerant genes of the isolates were also studied. Both psychrotolerant 16S rDNA fragments and enterotoxic genes could be detected among mesophilic and psychrotolerant strains. No relationship among psychrotolerance, presence of psychrotolerant 16S rDNA fragments and enterotoxic genes were found and the specific cspA fragment was only detected in a small fraction (9.5%) of the psychrotolerant isolates. One psychrotolerant isolate Bw2-1 was identified as B. weihenstephanensis, but no clear distinguishing characteristics between B. weihenstephanensis and psychrotolerant B. cereus were found. These results might be of importance for gaining further understanding of the growth properties of B. weihenstephanensis and psychrotolerant B. cereus as well as their contribution to food poisoning.

Conjugative transfer of insecticidal plasmid pHT73 from Bacillus thuringiensis to B. anthracis and compatibility of this plasmid with pXO1 and pXO2.

Bacillus anthracis, the etiologic agent of anthrax, is genetically close to and commonly shares a giant gene pool with B. cereus and B. thuringiensis. In view of the human pathogenicity and the long persistence in the environment of B. anthracis, there is growing concern about the effects of genetic exchange with B. anthracis on public health. In this work, we demonstrate that an insecticidal plasmid, pHT73, from B. thuringiensis strain KT0 could be efficiently transferred into two attenuated B. anthracis strains, Ba63002R (pXO1(+) pXO2(-)) and Ba63605R (pXO1(-) pXO2(+)), by conjugation in liquid medium in the laboratory, with transfer rates of 2.3 x 10(-4) and 1.6 x 10(-4) CFU/donor, respectively. The B. anthracis transconjugants containing both pHT73 and pXO1 or pXO2 could produce crystal protein Cry1Ac encoded by plasmid pHT73 and had high toxicity to Helicoverpa armigera larvae. Furthermore, the compatibility and stability of pHT73 with pXO1/pXO2 were demonstrated. The data are informative for further investigation of the safety of B. thuringiensis and closely related strains in food and in the environment.

[The synergism between Mtx1 from Bacillus sphaericus and Cyt1 Aa from Bacillus thuringiensis to Culex quinquefasciatus].

Mosquitocidal toxin 1 (Mtx1) was synthesized during vegetative phase of Bacillus sphaericus and it had been proved to have higher activity to Aedes spp. larvae and Binary toxin (Bin) resistance Culex larvae. The truncated 97 kDa Mtx1 with a deletion of the signal peptide and the Cyt1 Aa crystal protein, a 27.3 kDa delta-endotoxin from Bacillus thuringiensis subsp. israelensis (Bti), were purified from Escherichia coli and B. thuringiensis recombinant strains respectively. Both purified toxins had high toxicity against Culex quinquefasciatus larvae. Bioassay result revealed the purified Mtx1 toxin had high toxicity against the target mosquito larvae, with LCso of 45.2 ng/mL. However, the mixture of Mtx1 and Cytl Aa exhibited higher toxicity against the mosquito larvae, with a lowest LC50 value of 20.19 ng/mL at the ratio of 1:3. (Mtx1:Cyt1 Aa). The calculated synergistic factor of different mixtures suggested a strong synergistic effect between Cyt1 Aa toxin and Mtx1. Furthermore, the presence of Cyt1Aa in the mixture could induce early larval mortality, enhancing the activity of Mtx1 to the target mosquito larvae. The synergistic effect of Cyt1 Aa on mortality of Mtx1 to mosquito larvae might be caused by the damage of the larval midgut-hemocoel barrier induced by the activated CytlAa toxin, which enhanced the specific pathogenesis of Mtx1 on mosquito larvae. It is suggested that the co-application of Mtx1 and Cyt1 Aa in future will be integrated for mosquito management.

Lack of cross-resistance to Mtx1 from Bacillus sphaericus in B. sphaericus-resistant Culex quinquefasciatus (Diptera: Culicidae).

The toxicities of Mtx1 toxin against dipteran and lepidopteran species have been evaluated in this study. It was shown that Mtx1 has little or no toxicity to the tested lepidopteran species, but has moderate-level toxicity to Aedes albopictus Skuse (Diptera: Culicidae) and high-level toxicity to both susceptible and binary toxin-resistant Culex quinquefasciatus Say (Diptera: Culicidae). The LC(50) values of Mtx1 against a susceptible C. quinquefasciatus colony SLCq and two resistant colonies RLCq1/C3-41 and RLCq2/IAB59 selected in the laboratory with Bacillus sphaericus (Mayer & Neide) strains C3-41 and IAB59 respectively were 0.508, 0.854 and 0.675 mg L(-1) respectively. The data indicate that Mtx1 has a different mode of action from the binary toxin, and that it could be an alternative toxin to delay or overcome resistance development to binary toxin in C. quinquefasciatus.

Mosquitocidal toxin from Bacillus sphaericus induces stronger delayed effects than binary toxin on Culex quinquefasciatus (Diptera: Culicidae).

We studied the toxicity and delayed effects of a mosquitocidal toxin (Mtx1) and a binary toxin (Bin) produced in Escherchia coli E-TH21 and Bacillus thuringiensis B-CW1, respectively, on Culex quinquefasciatus (Diptera: Culicidae). Bioassay results showed that both E-TH21 powder and B-CW1 sporulated culture were highly toxic against susceptible Cx. quinquefasciatus, with LC50 values of 0.65 and 1.70 mg/liter against third and fourth instars at 48 h, respectively. After initial 48-h exposure of larvae to different concentrations of Mtx1 and Bin, significant continued mortality could be observed in larval, pupal, and emergence stages of Cx. quinquefasciatus. Importantly, the Mtx1 could induce higher cumulative larval and preadult mortalities than Bin toxin on the target mosquito. This finding is important for understanding the mode of action of Mtx1 and Bin toxins and for developing a new bioassay procedure for evaluation of toxicity of Bacillus sphaericus Neide, some strains of which produce Mtx1 and Bin, in the laboratory and field.

[Domain swapping of Cry1Aa and Cry1Ca from Bacillus thuringiensis influence crystal formation and toxicity].

Bacillus thuringiensis has been successfully used for agricultural pest and medical insect control with its significant benefits based on environmental and safety considerations. However, the deficiency of this pesticide, such as limited spectrum of insecticidal activity, low toxicity to the targets and the inducement of insect resistance, results in the urgent need to exploit new resources of B. thuringiensis or to modify known strains by genetic engineering. In this study, six recombinant Bacillus thuringiensis BT-ACC, BT-AAC, BT-ACA, BT-CAA, BT-CCA and BT-CAC were constructed through the domain swapping between crystal protein CrylAa and CrylCa. SDS-PAGE and Western blot revealed that only the recombinant BT-CAA and BT-CCA produced a 135kDa chimeric protein CrylCAA and CrylCCA respectively, but the production level was lower than the native protein CrylAa and CrylCa. These chimeric crystal proteins could be activated by trypsin, giving a 65kDa protease-resistant core toxin as the native crystal proteins CrylAa and CrylCa. Electron microscopy study indicated that the two chimeric proteins could be produced and accumulated as spherical or granules crystals during sporulation of BT-CAA and BT-CCA, whereas native CrylAa and CrylCa were accumulated as bipyramidal crystals. Unexpectedly, the toxicity of purified chimeric crystal proteins Cryl CAA and Cryl CCA was 3 - 5 times lower to Spodoptera exigua, and 190 - 260 times lower to Helicoverpa armigera than that of native CrylAa and CrylCa. These data suggested that the domain swapping of different crystal proteins might influence the formation and toxicity to targets of chemeric crystal proteins.