Neonicotinoid’s resistance monitoring, diagnostic mechanisms and cytochrome P450 expression in green peach aphid [Myzus persicae (Sulzer) (Hemiptera: Aphididae)]

Green peach aphid [Myzus persicae (Sulzer) (Hemiptera: Aphididae)] is a significant pest with a known history of insecticide resistance. Neonicotinoids could manage this pest; however, their frequent use led to the evolution of resistance in field populations of M. persicae. Toxicity data for neonicotinoid insecticides synergized with pipernyl butoxide (PBO) in a field population (FP) were collected and compared to a laboratory susceptible clone (SC) of aphids. The enhanced expression of metabolic resistance-related cytochrome P450 gene CYP6CY3 and an arginine-threonine substitution were detected in FP, causing a single point mutation (R81T) at β1 subunit of nicotinic acetylcholine receptor (nAChR) within D loop. High level of resistance to imidacloprid was developed in FP with 101-fold resistance ratio and moderate resistance level (10.9-fold) to acetamiprid. The results of PBO synergized bioassay suggested that cytochrome P450 enzymes were involved in the resistance to neonicotinoids. The mRNA transcriptional level of CYP6CY3 gene was significantly higher (3.74 fold) in FP compared to SC. The R81T mutation associated with neonicotinoid resistance had 26% resistant allele frequency in FP. Both P450 enzymes and R81T mutation of nAChR were found in field-evolved neonicotinoid resistance. It is concluded that field-evolved resistance in green peach aphid could be managed by using appropriate synergists such as PBO.

Comparative Study of the Effect of Solvents on the Efficacy of Neonicotinoid Insecticides Against Malaria Vector Populations Across Africa

Background New insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health by WHO. Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations. In this study, we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.MethodsMosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon (Njombe-Penja, Nkolondom, and Mangoum), the Democratic Republic of Congo (Ndjili-Brasserie), Ghana (Obuasi), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, acetone, MERO) on the efficacy of neonicotinoids against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. gambiae and to evaluate potential cross-resistance between pyrethroids and clothianidin.ResultsLower mortality were observed when using absolute ethanol or acetone alone as solvent (11.4- 51.9% mortality in Nkolondom, 31.7- 48.2% in Mangoum, 34.6- 56.1% in Mayµge, 39.4- 45.6% in Obuasi, 83.7- 89.3% in Congo and 71.05- 95.9% in Njombe pendja) compared to acetone + MERO for which 100% mortality were observed for all the populations. Synergist assays (PBO, DEM and DEF) revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility. A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors (OR=0.5; P=0.02). However, the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes (OR=2.10; P=0.013). ConclusionsThis study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting to lower mortality, thus possibly overestimating resistance, whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development. Therefore, we recommend monitoring the susceptibility using both acetone alone and acetone+MERO (8-10µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.

Comparative Study of the Effect of Solvents on the Efficacy of Neonicotinoid Insecticides Against Malaria Vector Populations Across Africa

Background: New insecticides with novel modes of action such as neonicotinoids have recently been recommended for public health use by WHO. Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations. In this study, we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.Methods: Mosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon (Njombe-Penja, Nkolondom, and Mangoum), the Democratic Republic of Congo (Ndjili-Brasserie), Ghana (Atatam), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, acetone, acetone+MERO) on the efficacy of neonicotinoids against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. gambiae and to evaluate potential cross-resistance between pyrethroids and clothianidin.Results: Lower mortalities were observed for all populations when using absolute ethanol or acetone alone as solvent (11.4- 51.9% mortality for Nkolondom, 31.7- 48.2% for Mangoum, 34.6- 56.1% for Mayuge, 39.4- 45.6% for Atatam, 83.7- 89.3% for Congo and 71.05- 95.9% for Njombe pendja) compared to acetone + MERO for which 100% mortality was observed for all the populations. Synergist assays (PBO, DEM and DEF) revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility. A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors (OR=0.5; P=0.02). However, the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes (OR=2.10; P=0.013). Conclusions: This study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting in lower mortality, thus possibly overestimating resistance, whereas the addition of MERO consistently increased the efficacy of neonicotinoids in terms of percentage mortalities and time to final mortality. The addition of MERO could however prevent the early detection of resistance development. We therefore recommend monitoring susceptibility using both acetone alone and acetone+MERO (8-10µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.

Simultaneous overexpression of multiple herbicide-metabolizing genes for broad-spectrum resistance in an agricultural weed Echinochloa phyllopogon

The use of herbicides in agricultural fields has driven the evolution of weeds for resistance, causing a grave threat to the current agriculture. One big mystery of weed resistance involves multiple-herbicide resistance (MHR) concomitant to enhanced herbicide metabolism. Previous research unveiled that the overexpression of catalytically promiscuous cytochrome P450s underlies the metabolism-based cross-resistance in multiple species. However, the concept of activation of promiscuous enzymes does not fully accommodate the resistance to diverse herbicides in MHR Echinochloa phyllopogon although the genetic inheritance of MHR was suggested as under a single gene control. Here, we show that the high-level resistance to diclofop-methyl in E. phyllopogon is caused by the simultaneous overexpression of CYP81A12/21, the previously identified promiscuous P450s, and a novel P450 CYP709C69. We found that the MHR line rapidly produced two distinct hydroxylated-diclofop-acid, only one of which was the major metabolite produced by CYP81As. RNA-seq followed by real-time PCR in the crossed progeny of MHR and sensitive lines identified several P450 genes whose overexpressions were associated with MHR. Gene functional characterization revealed that only CYP709C69 conferred diclofop-methyl resistance in rice calli and produced another hydroxylated-diclofop-acid in yeast, reinforcing the relatively low activity of CYP81As to diclofop-methyl. Plants transformed with CYP709C69 had unchanged sensitivity to 46 herbicides except for clomazone, where transgenic plants became more susceptible. The present findings establish a novel concept that simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the profile of metabolic resistance in weeds.

Sarcopenia and nutritional support for elderly and senile patients

Демографическое старение населения приводит к значительному увеличению доли лиц пожилого и старческого возраста как на амбулаторном, так и стационарном этапах лечения. За последнее десятилетие средний возраст основного контингента больных в отделениях реанимации и интенсивной терапии составляет более 65 лет, с тенденцией к увеличению, что предопределяет стратегию более тщательной оценки подходов к проведению лечебных мероприятий с учетом полиморбидности и функциональных возможностей пациентов старших возрастных групп как в период пребывания в стационаре, так и на дальнейших этапах восстановления и реабилитации. Известно, что в целом длительность пребывания в отделениях интенсивной терапии пожилых пациентов значительно выше, а результаты лечения и реабилитации достоверно ниже, чем у более молодых. Это связано с сочетанием целого ряда факторов: коморбидности/полиморбидности пациентов пожилого и старческого возраста, снижения функциональных резервов организма, синдрома старческой астении, главными составляющими которого являются саркопения, недостаточность питания. Связь старческой астении с увеличением длительности госпитализации, снижением физической активности, развитием функциональных дефицитов, увеличением риска падений, переломов и смертности достаточно хорошо изучена, однако в рутинной клинической практике оценке нутритивного статуса, диагностике недостаточности питания и решению проблемы недостаточности питания пациентов с синдромом старческой астении и саркопении уделяется недостаточное внимание, несмотря на известные факты о взаимосвязи недостаточности питания, снижения массы скелетных мышц, в том числе их силы или функции с неблагоприятным исходами заболевания. В амбулаторном этапе, кроме малоподвижного образа жизни, развитию и прогрессированию саркопении часто способствуют наличие тяжелого заболевания, сопровождающегося системным воспалением, метаболическая резистентность, нутритивная недостаточность и депрессия, характерные для данной категории больных. Современные подходы к лечению пациента пожилого и старческого возраста подразумевают проведение регулярной оценки нутритивного статуса и консультирование по вопросам рационального питания с внедрением методов нутритивной поддержки в комплексную программу ведения пожилого пациента наряду с основными лечебными и реабилитационными мероприятиями с целью замедления прогрессирования синдрома астении и саркопении. Demographic aging of the population leads to a significant increase in the proportion of elderly and senile people, both at the outpatient and inpatient stages of treatment. Over the past decade, the average age of the main contingent of patients in intensive care and intensive care units is more than 65 years, with a tendency to increase, which predetermines the strategy for a more thorough assessment of approaches to treatment measures, taking into account polymorbidity and functional capabilities of patients of older age groups, as the period of stay in hospital, and at further stages of recovery and rehabilitation. It is known that, in general, the duration of stay in intensive care units for elderly patients is much longer, and the results of treatment and rehabilitation are significantly lower than that of younger patients. This is due to a combination of a number of factors: comorbidity/polymorbidity of elderly and senile patients, decreased functional reserves of the body, senile asthenia syndrome, the main components of which are sarcopenia, and malnutrition. The relationship of senile asthenia with an increase in the duration of hospitalization, a decrease in physical activity, the development of functional deficits, an increase in the risk of falls, fractures and mortality is well understood, however, in routine clinical practice, assessing nutritional status, diagnosing malnutrition and solving the problem of malnutrition in patients with senile asthenia syndrome and Insufficient attention is paid to sarcopenia, despite the known facts about the relationship of malnutrition, a decrease in skeletal muscle mass, including their strength or function, with unfavorable outcomes of the disease. In the outpatient phase, in addition to a sedentary lifestyle, the development and progression of sarcopenia is often facilitated by the presence of a severe illness accompanied by systemic inflammation, metabolic resistance, nutritional deficiency and depression characteristic of this category of patients. Modern approaches to the treatment of an elderly and senile patient imply a regular assessment of the nutritional status and counseling on rational nutrition with the introduction of nutritional support methods into a comprehensive management program for an elderly patient, along with basic treatment and rehabilitation measures in order to slow the progression of asthenia and sarcopenia syndrome.

Bacillus thuringiensis and Chlorantraniliprole Trigger the Expression of Detoxification-Related Genes in the Larval Midgut of Plutella xylostella

Background: Diamondback moth (DBM), Plutella xylostella (L.), has developed resistance to many insecticides. The molecular mechanism of DBM resistance to Bt-G033A combined with chlorantraniliprole (CL) remains undefined.Methods: In this study, field-resistant strains of Plutella xylostella to three pesticides, namely, Bacillus thuringiensis (Bt) toxin (Bt-G033A), CL, and a mixture of Bt + CL, were selected to evaluate the resistance level. Additionally, transcriptomic profiles of a susceptible (SS-DBM), field-resistant (FOH-DBM), Bt-resistant (Bt-DBM), CL-resistant (CL-DBM), and Bt + CL-resistant (BtC-DBM) strains were performed by comparative analysis to identify genes responsible for detoxification.Results: The Bt-G033A was the most toxic chemical to all the DBM strains among the three insecticides. The comparative analysis identified 25,518 differentially expressed genes (DEGs) between pairs/combinations of strains. DEGs were enriched in pathways related to metabolic and catalytic activity and ABC transporter in resistant strains. In total, 17 metabolic resistance-related candidate genes were identified in resistance to Bt-G033A, CL, and Bt + CL by co-expression network analysis. Within candidate genes, the majority was upregulated in key genes including cytochrome P450, glutathione S-transferase (GST), carboxylesterase, and acetylcholinesterase in CL- and BtC-resistant strains. Furthermore, aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, trypsin, and ABC transporter genes were eminent as Bt-resistance-related genes. Expression patterns of key genes by the quantitative real-time PCR (qRT-PCR) proved the credibility of transcriptome data and suggest their association in the detoxification process.Conclusion: To date, this study is the most comprehensive research presenting functional transcriptome analysis of DBM using Bt-G033A and CL combined insecticidal activity.

Evaluation of the interaction between insecticide resistance-associated genes and malaria transmission in Anopheles gambiae sensu lato in central Côte d’Ivoire

Background There is evidence that the knockdown resistance gene (Kdr) L1014F and acetylcholinesterase-1 gene (Ace-1R) G119S mutations involved in pyrethroid and carbamate resistance in Anopheles gambiae influence malaria transmission in sub-Saharan Africa. This is likely due to changes in the behaviour, life history and vector competence and capacity of An. gambiae. In the present study, performed as part of a two-arm cluster randomized controlled trial evaluating the impact of household screening plus a novel insecticide delivery system (In2Care Eave Tubes), we investigated the distribution of insecticide target site mutations and their association with infection status in wild An. gambiae sensu lato (s.l.) populations. Methods Mosquitoes were captured in 40 villages around Bouaké by human landing catch from May 2017 to April 2019. Randomly selected samples of An. gambiae s.l. that were infected or not infected with Plasmodium sp. were identified to species and then genotyped for Kdr L1014F and Ace-1R G119S mutations using quantitative polymerase chain reaction assays. The frequencies of the two alleles were compared between Anopheles coluzzii and Anopheles gambiae and then between infected and uninfected groups for each species. Results The presence of An. gambiae (49%) and An. coluzzii (51%) was confirmed in Bouaké. Individuals of both species infected with Plasmodium parasites were found. Over the study period, the average frequency of the Kdr L1014F and Ace-1R G119S mutations did not vary significantly between study arms. However, the frequencies of the Kdr L1014F and Ace-1R G119S resistance alleles were significantly higher in An. gambiae than in An. coluzzii [odds ratio (95% confidence interval): 59.64 (30.81–131.63) for Kdr, and 2.79 (2.17–3.60) for Ace-1R]. For both species, there were no significant differences in Kdr L1014F or Ace-1R G119S genotypic and allelic frequency distributions between infected and uninfected specimens (P > 0.05). Conclusions Either alone or in combination, Kdr L1014F and Ace-1R G119S showed no significant association with Plasmodium infection in wild An. gambiae and An. coluzzii, demonstrating the similar competence of these species for Plasmodium transmission in Bouaké. Additional factors including behavioural and environmental ones that influence vector competence in natural populations, and those other than allele measurements (metabolic resistance factors) that contribute to resistance, should be considered when establishing the existence of a link between insecticide resistance and vector competence. Graphical Abstract

Crystal structure of Arabidopsis thaliana HPPK/DHPS, a bifunctional enzyme and target of the herbicide asulam

Herbicides are vital for modern agriculture, but their utility is threatened by genetic or metabolic resistance in weeds as well as heightened regulatory scrutiny. Of the known herbicide modes of action, 6-hydroxymethyl-7,8-dihydropterin synthase (DHPS) which is involved in folate biosynthesis, is targeted by just one commercial herbicide, asulam. A mimic of the substrate para-aminobenzoic acid, asulam is chemically similar to sulfonamide antibiotics - and while still in widespread use, asulam has faced regulatory scrutiny. With an entire mode of action represented by just one commercial agrochemical, we sought to improve the understanding of its plant target. Here we solve a 2.6 Å resolution crystal structure for Arabidopsis thaliana DHPS that is conjoined to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and reveal a strong structural conservation with bacterial counterparts at the sulfonamide-binding pocket of DHPS. We demonstrate asulam and the antibiotics sulfacetamide and sulfamethoxazole have herbicidal as well as antibacterial activity and explore the structural basis of their potency by modelling these compounds in mitochondrial HPPK/DHPS. Our findings suggest limited opportunity for the rational design of plant selectivity from asulam and that pharmacokinetic or delivery differences between plants and microbes might be the best approaches to safeguard this mode of action.