Putative pleiotropic effects of the knockdown resistance (L1014F) allele on the life-history traits of Anopheles gambiae

Background Existing mechanisms of insecticide resistance are known to help the survival of mosquitoes following contact with chemical compounds, even though they could negatively affect the life-history traits of resistant malaria vectors. In West Africa, the knockdown resistance mechanism kdrR (L1014F) is the most common. However, little knowledge is available on its effects on mosquito life-history traits. The fitness effects associated with this knockdown resistance allele in Anopheles gambiae sensu stricto (s.s.) were investigated in an insecticide-free laboratory environment. Methods The life-history traits of Kisumu (susceptible) and KisKdr (kdr resistant) strains of An. gambiae s.s. were compared. Larval survivorship and pupation rate were assessed as well as fecundity and fertility of adult females. Female mosquitoes of both strains were directly blood fed through artificial membrane assays and then the blood-feeding success, blood volume and adult survivorship post-blood meal were assessed. Results The An. gambiae mosquitoes carrying the kdrR allele (KisKdr) laid a reduced number of eggs. The mean number of larvae in the susceptible strain Kisumu was three-fold overall higher than that seen in the KisKdr strain with a significant difference in hatching rates (81.89% in Kisumu vs 72.89% in KisKdr). The KisKdr larvae had a significant higher survivorship than that of Kisumu. The blood-feeding success was significantly higher in the resistant mosquitoes (84%) compared to the susceptible ones (34.75%). However, the mean blood volume was 1.36 µL/mg, 1.45 µL/mg and 1.68 µL/mg in Kisumu, homozygote and heterozygote KisKdr mosquitoes, respectively. After blood-feeding, the heterozygote KisKdr mosquitoes displayed highest survivorship when compared to that of Kisumu. Conclusions The presence of the knockdown resistance allele appears to impact the life-history traits, such as fecundity, fertility, larval survivorship, and blood-feeding behaviour in An. gambiae. These data could help to guide the implementation of more reliable strategies for the control of malaria vectors.

Modeling homing suppression gene drive in haplodiploid organisms

Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study the characteristics of homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids. They are substantially more vulnerable to high resistance allele formation in the embryo due to maternally deposited Cas9 and gRNA and also to somatic cleavage activity. Examining models of continuous space where organisms move over a landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly due to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations.

Markov chain Monte Carlo Gibbs sampler approach for estimating haplotype frequencies among multiple malaria infected human blood samples

Background Malaria patients can have two or more haplotypes in their blood sample making it challenging to identify which haplotypes they carry. In addition, there are challenges in measuring the type and frequency of resistant haplotypes in populations. This study presents a novel statistical method Gibbs sampler algorithm to investigate this issue. Results The performance of the algorithm is evaluated on simulated datasets consisting of patient blood samples characterized by their multiplicity of infection (MOI) and malaria genotype. The simulation used different resistance allele frequencies (RAF) at each Single Nucleotide Polymorphisms (SNPs) and different limit of detection (LoD) of the SNPs and the MOI. The Gibbs sampler algorithm presents higher accuracy among high LoD of the SNPs or the MOI, validated, and deals with missing MOI compared to previous related statistical approaches. Conclusions The Gibbs sampler algorithm provided robust results when faced with genotyping errors caused by LoDs and functioned well even in the absence of MOI data on individual patients.

Genetic Screening to Identify Candidate Resistance Alleles to Cry1F Corn in Fall Armyworm Using Targeted Sequencing

Evolution of practical resistance is the main threat to the sustainability of transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt crops). Monitoring of resistance to Cry and Vip3A proteins produced by Bt crops is critical to mitigate the development of resistance. Currently, Cry/Vip3A resistance allele monitoring is based on bioassays with larvae from inbreeding field-collected moths. As an alternative, DNA-based monitoring tools should increase sensitivity and reduce overall costs compared to bioassay-based screening methods. Here, we evaluated targeted sequencing as a method allowing detection of known and novel candidate resistance alleles to Cry proteins. As a model, we sequenced a Cry1F receptor gene (SfABCC2) in fall armyworm (Spodoptera frugiperda) moths from Puerto Rico, a location reporting continued practical field resistance to Cry1F-producing corn. Targeted sequencing detected a previously reported Cry1F resistance allele (SfABCC2mut), in addition to a resistance allele originally described in S. frugiperda populations from Brazil. Moreover, targeted sequencing detected mutations in SfABCC2 as novel candidate resistance alleles. These results support further development of targeted sequencing for monitoring resistance to Bt crops and provide unexpected evidence for common resistance alleles in S. frugiperda from Brazil and Puerto Rico.

Intensity and mechanisms of deltamethrin and permethrin resistance in Anopheles gambiae s.l. populations in southern Benin

Background Insecticide resistance is threatening the effectiveness of efforts to control malaria vectors in Benin. This study explores the levels and mechanisms of insecticide resistance in An. gambiae s.l. to pyrethroids. Methods Larvae were collected from August 2017 to July 2018 in five communes in southern Benin (Adjohoun, Allada, Bohicon, Cotonou, and Porto-Novo) representing diverse ecological regions, and were reared in Benin’s insectary. Two- to five-day-old female mosquitoes from each district were exposed to multiple doses of deltamethrin and permethrin (1×, 2×, 5×, and 10×) using the WHO insecticide resistance intensity bioassay. The effect of pre-exposure to the synergist, piperonyl butoxide (PBO), was also tested at different pyrethroid doses. Molecular allele frequencies of kdr (1014F) and ace-1R (119S) insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled from each study area. Results An. gambiae s.l. were resistant to pyrethroid-only exposure up to 10× the diagnostic doses in all the study sites for both deltamethrin and permethrin. Mortality was significantly higher in An. gambiae s.l. pre-exposed to PBO followed by exposure to deltamethrin or permethrin compared to mosquitoes exposed to deltamethrin or permethrin only (p < 0.001). The difference in mortality between deltamethrin only and PBO plus deltamethrin was the smallest in Cotonou (16–64%) and the greatest in Bohicon (12–93%). The mortality difference between permethrin only and PBO plus permethrin was the smallest in Cotonou (44–75%) and the greatest in Bohicon (22–72%). In all the study sites, the kdr resistance allele (1014F) frequency was high (75–100%), while the ace-1 resistance allele (G119S) frequency was low (0–3%). Analysis of the metabolic enzymatic activity of An. gambiae s.l. showed overexpression of nonspecific esterases and glutathione S-transferases (GST) in all study sites. In contrast to the PBO results, oxidase expression was low and was similar to the susceptible An. gambiae s.s. Kisumu strain in all sites. Conclusion There is high-intensity resistance to pyrethroids in southern Benin. However, pre-exposure to PBO significantly increased susceptibility to the pyrethroids in the different An. gambiae s.l. populations sampled. The use of PBO insecticide-treated bed nets may help maintain the gains in An. gambiae (s.l.) control in southern Benin. Graphical Abstract

Studies of Resistance to Sharka (Plum Pox) in Romanian Apricot Progenies

Sharka (Plum pox) is considered one of the most devastating diseases of stone fruits in terms of agronomic impact and economic importance. The response of several apricot hybrids was evaluated over the vegetative period of 2010 by visual monitoring of symptom development and by serological and molecular methods. After two cycles of study, all the replicates of ‘Mari de Cenad’, ‘Traian’ and ‘Tabriz’ showed sharka symptoms while the replicates of ‘Stark Early Orange’ and NJA 2 did not show any symptoms and were not ELISA-positive. The resistant progenitors (‘Stark Early Orange’, NJA17, NJA42 and NJA2) were able to transmit PPV resistance to their descendants, in agreement with previous results (DOSBA et al, 1994, MARTINEZ-GOMEZ et al, 2000; AUDERGON et al, 1994). The genetic control hypotheses for PPV resistance in apricot referenced by different authors considered the resistance allele as dominant. Romanian apricot F1 and F2 progenies evaluated were classified into two groups: susceptible to PPV and resistant to PPV. After that the most important genotypes were grafted onto infected GF 305 (considered as indicator for PPV) in greenhouse conditions. The final step is the introduction and development of the marker-assisted selection (MAS) in PPV resistance F1 and F2 progenies in apricot Romanian genotypes. DNA was isolated from all hybrids and parent genotypes. These samples will be used to implement the molecular markers for identification of genes linked to plant response to PPV infection.

Estimation of Cry3Bb1 resistance allele frequency in field populations of western corn rootworm using a genetic marker

Following the discovery of western corn rootworm (WCR; Diabrotica virgifera virgifera) populations resistant to the Bacillus thuringiensis (Bt) protein Cry3Bb1, resistance was genetically mapped to a single locus on WCR chromosome 8 and linked SNP markers were shown to correlate with the frequency of resistance among field-collected populations from the US Corn Belt. The purpose of this paper is to further investigate the relationship between one of these resistance-linked markers and the causal resistance locus. Using data from laboratory bioassays and field experiments, we show that one allele of the resistance-linked marker increased in frequency in response to selection, but was not perfectly linked to the causal resistance allele. By coupling the response to selection data with a genetic model of the linkage between the marker and the causal allele, we developed a model that allowed marker allele frequencies to be mapped to causal allele frequencies. We then used this model to estimate the resistance allele frequency distribution in the US Corn Belt based on collections from 40 populations. These estimates suggest that chromosome 8 Cry3Bb1 resistance allele frequency was generally low (&lt;10%) for 65% of the landscape, though an estimated 13% of landscape has relatively high (&gt;25%) resistance allele frequency.

HETEROZYGOUS ORYZA SATIVA L. DOUBLED HAPLOID GENERATED THROUGH IN VITRO ANDROGENESIS

In the course of marker-assessted selection for the blast resistance gene Pi-b, among 372 rice Oryza sativa L. doubled haploids DH0 geterozygous plant was identified. In the offspring (11 doubled haploids DH1), only Pi-b resistance allele was revealed. The reason for the appearance of false heterozygosity is discussed. By analogy with mixoploidy, an explanation is proposed for the phenomenon of somatic tissues mixogeny.

Assessment of strawberry varieties by anthracnose resistance gene

The results of marker-assisted identification of the Rca2 anthracnose resistance gene in promising strawberry varieties were shown. The DNA marker STS-Rca2_240, linked to the Rca2 anthracnose resistance allele was identified in varieties Albion and Aprica (F. × ananassa), and variety Dar (F. × anashata). Strawberry varieties Alpha, Bereginya, Kokinskaya Zarya, Pamyati Zubova, Rubinovy kaskad, Slavutich, Yuniol, Big King, Brilla, Joly, Rumba and Vivara are characterized by the absence of the marker STS-Rca2_240. In strawberry varieties with the identified marker STS-Rca2_240 (Dar, Albion and Aprica), the Rca2 gene can be in a dominant homozygous or heterozygous state, which makes it possible to recommend these genotypes for use in breeding for anthracnose resistance.

Hétérogénéité génétique et résistance des vecteurs du paludisme (Anopheles gambiae s.l) aux insecticides en zone cotonnière au Benin

La résistance des vecteurs du paludisme aux insecticides utilisés dans la lutte anti vectorielle est un obstacle majeur pour les programmes nationaux de lutte contre le paludisme. La présente étude avait pour but d’évaluer la diversité génétique des populations de Anopheles gambiae et la résistance aux insecticides (mutation Kdr et Ace-1) en zone cotonnière au Bénin. Deux méthodes de capture ont été utilisées pour la collecte d’échantillon. La capture sur appât humain et la récolte de la faune résiduelle matinale par spray. La PCR SINE a permis de distinguer deux espèces jumelles et d’obtenir les différents génotypes pour les deux gènes. Deux espèces du complexe An. gambiae s.l ont été trouvées en sympatrie (Anopheles coluzzii et Anopheles gambiae). La résistance kdr et Ace-1 connaissent un flux saisonnier. L’allèle résistant du gène kdr est fixé dans la population de vecteurs (Fis>0). Par contre, un déficit de l’allèle résistant est noté au niveau du gène Ace-1 (Fis<0).Mots clés : Hétérogénéité, génétique, résistance, vecteur, paludisme. English Title: Genetic heterogeneity and resistance to insecticides in malaria vector Anopheles gambiae s.l in the cotton growing area in Benin Resistance of malaria vectors to insecticides used in vector control is a major threat for national malaria control programs. The purpose of this study was to assess the genetic diversity of Anopheles gambiae populations and resistance to insecticides (Kdr Ace-1 mutation) in the cotton growing area in Benin. Two samples methods were used for mosquito collection: Human Landing catch (HLC) and the residual fauna collection by spray. The SINE PCR allow to identify species the different genotypes resistance genes. Two species of the An. gambiae s.l complex have been found in sympatry (Anopheles coluzzii and Anopheles gambiae). kdr and Ace-1 resistance had seasonal variation trend. The kdr resistance allele is fixed in the vector population (Fis> 0). Moreover, a deficit of the Ace-1 resistance allele was observed (Fis <0). Keywords: Heterogeneity, genetic, resistance, vector, malaria.