Cytochrome P450 Genes of the CYP4 Clan and Pyrethroid Resistance in Chagas Disease Vectors

he chemical control of Triatoma infestans, the major Chagas disease vector in southern South America, has been threatened in the last years by the emergence of pyrethroid-resistant bug populations. As an alternative approach, the efficacy of the entomopathogenic fungus Beauveria bassiana to control T. infestans populations (regardless their pyrethroid susceptibility) has been demonstrated. Growing research efforts on the interaction between T. infestans and B. bassiana by molecular, ecological, biochemical and behavioral traits has allowed framing such interaction as an evolutionary arms race. This review will focus on the relationships established in this particular host-pathogen system, compiling available data on the relevance of fungal pathogenesis, insect behavior, population dynamics and human intervention to favor fungal dissemination in bug populations. The current snapshot shows the fungus ahead in the evolutionary arms race and predicts a promissory landscape for the biological control of Chagas disease vectors.

Nutrient Scarcity in a New Defined Medium Reveals Metabolic Resistance to Antibiotics in the Fish Pathogen Piscirickettsia salmonis.

Extensive use of antibiotics has been the primary treatment for the Salmonid Rickettsial Septicemia, a salmonid disease caused by the bacterium Piscirickettsia salmonis. Occurrence of antibiotic resistance has been explored in various P. salmonis isolates using different assays; however, P. salmonis is a nutritionally demanding intracellular facultative pathogen; thus, assessing its antibiotic susceptibility with standardized and validated protocols is essential. In this work, we studied the pathogen response to antibiotics using a genomic, a transcriptomic, and a phenotypic approach. A new defined medium (CMMAB) was developed based on a metabolic model of P. salmonis. CMMAB was formulated to increase bacterial growth in nutrient-limited conditions and to be suitable for performing antibiotic susceptibility tests. Antibiotic resistance was evaluated based on a comprehensive search of antibiotic resistance genes (ARGs) from P. salmonis genomes. Minimum inhibitory concentration assays were conducted to test the pathogen susceptibility to antibiotics from drug categories with predicted ARGs. In all tested P. salmonis strains, resistance to erythromycin, ampicillin, penicillin G, streptomycin, spectinomycin, polymyxin B, ceftazidime, and trimethoprim was medium-dependent, showing resistance to higher antibiotic concentrations in the CMMAB medium. The mechanism for antibiotic resistance to ampicillin in the defined medium was further explored and was proven to be associated to a decrease in the bacterial central metabolism, including the TCA cycle, the pentose-phosphate pathway, energy production, and nucleotide metabolism, and it was not associated with decreased growth rate of the bacterium or with the expression of any predicted ARG. Our results suggest that nutrient scarcity plays a role in the bacterial antibiotic resistance, protecting against the detrimental effects of antibiotics, and thus, we propose that P. salmonis exhibits a metabolic resistance to ampicillin when growing in a nutrient-limited medium.

Evaluation of Resistance to Different Insecticides and Metabolic Detoxification Mechanism by Use of Synergist in the Common Bed Bug (Heteroptera: Cimicidae).

Bed bugs have become a common urban pest with consequences on human health and economic costs to the hotel and tourism sectors. Insecticide resistance is considered an important factor in the current bed bug resurgence, and multiple resistance mechanisms could be working in the resistant bed bug populations. In the present study, we determined the resistance profile to four insecticides with a different mode of action in Cimex lectularius L. (Heteroptera: Cimicidae) field-collected colonies from Argentina. Furthermore, the synergism effect of piperonyl butoxide (PBO) with deltamethrin was investigated to explore the contribution of detoxification metabolism to resistance. Our results showed that most of the field-collected colonies are extremely resistant to deltamethrin and propoxur, much more than to azametiphos and imidacloprid. The differences in resistance ratios among field-collected colonies could be associated with different modes of action of insecticides used in control pest and the mechanisms involved in the resistance. PBO pretreatment led to a significantly decreased RR in pyrethroid-resistant colonies, suggesting an upturn of monooxygenase activity for deltamethrin detoxification. However, the high RR detected could involve other mechanisms as part of the whole resistant phenotype in colonies of C. lectularius resistant to pyrethroids.

Mechanisms of pyrethroid resistance inHaematobia irritans (Muscidae) from Mato Grosso do Sul state, Brazil

Horn fly resistance to pyrethroid insecticides occurs throughout Brazil, but knowledge about the involved mechanisms is still in an incipient stage. This survey was aimed to identify the mechanisms of horn fly resistance to cypermethrin in Mato Grosso do Sul state, Brazil. Impregnated filter paper bioassays using cypermethrin, synergized or not with piperonyl butoxide (PBO) and triphenyl phosphate (TPP), were conducted from March 2004 to June 2005 in horn fly populations (n = 33) from all over the state. All populations were highly resistant to cypermethrin, with resistance factors (RF) ranging from 89.4 to 1,020.6. Polymerase chain reaction (PCR) assays to detect the knockdown resistance (kdr) mutation also were performed in 16 samples. The kdr mutation was found in 75% of the tested populations, mostly with relatively low frequencies ( 20%), and was absent in some highly resistant populations. Addition of TPP did not significantly reduce the LC50 in any population. However, PBO reduced LC50s above 40-fold in all tested populations, resulting in RFs 10 in most cases. Horn fly resistance to cypermethrin is widespread in the state, being primarily caused by an enhanced activity of P450 mono-oxygenases and secondarily by reduced target site sensitivity.

Resistência da mosca-dos-chifres a inseticidas piretróides ocorre em todo o país, entretanto, o conhecimento sobre os mecanismos envolvidos é ainda incipiente. Este estudo objetivou identificar os mecanismos de resistência desta mosca à cipermetrina em Mato Grosso do Sul. Bioensaios utilizando papéis impregnados com cipermetrina, isoladamente ou sinergizada por butóxido de piperonila (PBO) ou trifenil fosfato (TPP), foram realizados de março2004 a junho2005 em 33 populações. Todas as populações apresentaram elevada resistência à cipermetrina, com fatores de resistência (FR) variando de 89,4 a 1.020,6. Ensaios de reação em cadeia da polimerase (PCR) visando a detecção de kdr(knockdown resistance) foram realizados em 16 amostras. A mutação kdr foi detectada em 75% das populações, geralmente em baixas frequências ( 20%) e ausente em algumas populações resistentes. A adição de TPP não reduziu significativamente a CL50 em nenhuma população. Entretanto, o PBO reduziu em mais de 40 vezes a CL50 de todas as populações testadas, resultando em FR 10 na maioria dos casos. Resistência da mosca-dos-chifres à cipermetrina encontra-se disseminada no estado, sendo causada primariamente por um aumento da atividade de P450 mono-oxigenases e secundariamente pela redução da sensibilidade do sítio de ação do inseticida.

The classification of esterases: an important gene family involved in insecticide resistance - A review

The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.