Effects of temperature on baseline susceptibility and stability of insecticide resistance against Plutella xylostella (Lepidoptera: Plutellidae) in the absence of selection pressure

Plutella xylostella L. (Lepidoptera: Plutellidae) is an important pest causing significant losses to vegetables worldwide. Insecticides resistance in P. xylostella is a serious issue for scientists since last 30 years. However, deltamethrin and Bt Cry1Ac are commonly used insecticides against P. xylostella but studies involving development of resistance in P. xylostella against these two insecticides at different temperatures are lacking. The current study was aimed to find out the toxicity of deltamethrin and Bt Cry1Ac, and resistance development in P. xylostella. Results showed that the positive correlation between the temperature and toxicities of deltamethrin and Bt Cry1Ac. The results indicated −0.051, −0.049, −0.047, and −0.046 folds of deltamethrin resistance at 15 °C, 20 °C, 25 °C, and 30 °C temperatures, respectively from 1^st to 12^th generations. The toxicity of Bt Cry1Ac after 24 h was 2.2 and 4.8 folds on 1^st generation at 20 °C and 25 °C temperatures, respectively compared to the toxicity recorded at 15 °C (non-overlapping of 95% confidence limits). Based on the results of this study, it is concluded that the temperature has a positive correlation with the toxicity of deltamethrin and Bt Cry1Ac against the larvae of P. xylostella. This study suggests that deltamethrin and Bt Cry1Ac can be included in the management program of P. xylostella on many vegetable crops. The baseline susceptibility data might be helpful to understand the resistance mechanisms in P. xylostella.     

Downregulation of ubiquitin gene expression in the deltamethrin‐resistant diamondback moth (Plutella xylostella L.)

Plutella xylostella (L.) has long been regarded as a very important universal pest of cruciferous crops. It is therefore important to understand its mechanisms of insecticide resistance. In our previous study, we obtained a differently expressed sequence with relatively high homology to the ubiquitin (UBI) gene as assessed via cDNA representational difference analysis (RDA). To further study this product, we cloned UBI open reading frame (ORF) sequences identified from both sexes of the deltamethrin‐resistant strain (DRF and DRM) and the deltamethrin‐sensitive strain (SF and SM) of P. xylostella. The deduced protein sequence of DRM was different from the other samples. The expression level was measured using real‐time quantitative polymerase chain reaction (PCR) and Western blot. The results of both analyses revealed the expression levels, in order from highest to lowest, to be in SF, SM, DRF and DRM. This indicates that the long‐term use of deltamethrin on P. xylostella results in decreased expression of the UBI gene.     

MH-DAB gene polymorphism and disease resistance to Flavobacterium columnare in grass carp (Ctenopharyngodon idellus)

In this study, the association between MH-DAB gene polymorphism and disease resistance was evaluated by challenging grass carp (Ctenopharyngodon idellus) with Flavobacterium columnare. Eight genotypes and six alleles were found, and named by common nomenclature. The genotypes AA, BB, EE, and DE, and the alleles Ctid-DAB1*0101, Ctid-DAB1*0201 and Ctid-DAB1*0401 were more preponderant in fish. The genotype BB was associated with higher resistance to F. columnare, as well as two alleles Ctid-DAB*0101 and Ctid-DAB*0201. Allele Ctid-DAB*0102 has decreased resistance to F. columnare. The expression of MH-DAB gene was decreased in the liver, kidney, and intestine but not in the spleen, gill, and skin at 2 days post infection (dpi), versus to that in the control group. MH-DAB gene expression was up-regulated in most tissues but remained at normal levels in the intestine at 15 days post infection. Our data suggested that MH-DAB polymorphism can be used as a potential genetic marker for disease resistance breeding of grass carp in the future.     

Transient expression of specific Cotesia plutellae bracoviral segments induces prolonged larval development of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses a segmented and dispersed genome that is located on chromosome(s) of its symbiotic endoparasitic wasp, C. plutellae. When the host wasp parasitizes larvae of the diamondback moth, Plutella xylostella, at least 27 viral genome segments are delivered to the parasitized host along with the wasp egg. The parasitized P. xylostella exhibits significant immunosuppression and a prolonged larval development. Parasitized larvae take about 2 days longer than nonparasitized larvae to develop until the wandering stage of the final larval instar, and die after egress of the full grown wasp larvae. Developmental analysis using juvenile hormone and ecdysteroid analogs suggests that altering endocrine signals could induce the retardation of larval developmental rate in P. xylostella. In this study we used a transient expression technique to micro-inject individual CpBV genome segments, and tested their ability to induce delayed larval development of P. xylostella. We demonstrated that a CpBV segment was able to express its own encoded genes when it was injected into nonparasitized larvae, in which the expression patterns of the segment genes were similar to those in the larvae parasitized by C. plutellae. Twenty three CpBV genome segments were individually cloned and injected into the second instar larvae of P. xylostella and their effects assessed by measuring the time taken for host development to the cocooning stage. Three CpBV genome segments markedly interfered with the host larval development. When the putative genes of these segments were analyzed, it was found that they did not share any common genes. Among these segments able to delay host development, segment S27 was predicted to encode seven protein tyrosine phosphatases (CpBV-PTPs), some of which were mutated by insertional inactivation with transposons, while other encoded gene expressions were unaffected. The mutant segments were unable to induce prolonged larval development of P. xylostella. These results suggest that CpBV can induce prolonged larval development of P. xylostella, and that at least some CpBV-PTPs may contribute to the parasitic role probably by altering titers of developmental hormones.     

Phytotoxic substances with allelopathic activity may be central to the strong invasive potential of Brachiaria brizantha

The grass Brachiaria brizantha, native to eastern Africa, becomes naturalized and dominant quickly in the non-native areas. It was hypothesized that phytotoxic chemical interaction between this plant and native plants may play an important role in the invasion of B. brizantha. However, no potent phytotoxic substance has been reported in this species. Therefore, we investigated possible allelopathic activity and searched for phytotoxic substances with allelopathic activity in B. brizantha. An aqueous methanol extract of B. brizantha inhibited the growth of roots and shoots of garden cress (Lepidium sativum), lettuce (Lactuca sativa), timothy (Phleum pratense) and ryegrass (Lolium multiflorum) seedlings. The extract was purified by several chromatographic runs and three allelopathically active substances were isolated and identified by spectral analysis as (6R,9R)-3-oxo-α-ionol, (6R,9S)-3-oxo-α-ionol and 4-ketopinoresinol. (6R,9R)-3-Oxo-α-ionol and (6R,9S)-3-oxo-α-ionol inhibited root and shoot growth of garden cress at concentrations greater than 30 and 10 μM, respectively. The activity of (6R,9S)-3-oxo-α-ionol was 5.3- to 6.2-fold that of (6R,9R)-3-oxo-α-ionol. The stereochemistry of the hydroxyl group at position C-9 may be important for the inhibitory activities of those compounds. 4-Ketopinoresinol inhibited root and shoot growth of garden cress at concentrations greater than 30 μM. The growth inhibitory activity of (6R,9S)-3-oxo-α-ionol was the greatest and followed by 4-ketopinoresinol and (6R,9R)-3-oxo-α-ionol. These results suggest that those phytotoxic substances may contribute to the allelopathic effect caused by B. brizantha and may be involved in the invasion of B. brizantha.     

Efficacy of entomopathogenic nematode,Steinernema carpocapsae against the diamondback moth,Plutella xylostella (L.) in laboratory condition

In vitro studies were carried out on the diamondback moth, Plutella xylostella larvae using an insect entomopathogenic nematode isolate, Steinernema carpocapsae obtained from the Koppert company, the Netherlands. Larvae of P. xylostella were collected from cabbage farms around Mashhad city of Iran. During the study, the responses of larvae at 25?°C for three periods of 24, 48 and 72?h with different concentrations of 0, 5, 10, 20, 40, 80, 160 and 320 third instar larvae of nematode (infective stage?=?IJs) per insect into 10?cm Petri dishes containing filter paper soaked with 1?ml of nematodes suspension were compared. Maximum mortality caused by S. carpocapsae nematode was 88% at 24?h, and it was 100% at 48 and 72 h. With increasing nematode population level and exposure time (ET in hour), mortality of P. xylostella larvae was increased. Based on probit analysis, LC₅₀ values of S. carpocapsae nematode in three test periods were 45.61, 12.02 and 40.80 IJs per insect, respectively. Initial ANOVA was performed for S. carpocapsae nematode. The effect of both nematode population levels (IJ) and ET on third instar larvae of the diamondback moth, P. xylostella and interaction between IJ and ET were significant. In general, it is recommended to apply this nematode in suitable condition for controlling diamondback moth.     

Comparative bioassay of different isolates of Bacillus thuringiensis subsp. kurstaki on the third larval instars of diamondback moth,Plutella xylostella (L.) (Lep.: Plutellidae)

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most important pests of cruciferous plants throughout the world. In recent years, this insect has been a serious pest for cabbage fields in Tehran province. Resistance of P. xylostella to all main groups of insecticides has been recorded and it is ranked in the 20 most resistant pest species reported up to now. According to many researchers, to eliminate the problem of pest resistance to chemical pesticides, an integrated pest management programme should be used. In line with this, the uses of microbial control agents (MCAs) are discussed. The bacterium, Bacillus thuringiensis (Bt) is one of microbial control agents of pests. It is characterised by its ability to produce proteic crystalline inclusions during sporulation. Cry1 protein has insecticidal activity and is highly specific to certain insects and not toxic to unrelated insects, plants or vertebrates. In this work, the pathogenicity of some Bt isolates, including Dipel, 20, 29, 79 and 87, was tested against P. xylostella and the lethal concentrations (LC₅₀) of their crystal proteins to P. xylostella third larval instar was determined. The experiment was designed in factorial in randomised complete design with 5 treatments (different concentrations including 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ CFU/ml and 5 replications and with 10 third larval instars. Spore–crystal complex was applied to the surface of natural diets (cabbage leaves) and the mortality of P. xylostella larvae was assessed 120?h after exposure of Bt toxin in each treatment. Results showed that percentage of survival was significantly higher for control treatment. Results also showed that after 5?days, LC₅₀ for isolates of Dipel, 20, 29, 79 and 87 were equal to 1?×?10⁶, 1?×?10⁵, 5?×?10⁵, 4?×?10⁵ and 1?×?10⁴ CFU/ml, respectively. LT₅₀ were equal to 93.71, 48.04, 71, 40.49 and 75.28?h. Of and most the percentage larval mortality relate to attendance 87 and also at least percentage mortality is related to the groom Dipel.     

Reduction in swimming performance in juvenile rainbow trout (Oncorhynchus mykiss) following sublethal exposure to pyrethroid insecticides

While the lethal toxicity of pyrethroid insecticides to fish is well documented, their sublethal physio-behavioral effects remain poorly characterized. Known pyrethroid-associated changes to insect neuromuscular function may translate into similar effects in fish, thereby altering swimming ability and affecting foraging, predator avoidance, and migration. Three experiments were conducted using critical (U_crit) and burst (U_max) swimming speeds to assess the sublethal effects of the pyrethroids permethrin and deltamethrin in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to deltamethrin (100, 200, or 300 ng/L) or permethrin (1, 2, or 3 μg/L) in water for 4 d, and assessed for swimming performance. Deltamethrin (200 and 300 ng/L) reduced U_crit, but not U_max, while both swim performance measurements were unaffected by permethrin. Subsequent experiments used only U_crit to assess deltamethrin exposure. In a time course experiment, deltamethrin (300 ng/L) reduced U_crit after 1 and 4 d of exposure, but after 7 d of exposure U_crit was fully recovered. Finally, deltamethrin (1, 2, or 3 μg/L) reduced U_crit after 1 h bath exposures similar to recommended protocols for deltamethrin based sea-lice treatment in aquaculture. The real-world implications of the revealed pyrethroid-associated swimming ability reductions in salmon may be important in areas close to aquaculture facilities.     

Ubiquitin chains in the Dsk2 UBL domain mediate Dsk2 stability and protein degradation in yeast

Ubiquitin-like (UBL)–ubiquitin-associated (UBA) proteins, including Dsk2 and Rad23, act as delivery factors that target polyubiquitinated substrates to the proteasome. We report here that the Dsk2 UBL domain is ubiquitinated in yeast cells and that Dsk2 ubiquitination of the UBL domain is involved in Dsk2 stability, depending on the Dsk2 UBA domain. Also, Dsk2 lacking ubiquitin chains impaired ubiquitin-dependent protein degradation and decreased the interaction of Dsk2 with polyubiquitinated proteins in cells. Moreover, Dsk2 ubiquitination affected ability to restore the temperature-sensitive growth defect of dsk2Δ. These results indicate that ubiquitination in the UBL domain of Dsk2 has in vivo functions in the ubiquitin–proteasome pathway in yeast.     

Differential host growth regulation by the solitary endoparasitoid, Meteorus pulchricornis in two hosts of greatly differing mass

Solitary koinobiont endoparasitoids generally reduce the growth of their hosts by a significant amount compared with healthy larvae. Here, we compared the development and host usage strategies of the solitary koinobiont endoparasitoid, Meteorus pulchricornis, when developing in larvae of a large host species (Mythimna separata) and a much smaller host species (Plutella xylostella). Caterpillars of M. separata were parasitized as L2 and P. xylostella as L3, when they weighed approximately 2 mg. The growth of parasitized M. separata larvae was reduced by almost 95% compared with controls, whereas parasitized P. xylostella larvae grew some 30% larger than controls. Still, adult wasps emerging from M. separata larvae were almost twice as large as wasps emerging from P. xylostella larvae, had larger egg loads after 5 days and produced more progeny. Survival to eclosion was also higher on M. separata than on P. xylostella, although parasitoids developed significantly faster when developing on P. xylostella. Our results provide evidence that koinobionts are able to differentially regulate the growth of different host species. However, there are clearly also limitations in the ability of parasitoids to regulate phenotypic host traits when size differences between different host species are as extreme as demonstrated here.     

The dihydrolipoyl acyltransferase gene BCE2 participates in basal resistance against Phytophthora infestans in potato and Nicotiana benthamiana

Dihydrolipoyl acyltransferase (EC 2.3.1.12), a branched-chain α-ketoacid dehydrogenase E2 subunit (BCE2), catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. However, the role of BCE2 responding to biotic stress in plant is not clear. In this study, we cloned and characterized a BCE2 gene from potato, namely StBCE2, which was previously suggested to be involved in Phytophthora infestans–potato interaction. We found that the expression of StBCE2 was strongly induced by both P. infestans isolate HB09-14-2 and salicylic acid. Besides, when the homolog of StBCE2 in Nicotiana benthamiana named NbBCE2 was silenced, plants showed increased susceptibility to P. infestans and reduced accumulation of hydrogen peroxide (H₂O₂). Furthermore, we found that a marker gene NbrbohB involved in the production of reactive oxygen species, was also suppressed in NbBCE2-silenced plants. However, silencing of NbBCE2 had no significant effect on the hypersensitive responses trigged by INF1, R3a-AVR3a^KI pair or Rpi-vnt1.1-AVR-vnt1.1 pair. Our results suggest that BCE2 is associated with the basal resistance to P. infestans by regulating H₂O₂ production.     

Transient transcription of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp and encodes two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28-50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. Their expressions were detected in all tested tissues including hemocyte, fat body, gut, and epidermis. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to nonparasitized larvae of P. xylostella, in which the two genes were expressed at least for 4 days post-injection. The larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression of the viral segment-treated larvae, while dsRNA specific to ORF302 did not. These results suggest that a putative RNase fused with a BEN domain encoded in CpBV-S3 plays a parasitic role in inducing host immunosuppression in the parasitism.     

Suppression of stop codon UGA in acrB can contribute to antibiotic resistance in Klebsiella pneumoniae ATCC10031

We previously reported that Klebsiella pneumoniae MGH78578 exhibited higher resistance against various antimicrobials than K. pneumoniae ATCC10031. In this study, we showed that the plasmid, pKPN5, in K. pneumoniae MGH78578 played an important role in resistance against aminoglycosides, ampicillin, tetracycline, and chloramphenicol, while genome-derived β-lactamases and drug efflux pumps appeared to be more important in resistance to cloxacillin. acrAB, encoding a potent multidrug efflux pump, was cloned from K. pneumoniae MGH78578 and ATCC10031, to investigate reasons for the high drug resistance of K. pneumoniae MGH78578, and the results revealed that AcrAB from K. pneumoniae ATCC10031 conferred weaker drug resistance than AcrAB from K. pneumoniae MGH78578. DNA sequencing revealed that acrB from K. pneumoniae ATCC10031 carried the nonsense mutation, UGA, which was not found in acrB from K. pneumoniae MGH78578. However, acrB from K. pneumoniae ATCC10031 conferred slightly elevated resistant levels to several antimicrobials. The intact length of AcrB was detected in K. pneumoniae ATCC10031 by Western blot analysis, even though its quantity was small. Therefore, the stop codon UGA in acrB was thought to be overcome to some extent in this strain. We artificially introduced the nonsense mutation, UGA to the cat gene on pACYC184, and the plasmid also elevated the MIC of chloramphenicol in K. pneumoniae ATCC10031. These results suggest that a mechanism to overcome the nonsense mutation in acrB sustained resistance against a few β-lactams, dyes, and cholic acid in K. pneumoniae ATCC10031.     

Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål)

To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella.     

Accord insertion in the 5′ flanking region of CYP6G1 confers nicotine resistance in Drosophila melanogaster

What has driven the sweep of the Accord retrotransposon insertion allele of CYP6G1 in the natural populations of Drosophila melanogaster is unknown. Previous studies on the DDT selection hypothesis produced conflicting data. To reexamine the DDT selection hypothesis and search for alternative explanations, we conducted a series of correlation and genetic linkage experiments with eight D. melanogaster natural populations collected from California (CM1, CM2, CM3, and CM7) and Africa (AM2, AM3, AM4, AM7). Diagnostic PCR showed that CM1, CM2, CM7, and AM3 have the Accord insertion in the CYP6G1 locus, whereas the other four strains do not. RT-PCR analysis exhibits a 100% correlation between Accord insertion and CYP6G1 overexpression. However, among the four strains with Accord-mediated CYP6G1 overexpression only CM1 and CM7 are resistant to DDT, and the other two strains (CM2 and AM3), like the four Accord-free strains, are susceptible to DDT. By contrast, all the four strains with Accord-mediated CYP6G1 overexpression are resistant to nicotine, a plant allelochemical. Genetic crosses between DDT resistant and susceptible Accord-insertion strains, as well as crosses between Accord-insertion and Accord-free strains demonstrated that Accord insertion and CYP6G1 overexpression are genetically linked to nicotine resistance rather than DDT resistance. These results suggest that naturally-occurring allelochemicals such as nicotine are the initial driving force for the worldwide prevalence of the Accord insertion allele of CYP6G1 in D. melanogaster natural populations.