Larvae Of Strain Research Articles

Resistance to a chemical pesticide increases vulnerability to a biopesticide: Effects on direct mortality and mortality by predation

Pesticide mixtures are increasingly used to fight pest species that developed resistance to pesticides. To assess the pesticide control efficiency and to reduce ecological damage to non-target species, it is important to quantify the effect of these mixtures and compare them with the effect of their single pesticides on pest species, non-target species and their predator-prey interactions. We studied the effects of the chemical pesticide chlorpyrifos (CPF), the biopesticide Bacillus thuringiensis israelensis (Bti) and their mixture both on the direct mortality and on the mortality by predation. We focused on larvae of a CPF-resistant and a non-resistant strain of the vector mosquito Culex quinquefasciatus and its predator, the pygmy backswimmer Plea minutissima. In the CPF-Bti mixture, both pesticides interacted antagonistically for direct mortality. Exposure to the mixture caused equal direct mortality and equal mortality by predation in both strains. As expected, exposure to CPF resulted in less direct mortality and less mortality by predation in the CPF-resistant mosquito strain compared to the non-resistant strain. Notably, Bti caused a higher mortality in the mosquito larvae of the CPF-resistant strain compared to the non-resistant strain. Furthermore, the predator killed more mosquito larvae of the resistant strain compared to the non-resistant strain when exposed before to Bti alone. These observations identify a novel cost of resistance to a chemical pesticide in terms of increased vulnerability to a biopesticide.

Tolerance of three Aedes albopictus strains (Diptera: Culicidae) from different geographical origins towards winter temperatures under field conditions in northern Germany.

The continuing spread of the Asian tiger mosquito Aedes albopictus, a vector of many arboviruses and some dirofilarial worms, in Europe calls for advanced investigations on its ecological ability to establish and overwinter in temperate, more northern geographic regions. To meet this purpose, eggs of Ae. albopictus laboratory strains of tropical, subtropical and temperate origin were exposed to field conditions during one or two winter seasons in northeastern Germany. After 1 to 16 weeks of outdoor exposure, eggs were flooded in the laboratory, and the hatching rates were determined. During the winter season 2015/2016, when temperatures reached –10°C, the subtropical strain showed hatching after all time periods while the tropical strain displayed hatching only until two weeks of cold exposure. In the winter season 2016/2017, with temperatures as low as ‒6°C, all three strains produced hatching larvae after all time periods. Both the hatching rates and the hatching behaviour differed between the strains. Larvae of the subtropical and temperate strains hatched in installments over a period of four weeks while the larvae of the tropical strain hatched within a short time period, often one week. The results of the study demonstrate that Ae. albopictus strains of different, even tropical, origin might be able to survive a central European winter, although this is likely to depend on the specific course of the temperatures. Further studies with different temperature regimes and different mosquito strains are needed to specify these findings.

Negative cross resistance of Spodoptera litura Fabricius population of tomato to newer molecule spinetoram

Field population of Spodoptera litura from tomato ( resistant to the majority of the conventional insecticide molecules) were subjected to the in vivo toxicity of spinetoram 12 SC to assess whether cross resistance exists or not. Untreated larvae of both field and laboratory strains showed no mortality during 48 hours of feeding. After 48 hours of feeding on spinetoram 12 SC treated leaves, LC50s of field larvae were 0.28, 0.93, 3.71 and 7.11 ppm for the 2nd, 3rd, 4th and 5th instars of S. litura respectively. However, in the laboratory strain these values were 1.12, 5.86, 36.72 and 91.55 ppm for 2nd, 3rd, 4th and 5th instars of S. litura respectively. Resistance ratio was 0.25, 0.16, 0.10 and 0.08 for the 2nd instar up to the 5th instar of S. litura.

The biological role of the enigmatic C3larvinAB toxin of the honey bee pathogenic bacterium Paenibacillus larvae.

Paenibacillus larvae is the causative agent of the notifiable epizootic American foulbrood, a fatal bacterial disease of honey bee larvae. The species P. larvae has been classified into four differentially virulent and prevalent genotypes (ERIC I-IV), which also differ in their virulence factor equipment. Recently, a novel P. larvae toxin, the C3-like C3larvin, has been described. Genome analysis now revealed that the C3larvin gene is actually a part of a toxin locus encompassing two genes encoding a binary AB toxin with the A subunit being C3larvin (C3larvinA) and a putative B subunit (C3larvinB) encoded by the second gene. Sequence and structural analyses demonstrated that C3larvinB is a homologue of the Bacillus anthracis protective antigen (PA), the B subunit of anthrax toxin. The C3larvinAB toxin locus was interrupted by point mutations in all analysed P. larvae ERIC I and ERIC II strains. Only one P. larvae ERIC III/IV strain harboured an uninterrupted toxin locus comprising full-length genes for C3larvinA and B. Exposure bioassays did not substantiate a role as virulence factor for C3larvinAB in P. larvae ERIC I/II. However, the PA homologue C3larvinB had an influence on the virulence of the unique P. larvae strain expressing the functional C3larvinAB locus.

The Effectiveness of Derris elliptica (Wall.) Benth Root Extract Against Temephos-resistant Aedes aegypti Larvae

Derris elliptica (Wall.) Benth is a wild plant or weed in agricultural fields that was traditionally used for poisoning fish and plant pests. Several studies showed the low lethal concentration of Derris elliptica root extract to control the laboratory strain Aedes aegypti larvae. This study aimed to evaluate the effectiveness of Derris elliptica root extract against the temephos-resistant Aedes aegypti larvae from the Dengue endemic areas. This experimental study used post-test only control group design with five different concentrations of methanolic Derris elliptica root extract, namely 0.09%, 0.13%, 0.17%, 0.21%, and 0.25%, respectively. Twenty-five of F1 temephos-resistant of wild-caught Aedes aegypti larvae were subjected to each group of the five different concentrations, and two control groups with aquadest. The temephos-resistant strain of Aedes aegypti larvae was determined by bioassay test with temephos -0.02 ppm compound. Mortality rate and lethal concentration (LC50 and LC90) of the larvae were calculated after 24-h contact. Average of mortality rate in each concentration after 24-h contact with Derris elliptica root extract were 32.8%, 49.6%, 72.0%, 95.2%, and 100% with LC50 and LC90 were 1,600 and 2,040 ppm, respectively. The methanolic Derris elliptica root extract have larvacidal potential to control the temephos-resistant Aedes aegypti larvae. Further research to obtain secondary metabolites is needed.

Repeated phenotypic selection for cuticular blackness of armyworm larvae decreased stress resistance

Armyworm Mythimna separata larvae show changes in cuticle darkening depending on population densities and are roughly categorized into two phenotypes, a pale brown solitary type and black-colored gregarious type. Although the color difference in both larval types is apparent, it remains ambiguous whether any change in physiological traits accompanies the cuticle darkening. To answer this query, we repeated genetic selection of the blackness phenotype over one hundred generations in our laboratory colony and produced a black-colored (BL) strain. Comparison between non-selected control (CTL) and BL strains revealed an increased fecundity and adult life span in the BL strain compared with the CTL strain. In contrast, BL strain larvae were found to be significantly more sensitive to heat stress than those in the CTL strain. Hemolymph reactive oxygen species (ROS) levels were higher in the BL strain than in the CTL strain under both non-stress and heat stress conditions. Antioxidant activities of the hemolymph were not significantly different between the two strains under non-stress condition, but the activities increased to higher levels in the CTL strain than those in the BL strain after heat stress. Activities and gene expression levels of antioxidant enzymes such as catalase and superoxide dismutase (SOD) in the fat body were significantly higher in CTL strain larvae than in BL strain larvae after heat treatment. Thermal stress tolerance of the offspring of crossings between the two strains showed a tolerance level almost equivalent to the maternal one: the cross between CTL females and BL males produced offspring with the higher tolerance compared with the oppositely crossed offspring. Expression levels of the antioxidant enzyme genes of the former offspring were found to be similar to those of CTL strain. These results indicate a trade-off between reproductive activity and stress resistance: the BL strain had acquired high reproductivity but had lost stress tolerance through repeated genetic selection. Furthermore, the present genetic analyses demonstrated that the phenotype of stress tolerance is derived from the maternal parent.

Selection and characterization of the inheritance of resistance of Spodoptera frugiperda (Lepidoptera: Noctuidae) to chlorantraniliprole and cross-resistance to other diamide insecticides.

Understanding the genetic basis of insect resistance to insecticides can help to implement insecticide resistance management (IRM) strategies. In this study, we selected a strain of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) resistant to chlorantraniliprole using the F2 screen method, characterized the inheritance of resistance and evaluated patterns of cross-resistance to other diamide insecticides. The chlorantraniliprole-resistant strain (Chlorant-R) was selected from a field-collected population with an estimated allele frequency of 0.1316. The estimated median lethal concentration (LC50 ) values were 0.011 and 2.610 µg a.i. cm-2 for the susceptible (Sus) and Chlorant-R strains, respectively, resulting in a resistance ratio (RR) of 237-fold. The LC50 values of the reciprocal crosses were 0.155 and 0.164 µg a.i. cm-2 , indicating that resistance is autosomally inherited. Resistance was characterized as incompletely recessive and monogenic at concentrations close to the recommended field rates of chlorantraniliprole. Survival of the resistant strain and heterozygous larvae in maize plants treated at the field rate was ∼ 60 (similar to the susceptible strain on untreated maize) and 15%, respectively. The Chlorant-R strain showed low cross-resistance to cyantraniliprole (RR ∼ 27-fold) and very high cross-resistance to flubendiamide (RR > 42 000-fold). The frequency of chlorantraniliprole resistance allele was moderately high in a field-collected population of S. frugiperda. The inheritance of chlorantraniliprole resistance was characterized as autosomal, incompletely recessive and monogenic. S. frugiperda showed cross-resistance to other diamide insecticides. These results provide essential information for the implementation of IRM programs to preserve the useful life of diamide insecticides for controlling S. frugiperda in Brazil. © 2019 Society of Chemical Industry.

Resveratrol Ameliorates Glucocorticoid-Induced Bone Damage in a Zebrafish Model.

Resveratrol (Res) is a multi-functional polyphenol compound that has protective functions in cardiovascular and neurodegenerative diseases. This study aimed to determine the effect of Res on osteogenic differentiation and bone mineralization in zebrafish (Danio rerio) with dexamethasone (Dex)-induced bone damage. Our results showed that Dex exposure (15 μmol/l) decreased the green fluorescence areas and the integrated optic density (IOD) values in the skull bones of zebrafish larvae of the TG(SP7:EGFP) strain in a dose-dependent manner (p < 0.01). Furthermore, Dex exposure decreased the alizarin red S-stained areas (bone mineralization area) in the skeleton and spinal bones of zebrafish larvae of the AB strain in a dose-dependent manner (p < 0.01). By contrast, Res treatment (150 μmol/l) significantly increased both the green fluorescence and bone mineralization area in Dex-exposed zebrafish larvae. Thus, our data show that Res improves bone mineralization after glucocorticoid-induced bone damage in a zebrafish model. Res may be a candidate drug for the prevention of osteoporosis.

Survival Rate and Early Development of Giant Gourami Padang Strain Osphronemus gouramy (Perciformes: Osphronemidae)

Aquaculture of gourami in Indonesia is still carried out traditionally with a simple touch of technology that makes the mortality rate of larvae is still very high. This study aimed to determine the survival rate and development of giant gourami Padang strain larvae Osphronemus gouramy reared in a traditional way and to gain insight into the cause of mass death during the larval period. The larvae were obtained from six pairs of broodstocks that consisted of three categories, which is the grey broodstocks that spawned the previous month, the grey broodstocks that did not spawn the previous month, and the pink broodstocks that did not spawn the previous month, and were named after the body colour of their respective parents. The study was conducted in duplicate and the larvae were sampled for photography every day and the larvae development was described from Day 1 to Day 10. The eggs began to hatch on Day 2, and all eggs hatched on Day 3 that marked the end of the embryonic stage. Pale melanophores became more intense on Day 6 and the yolk sac was still visible on Day 10. The mass deaths during embryogenesis are thought to be influenced by factors such as broodstock quality, while the ones occurring in the late larval period are more likely related to water quality and stocking density. Ammonia excretion subsequently oxidized into more toxic nitrite was the only possible pollutant to be present in the rearing water. In the Grey II.1 and Grey II.2 basins where the mass death occurred on Day 10, the mean of survival rates declined significantly from 83.45% on Day 9 to 32.15% on Day 10. Thus, this study confirmed that the basins with larvae density of above 30 individuals per litre can support the larvae rearing for eight days and on Day 9 the larvae must be transferred to another tank. The success of larval production depends not only on the welfare and the feed quality of the broodstocks but also on the optimal environment in larval rearing. In addition, the nomenclature for the larval stage of gourami was also discussed and determined.

10-HDA, A Major Fatty Acid of Royal Jelly, Exhibits pH Dependent Growth-Inhibitory Activity Against Different Strains of Paenibacillus larvae.

Paenibacillus larvae (P. larvae) is a bacterial pathogen causing American foulbrood (AFB), the most serious disease of honeybee larvae. The food of young larvae could play an important role in the resistance of larvae against AFB. It contains antibacterial substances produced by honeybees that may inhibit the propagation of the pathogen in larval midguts. In this study, we identified and investigated the antibacterial effects of one of these substances, trans-10-hydroxy-2-decenoic acid (10-HDA), against P. larvae strains including all Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes. Its inhibitory activities were studied by determining the minimum inhibitory concentrations (MICs). It was found that 10-HDA efficacy increases substantially with decreasing pH; up to 12-fold differences in efficacy were observed between pH = 5.5 and pH = 7.2. P. larvae strains showed different susceptibility to 10-HDA; up to 2.97-fold differences existed among various strains with environmentally important ERIC I and ERIC II genotypes. Germinating spores of the pathogen were generally more susceptible to 10-HDA than vegetative cells. Our findings suggest that 10-HDA could play significant role in conferring antipathogenic activity to larval food in the midguts of young larvae and contribute to the resistance of individual larvae to P. larvae.

Evaluation of Quantitative PCR (qPCR) Paenibacillus larvae Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris.

The application of quantitative PCR (qPCR) as a routine method to detect and enumerate Paenibacillus larvae in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of Apis mellifera. However, none of the qPCR tests described so far has been officially proposed as a standard procedure for P. larvae detection and enumeration for surveillance purposes. Therefore, in this study, inclusivity, exclusivity and sensitivity of detection of P. larvae spores directly in samples of honey and hive debris were re-evaluated for the previously published qPCR methods. To this aim, recently acquired P. larvae sequence data were considered to assess inclusivity in silico and more appropriate non-target species were used to verify exclusivity experimentally. This led to the modification of a previously described method by shortening the forward primer, designing a new reverse primer and using more stringent amplification conditions. The new test allowed the detection of P. larvae spores in honey and hive debris down to 1 CFU/g. The qPCR test optimized in this study proved suitable for quantification and also for identification of field P. larvae strains and real contaminated samples. Therefore, it is proposed for reliable detection and quantification of P. larvae in honey and hive debris, thus circumventing the disadvantages of late AFB diagnosis based on clinical symptoms and possible underestimation of spore numbers that is the main drawback of culture-dependent procedures.

Characterization and expression profiling of microRNAs in response to plant feeding in two host-plant strains of the lepidopteran pest Spodoptera frugiperda

BackgroundA change in the environment may impair development or survival of living organisms leading them to adapt to the change. The resulting adaptation trait may reverse, or become fixed in the population leading to evolution of species. Deciphering the molecular basis of adaptive traits can thus give evolutionary clues. In phytophagous insects, a change in host-plant range can lead to emergence of new species. Among them, Spodoptera frugiperda is a major agricultural lepidopteran pest consisting of two host-plant strains having diverged 3 MA, based on mitochondrial markers. In this paper, we address the role of microRNAs, important gene expression regulators, in response to host-plant change and in adaptive evolution.ResultsUsing small RNA sequencing, we characterized miRNA repertoires of the corn (C) and rice (R) strains of S. frugiperda, expressed during larval development on two different host-plants, corn and rice, in the frame of reciprocal transplant experiments. We provide evidence for 76 and 68 known miRNAs in C and R strains and 139 and 171 novel miRNAs. Based on read counts analysis, 34 of the microRNAs were differentially expressed in the C strain larvae fed on rice as compared to the C strain larvae fed on corn. Twenty one were differentially expressed on rice compared to corn in R strain. Nine were differentially expressed in the R strain compared to C strain when reared on corn. A similar ratio of microRNAs was differentially expressed between strains on rice. We could validate experimentally by QPCR, variation in expression of the most differentially expressed candidates. We used bioinformatics methods to determine the target mRNAs of known microRNAs. Comparison with the mRNA expression profile during similar reciprocal transplant experiment revealed potential mRNA targets of these host-plant regulated miRNAs.ConclusionsIn the current study, we performed the first systematic analysis of miRNAs in Lepidopteran pests feeding on host-plants. We identified a set of the differentially expressed miRNAs that respond to the plant diet, or differ constitutively between the two host plant strains. Among the latter, the ones that are also deregulated in response to host-plant are molecular candidates underlying a complex adaptive trait.

Efficacy of Gaseous Ozone Against Paenibacillus Larvae Spores on Hive Materials

American Foulbrood (AFB) is a highly contagious bacterial honey bee disease caused by Paenibacillus larvae. The elimination of P. larvae spores from contaminated hives is a key factor to achieve the long-term success in AFB control. The purpose of this study was to evaluate the efficacy of gaseous ozone in inactivating the P. larvae spores on wooden and plastic hive materials. Untreated pinewood and polyvinyl chloride (PVC) were chosen as representatives for the hive materials. Pinewood and PVC sticks inoculated with a spore cocktail of three P. larvae strains were treated up to 120 min with two different constant concentrations of gaseous ozone (9.8 and 17.1 mg/L) at room temperature. Ozonation at 17.1 mg/L for 120 min yielded over the 4 log reduction in the counts of spores on PVC sticks. Whereas, reduction of 2.3 log was obtained on pinewood sticks under the same experimental conditions. Reductions achieved in the levels of P. larvae spores on PVC sticks after 90 and 120 min of ozonation were significantly (p<0.05) higher than those on pinewood sticks. Our results suggest that gaseous ozone treatment is a promising candidate for the sterilization of plastic hives contaminated with P. larvae spores.

Detection of Paenibacillus larvae by Real-Time PCR

Background: : : is the agent of the American Foulbrood disease (AFB), which may determine the death of the hive. The detection strategy for its diagnosis is based on clinical signs of disease, isolation and identification of P. larvae, which usually employs microbiological and biochemical methods. Recently, molecular methods based on analysis of 16S rDNA by conventional PCR have been adopted, providing greater security and analytical speed. The rapid diagnosis is important to minimize economic losses and assess routes of spread of the pathogen. Despite the strong existing sanitary control, P. larvae was recently identified in the Brazilian states of Rio Grande do Sul and Paraná. After that, outbreaks have been reported in neighboring countries. This investigation was conducted to develop a protocol for detection of P. larvae by real-time PCR, allowing the reduction in the time of diagnosis, without loss of robustness found in the conventional PCR methods. Materials, Methods & Results: Twenty-nine (29) P. larvae strains were evaluated by real-time PCR using SYBR Green. The primers Pltr-F/R were designed according to the sequence X60619 of 16S rDNA gene published in GenBank, to amplify a fragment of 74 base pairs. The target gene is highly conserved and specific to P. larvae. The amplification conditions consisted of 1 cycle of 50°C for 2 minutes and 1 cycle of 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The fluorescence was monitored during the annealing at 60°C. The reactions were conducted in a 7500 Real Time PCR System equipment, using SYBRGreen PCR master mix (both Applied Biosystems), containing 2X Platinum SYBRGreen qPCR Supermix-UDG. The concentrations of primers were 1, 10 and 100 mM, and different concentrations of MgCl2 (0,0mM de MgCl2, 1.0mM de MgCl2 , 2.0mM de MgCl2 and 3.0mM de MgCl2) were tested, with a final volume of 50mL; 25mL and 15mL, containing a 5mL sample. The analysis of the melting curve was made based on a 95°C for 15 seconds and 60°C for 20 seconds and one cycle with temperature ranging between 60°C and 95°C for 20 minutes. The best results of sensitivity and specificity in the reaction with SYBR Green were obtained with primer concentration set as 100 mM. The different concentrations of MgCl2 tested did not affect the performance of the reaction. No amplification was observed with DNA obtained from Paenibacillus alvei or Bacillus species. The limit of detection was set as 6 pg of DNA template. The regression analysis of the CT values of the PCR products showed a linear relationship between the initial amounts of DNA template and the values of CT (R2 = 0.9982), indicating that the test is highly precise. Discussion: The protocol developed allowed the unequivocal identification of P. larvae, as all strains were detected by this approach. The amplification of the expected 16S rDNA gene fragment was verified by amplification with the primers Pltr F/R only for chromosomal DNA of P. larvae. In addition, the amplicon specificity was verified by sequencing and no amplification was observed when the primers were tested with DNA from other bacterial species. The protocol developed in this study proved to be sensitive and specific, providing a rapid and accurate diagnostic tool. The results showed that the analysis by real-time PCR of partial 16S rDNA gene of P. larvae represents an important alternative for rapid diagnosis of AFB disease. The use of this methodology may represent an advance for rapid confirmation of the presence of this bacterium, what will allow the adoption of control measures against AFB, which can avoid its spreading in Brazilian territory.Paenibacillus larvae

Laurus nobilis L. Extracts against Paenibacillus larvae: Antimicrobial activity, antioxidant capacity, hygienic behavior and colony strength.

The aim of this work was to compare the antimicrobial activity against Paenibacillus larvae and the antioxidant capacity of two Laurus nobilis L. extracts obtained by different extraction methods. The hydroalcoholic extract was moreover added as supplementary diet to bees in field conditions to test behavioural effects and colony strength. Both laurel extracts were subjected to different phytochemical analysis to identify their bioactive compounds. Antimicrobial activity was analyzed by the minimal inhibitory concentration (MIC) determination by means the agar dilution method. The hydroalcoholic extract (HE) was able to inhibit the bacterial growth of all P. larvae strains, with 580 µg/mL mean value. This better antibacterial activity in relation to the essential oil (EO) could be explained by the presence of some phenolic compounds, such as flavonoids, evidenced by characteristic bands resulting from the Fourier Transform Infrared Spectroscopy (FTIR) analysis. Antioxidant activities of the extracts were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging ability and ferric reducing antioxidant power (FRAP) assays. The HE showed the highest antioxidant activity as measured by DPPH, with IC50 values of 257 ± 12 μg/mL. The FRAP assay method showed that the HE was 3-fold more effective reducing agent than the EO. When the bee colonies were supplied with laurel HE in sugar paste an improvement in their general condition was noticed, although neither the hygienic behavior nor the proportions of the breeding cells varied statistically due to the treatment. In conclusion, the inhibition power against P. larvae attributable to the phenolic compounds, the antioxidant capacity of the HE, and the non-lethal effects on adult honey bees on field trials suggest the HE of laurel as a promising substance for control American foulbrood disease.

Antibacterial Activity of Bees Gut Lactobacilli against Paenibacillus Larvae In Vitro

Abstract The aim of this study was to evaluate antimicrobial activity of bees gastrointestinal Lactobacillus spp. of against Paenibacillus larvae. Content of the intestinal tract was cultured for isolation of Lactobacillus spp. Gut homogenates were plated on de Man, Rogosa and Sharpe agar (MRS, Oxoid) plates and incubated for 48-72h at 30°C anaerobically. Then, the identification of isolates with MALDI-TOF MS Biotyper was done. The bacterial strains Lactobacillus gasseri, L. amylovorus, L. kunkeei, L. fructivorans, Paenibacillus larvae were isolated from gut content of bees. The disc diffusion method was used for the determination of antimicrobial activities of the Lactobacillus supernatant against two strains of Paenibacillus larvae. The best antimicrobial activity of Lactobacillus against Paenibacillus larvae from gut was found in L. gasseri supernatant. Lesser degree of antimicrobial activity against P. larvae was found in L. kunkeei supernatant. The strongest antibacterial activity against P. larvae CCM 4438 was found in L. gasseri and L. amylovorus and the least antibacterial activity was found in L. fructivorans.

ACTIVITY OF HYDROLYSIS ENZYMES IN DIFFERENT INSTAR LARVAE OF THE AMERICAN BOLLWORM, HELICOVERPA ARMIGERA (HUBNER)

Enzymes activity included acetylcholinesterase, non-specific α and β- esterases and alkaline phosphatase were determined calorimetrically in the whole homogenates of the different instar larvae; 3rd, 4th, 5th and 6th of the field strain as well as the baseline, laboratory strain of American bollworm, Helicoverpa armigera. The results indicated that the enzymes activity levels in the different instar larvae of the field strain were markedly higher than that in the laboratory strain at the respective instar larvae. The enzymatic activity levels increased steady from the 3rd to the 6th instar larvae. A sharp increase in the enzymatic activity levels were determined in the 6th instar larvae. The average levels of the enzymatic activity in both field colony and laboratory strain were 11.630 and 3.627 µg acetyl bromide released / min. /individual for A.Ch.E.; 8.819 and 3.430 µg α-naphthol released/ min/ individual and the corresponding values were ranged between 5.232 and 3.218 µg β– naphthol released / min/ larva for beta esterase; respectively and157.669 and 50.428 unit X 103/larva for alkaline phosphatase; respectively

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm.

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for pest resistance management in China.

Paenibacillus larvae subspecies with dissimilar virulence patterns also group by vegetative growth characteristics and enolase isozyme biochemical properties

Paenibacillus larvae – the causal agent of American foul brood disease in Honey bees – group to different subspecies based upon disease progression and virulence as well as by molecular genotype. Vegetative growth studies reveal that virulence-grouped subspecies arrive at different saturated cell densities. In addition, strains segregating based upon virulence phenotype contain different genotypes in the locus encoding for the key glycolytic enzyme enolase. DNA sequence comparison of enolase loci from 7 Paenibacillus larvae strains identified 6 single-nucleotide polymorphisms (SNP) that segregated based on subspecies virulence classification. Only one polymorphism represented a change in amino acid coding (glycine or alanine) at position 331 of the protein. The kinetic properties of two recombinant enolase proteins expressed from enolase alleles isolated from different virulence classed strains (P. larvae ATCC 9545 and SAG 10367) yielded a Km and of 4.2μM and 1.5μM and Vmax of 16.2μmolmin−1mg−1 and 10.8μmolmin−1mg−1, respectively. Enolase from P. larvae SAG 10367 had a maximum reaction velocity lower than and a specificity constant approximately 1.6× higher than that of P. larvae ATCC 9545.

In-vitro efficacy of a botanical acaricide and its active ingredients against larvae of susceptible and acaricide-resistant strains of Rhipicephalus (Boophilus) microplus Canestrini (Acari: Ixodidae)

Ticks and tick-borne diseases are a major constraint for a sustainable cattle industry in the tropical and subtropical regions of the world. The development of resistance to most of the commonly used acaricides has led to an assessment of herbal products for acaricidal activity as an eco-friendly tick control alternative. A botanical product, Essentria® IC-3 insect concentrate containing rosemary oil (10%), geraniol (5%) and peppermint oil (2%), acts on target pests by blocking octopamine. Essentria® IC-3 and its active components were evaluated for larvicidal activity against several susceptible and acaricide-resistant strains of the cattle fever tick, Rhipicephalus (Boophilus) microplus Canestrini by Larval Packet test using 14–21 d old unfed larvae. The efficacy was assessed by measuring percent larval mortality and estimating lethal concentrations at 50% (LC50) and 95% (LC95) with 95% confidence limits (CL) using probit analysis. The LC50 and LC95 (95% CL) values for Essentria® IC-3 against the susceptible strain were estimated as 0.647% (0.59–0.69) and 1.033% (0.94–1.19), respectively, whereas, LC50 and LC95 values for other strains were variable, ranging from 0.597-1.674% and 0.927–2.236%, respectively. Among the various active ingredients, the larvicidal property of Essentria® IC-3 seem to be attributable mainly to geraniol and the LC50 and LC95 (95% CL) values for geraniol against the susceptible Deutch strain were estimated as 0.656% (0.61–0.69) and 1.114% (1.03–1.25), respectively. The comparison of LC50 and LC95 values of acaricide-resistant strains showed susceptibility comparable to Deutch against geraniol except for the Las Palmas strain. We report a low level of resistance in some of the acaricide-resistant strains against the herbal acaricide in the cattle tick for the first time, possibly due to cross-resistance to chemical acaricides.