Genetic characterization of the Dyscalc locus.

The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is an important cosmopolitan and extremely polyphagous species capable of causing direct and indirect damage to various crops. Insecticide resistance in melon aphids is of particular concern. To determine the basis of resistance, organophosphate (OP)-resistant strains of A. gossypii were obtained by continuous selection with dimethoate in the laboratory, and resistance mechanisms were investigated along with susceptible strains. Three resistant strains LKR-1, LKR-2, and LKR-3 exhibiting 270-, 243-, and 210-fold resistance obtained after 30 generations of selection with dimethoate, respectively, were utilized in this study. The role of acetylcholinesterase (AChE), a target enzyme for OPs and carbamates (CMs), was investigated. AChE enzyme assay revealed that there was no significant change in the activities of AChE in resistant and susceptible strains. However, AChE inhibitory assay showed that 50% of the enzyme activity in resistant strains was inhibited at significantly higher concentration of dimethoate (131.87, 158.65, and 99.29 µmolL(−1)) as compared with susceptible strains (1.75 and 2.01 µmolL(−1)), indicating AChE insensitivity owing to altered AChE. Molecular diagnostic tool polymerase chain reaction-restriction fragment length polymorphism revealed the existence of two consistent non-synonymous point mutations, single-nucleotide polymorphism, viz., A302S (equivalent to A201 in Torpedo californica Ayres) and S431F (equivalent to F331 in T. californica), in the AChE gene Ace2 of resistant strains. Further, cloning and sequencing of a partial fragment of Ace2 (897 bp) gene from susceptible and resistant strains revealed an additional novel mutation G221A in resistant strains, LKR-1 and LKR-2. Susceptible Ace2 genes shared 99.6 and 98.9% identity at the nucleic acid and amino acid levels with resistant ones, respectively. Functional analysis of these point mutations was assessed by in silico docking studies using the modeled wild-type and naturally mutated AChE2. Computational analysis showed that the conformational changes in AChE2 active site due to structural gene substitutions (A302S, S431F, and G221A) significantly reduced the level of ligand (OP-dimethoate, omethoate, and CM-pirimicarb) binding, suggesting that they are potentially associated with resistance development. These results unambiguously suggested that multiple mutations located in the enzyme active site are responsible for AChE insensitivity to dimethoate and are likely the molecular basis for dimethoate resistance in these selected field populations of A. gossypii.

Susceptibility to diabetogenic virus: Host versus pancreatic factors

The Plasmacytoma Resistance Gene, Pctr2, Delays the Onset of Tumorigenesis and Resides in the Telomeric Region of Chromosome 4

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

A novel approach to the control of anthelmintic-resistant Haemonchus contortus in sheep

Triazophos resistance mechanisms in the rice stem borer ( Chilo suppressalis Walker)

Aedes aegypti Linnaeus (Diptera: Culicidae) is an important vector of mosquito-borne viruses, including dengue, Zika and chikungunya. In the case of dengue, approximately half of the world’s population is at risk, and an estimated 100–400 million new infections are reported each year. To control Ae. aegypti populations, mosquito management programmes rely heavily on insecticides, which have been the mainstay of mosquito management programmes for almost 50 years. The most common class of insecticide used to control mosquito vectors of human disease are the pyrethroids. These target the voltage-gated sodium channel, an essential protein that facilitates electrical signalling within the nervous system. Due to the intense selection pressures applied by public health programmes and the incidental exposure of mosquitoes to agricultural and domestic insecticides, pyrethroid-resistant Ae. aegypti populations are now common.Target site resistance, one of the common mechanisms associated with resistance to pyrethroids, occurs most frequently in mosquitoes through non-synonymous mutations in the voltage-gated sodium channel gene. These point mutations, commonly known as knockdown resistance (kdr) mutations, can cause a change in the shape of the voltage-gated sodium channel, which decreases the efficacy with which pyrethroids bind to them. Knockdown resistance mutations in Ae. aegypti populations have been reported from many parts of the world and reflect the increasingly common reports of phenotypic resistance to pyrethroids on a global scale. The geographically widespread distribution of pyrethroid-resistant Ae. aegypti has emphasised the requirement for insecticide resistance management strategies that conserve susceptibility and ensure the availability of effective insecticidal chemistries to control mosquito-borne disease. Insecticide Resistance Management (IRM) strategies rely on reducing selection pressure, which can result in the immigration of susceptible genotypes and, where they exist, allow fitness costs to reduce the frequency of resistance mutations. With regard to the latter, our understanding of the consequences of kdr mutations on the fitness and behaviour of Ae. aegypti, in the absence of pyrethroids, is limited. Furthermore, we do not have a clear understanding of how Ae. aegypti with or without these mutations are affected by the sublethal doses of insecticide that are common in urban and peri-urban habitats.I backcrossed a pyrethroid-resistant strain of Ae. aegypti from Timor-Leste, harbouring the V1016G and S989P kdr mutations, with an insecticide susceptible strain from Queensland, Australia that does not carry kdr mutations. This produced a strain of Ae. aegypti that was homozygous for the V1016G and S989P kdr mutations in an otherwise susceptible genetic background. Using double-digest restriction associated sequencing, I confirmed that >99.9% of the backcrossed strain’s genome originated from the susceptible parental strain. The creation of the backcrossed strain allowed me to compare differences in life-history traits and behaviour between the backcrossed-resistant and susceptible parent strain, in the absence or presence of permethrin, and to directly attribute those distinctions to the effects of the homozygous V1016G/S989P genotype.The results presented within this thesis demonstrate that, in comparison to the susceptible strain, mosquitoes with the V1016G/S989P genotype exhibited longer larval development times, had smaller wing lengths and females had a shorter average lifespan (Chapter II). Investigations into behavioural changes showed a significant reduction in mean wing beat frequency in males and a significant reduction in estimated male mating success, associated with the introgressed kdr genotype (Chapter III). These results suggest specific and significant fitness costs and behavioural changes associated with the double homozygous V1016G/S989P genotype, in the absence of insecticides. These likely consequences of resistance evolution may encourage reversion to susceptibility in the absence of insecticide selection pressures.I also examined the impact of sublethal permethrin exposure on the mosquito fitness parameters of survival, fecundity, fertility, blood-feeding, host location and mating success. For the first time, I demonstrated some expected, negative impacts of sublethal exposure on susceptible Ae. aegypti mosquitoes but also showed that detrimental effects on longevity and host location can occur even in highly resistant mosquitoes (Chapter IV). Overall, I demonstrated that significant differences in fitness exists between mosquitoes with susceptible and resistant genotypes, in the presence of sublethal doses of insecticides. The competitive advantage conferred to resistant mosquito strains in the presence of sublethal doses of permethrin from various sources, questions our ability to create and exploit untreated refugia or mosaics and therefore remove selection pressures and permit resistance-associated fitness costs to manifest.My results focus on the pleiotropic effects of a common kdr genotype on the biology and behaviour of Ae. aegypti in the absence and presence of insecticides. By cataloguing these effects, this thesis provides evidence to inform the introduction of specific IRM measures into vector control programmes and predict their potential impact by cataloguing these effects. My results are discussed in relation to reducing pyrethroid selection pressures and the subsequent impact on the frequency of resistant genotypes by temporarily removing pyrethroid selection pressures as part of IRM and encouraging the consequent recovery or conservation of pyrethroid susceptibility.

An integral membrane protein, Claudin 5 (CLDN5), is a critical component of endothelial tight junctions that control pericellular permeability. Breaching of endothelial barriers is a key event in the development of pulmonary edema during acute lung injury (ALI). A major irritant in smoke, acrolein can induce ALI possibly by altering CLDN5 expression. This study sought to determine the cell signaling mechanism controlling endothelial CLDN5 expression during ALI. To assess susceptibility, 12 mouse strains were exposed to acrolein (10 ppm, 24 h), and survival monitored. Histology, lavage protein, and CLDN5 transcripts were measured in the lung of the most sensitive and resistant strains. CLDN5 transcripts and phosphorylation status of forkhead box O1 (FOXO1) and catenin (cadherin-associated protein) beta 1 (CTNNB1) proteins were determined in control and acrolein-treated human endothelial cells. Mean survival time (MST) varied more than 2-fold among strains with the susceptible (BALB/cByJ) and resistant (129X1/SvJ) strains (MST, 17.3 ± 1.9 h vs. 41.4 ± 5.1 h, respectively). Histological analysis revealed earlier perivascular enlargement in the BALB/cByJ than in 129X1/SvJ mouse lung. Lung CLDN5 transcript and protein increased more in the resistant strain than in the susceptible strain. In human endothelial cells, 30 nM acrolein increased CLDN5 transcripts and increased p-FOXO1 protein levels. The phosphatidylinositol 3-kinase inhibitor LY294002 diminished the acrolein-induced increased CLDN5 transcript. Acrolein (300 nM) decreased CLDN5 transcripts, which were accompanied by increased FOXO1 and CTNNB1. The phosphorylation status of these transcription factors was consistent with the observed CLDN5 alteration. Preservation of endothelial CLDN5 may be a novel clinical approach for ALI therapy.

The rice weevil, Sitophilus oryzae (Linnaeus) is a cosmopolitan insect pest that can cause severe damage to stored grain with regard to both quality (loss of nutritional and aesthetic value) and quantity (loss of grain weight). Heavy reliance on the fumigant phosphine to disinfest stored products has led to resistance to phosphine in many insect species including S. oryzae. The frequency and level of phosphine resistance in S. oryzae has increased in the last fifty years in Asian and Africa countries and recently in Australia as well. A strongly phosphine resistant strain of S. oryzae was first detected in 2009 in Australia. I have determined the strong resistance trait in this strain to be incompletely recessive, autosomal and controlled by at least two major genes. The weakly resistant Australian strain shares a common resistance factor with the strongly resistant strain (So_rph1), which is responsible for weak resistance. I also compared the resistance trait of the Australian strain to that of collected from Vietnam and China. Complementation analysis revealed a shared genetic mechanism of phosphine resistance between the three strains that I tested. I then identified and sequenced the genes conferring phosphine resistance in this species. This process began with simply sequencing transcripts of the two genes known to be responsible for phosphine resistance in Rhyzopertha dominica and Tribolium castaneum (rph1/tc_rph1 and rph2/tc_rph2). rph2/tc_rph2 had been published and was known to encode the metabolic enzyme, dihydrolipoamide dehydrogenase (DLD). Variants of DLD (So_rph2) also cause strong resistance in S. oryzae when interacting with resistance variants of So_rph1. Therefore, rph2/DLD is responsible for strong resistance in each of the three species. A specific substitution mutation (N505T) in DLD is likely responsible for phosphine resistance as it is found in all strongly resistant strains of S. oryzae from Australia, Vietnam and China. A fitness cost associated with the resistance allele of So_rph2 was observed in the absence of selection in populations derive from the F1 progeny between a strongly resistant strain and strains lacking a resistance allele at So_rph2. As DLD is one of three subunits (E1, E2, E3) of a-ketoacid dehydrogenase complexes, I hypothesized that suppression of the activity of subunits other than DLD might also result in resistance to phosphine. Thiamine pyrophosphate is critical cofactor of the E1 subunit and is required by very few other enzymes. I reasoned that insects cultured on a diet of white rice, which is deficient in thiamine, would experience inhibition of the activity of E1. I expected to observe the change in response to phosphine in both the susceptible and resistance strains of S. oryzae when fed on white rice diet compared with the thiamine containing diet of whole grain (brown rice or whole wheat). Against my expectation, I found no significant affect of a thiamine deficient diet toward phosphine resistance in S. oryzae. A diet of white rice, however, did result in slowed developmental and a reduced number of progeny, but this effect was similar in both the phosphine resistant and susceptible strains. My work has demonstrated that the genetic mechanism for phosphine resistance is similar between S. oryzae and other pest insects studied so far. It will be straightforward to develop a molecular diagnostic to detect resistance alleles of S. oryzae in insect populations globally, facilitating resistance management.

Proteomic assessment of resistance to the fumigant phosphine in the lesser grain borer, Rhyzopertha dominica (F.)

Fitness in a malathion resistant Tribolium castaneum strain; feeding, growth and digestion

Three cDNAs, cloned from both pyrethroid-susceptible and -resistant strains of Lygus lineolaris, contained a 1548 nucleotide open reading frame encoding a 516 amino acid residue protein. Predicted cytochrome P450s from cDNAs were classified as the first three new members of subfamily CYP6X, CYP6X1v1 for a susceptible strain and CYP6X1v2 and CYP6X1v3 for two resistant strains. Putative cytochrome P450 CYP6X1s from L. lineolaris were highly similar (up to 42% amino acid sequence identity) to several insect CYP6s that are responsible for reduced sensitivity to pyrethroid insecticides. A total of twenty-six nucleotide substitutions were revealed between cDNAs of susceptible and resistant strains. Two nucleotide substitutions resulted in amino acid changes, Asp373 to Ala373 and Ser487 to Ala487, between susceptible and resistant strains. The resistant laboratory strain contained 2.1-fold higher cytochrome P450 mRNA per microgram total RNA than the susceptible laboratory strain. Topical treatment with 10 ng permethrin elevated cytochrome P450 mRNA levels by approximately 2-fold. The results of this study indicated that cytochrome P450 gene mutation, coupled with up-regulation, was present only in the pyrethroid resistant strains, and was possibly related to resistance development in the tarnished plant bug.

A novel F1552/C1552 point mutation in the Aedes aegypti voltage-gated sodium channel gene associated with permethrin resistance

Susceptibility to oxydemeton-methyl and imidacloprid, and the inhibitory effects of oxydemeton-methyl and some organophosphate compounds on acetylcholinesterase (AChE) and carboxylesterase activity were studied in two populations (Karaj and Rasht) of green peach aphids, Myzus persicae (Sulzer). Results show that the Karaj population was resistant to oxydemeton-methyl but susceptible to imidacloprid. The esterase activity of the resistant and susceptible populations suggests that one of the resistance mechanisms to oxydemeton-methyl was esterase-based. The inhibition assay shows that the AChE of the Karaj population is less sensitive to oxydemeton-methyl and paraoxon derivatives. Regarding the paraoxon derivatives, the smaller paraoxon side chain is more potent against the modified AChE than against the AChE from the susceptible strain. Fertility life table parameters of green peach aphid populations resistant and susceptible to oxydemeton-methyl also were studied under laboratory conditions. The standard errors of the population growth parameters were calculated using the Jackknife method. Results showed that susceptible strain exhibits a significantly higher rm than the resistant strain, probably because the resistant strain had a higher generation time than the susceptible strain. These results suggested that the resistant Karaj strain may be less fit than the susceptible strain.

Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two trypsin-like proteinases, which is found in both the Bt-susceptible and -resistant strains of the Indianmeal moth. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF064525 for the RC688 strain and AF064526 for HD198).