Identification and geographical distribution of pyrethroid resistance mutations in the poultry red mite Dermanyssus gallinae

The poultry red mite (PRM) Dermanyssus gallinae is the most common ectoparasite on poultry and causes high economic losses in poultry farming worldwide. Pyrethroid acaricides have been widely used for its control and, consequently, pyrethroid resistance has arisen. In this study we aim to investigate the occurrence of resistance and study the geographical distribution of pyrethroid resistance mutations across PRM populations in Europe

First evidence of resistance to pyrethroid insecticides in Italian Aedes albopictus populations after 26 years since invasion

Aedes albopictus has spread during the last decades all over the world. This has increased significantly the risk of exotic arbovirus transmission (e.g. Chikungunya, Dengue, and Zika) also in temperate areas, as testified by the Chikungunya 2007- and 2017-outbreaks in north-east and central Italy. Insecticides represent a main tool for limiting the circulation of these mosquito-borne viruses. The aim of the present study is to start filling the current gap of knowledge on pyrethroid insecticide resistance of European Ae. albopictus populations focusing on populations from Italy, Albania and Greece

Behavioural responses of fourth instar Aedes aegypti (Diptera:Culcidae) larvae to Plagiorchis elegans cercariae

Exposure of fourth instar Aedes aegypti larvae to graded concentrations of the entomopathogenic cercariae of the digenean Plagiorchis elegans, induced fundamental changes in the partitioning of time among a number of major behavioural activities. In the absence of the parasite, swimming, breathing and feeding remained stable over time. Grooming was rarely observed. In the presence of cercariae, larvae increased grooming 26-fold. Concomitantly, feeding was reduced to 33% of control values. Times spent swimming and breathing remained unchanged. The increase in grooming activity was attributable to the attachment of cercariae to the surface of the mosquito larvae, but was independent of infection. Grooming reduced cercarial penetration. Seventy-five percent of cercariae were lost to predation by larvae, but their caloric value was insignificant and failed to compensate for the loss of feeding time due to grooming. The findings are discussed in the context of their impact on larval growth, development and survival

Insecticide resistance status of mosquitoes of Greece

In the present study I investigated the insecticide resistance status of two medically important mosquito species Culex pipiens and Aedes albopictus collected from Greece. At first, the resistance status of thirteen Cx. pipiens populations from five regional units in Greece over a three years period was examined, against four insecticides used for its control. Bioassays demonstrated susceptibility of most populations to all insecticides, except temephos in some regions. We also monitored the frequency of the pyrethroid target site resistance mutations L1014F (kdr), as well as the G119S and F290V in the Ace1 gene. Ace1 insensitivity mutations were found at low frequencies and always in heterozygocity. However, the frequency of kdr pyrethroid resistance mutations was higher (up to 63% in Thessaloniki). Concerning ―Asian mosquito tiger‖ Ae. albopictus eggs were collected with oviposition traps from two different localities (Corfu, first species’ site of introduction in Greece and Rizoupoli, first detection site in capital city of Athens). Larvae and adult mosquitoes were tested in the lab using all registered products in Greece for mosquito control according to WHO guidelines. Population from Rizoupoli – Athens showed high resistance to temephos. Resistant Ratio (RR50=8,83) were high compared to other available data from literature. Additionally adult mosquitoes from the same population showed reduced LT50 when exposed to deltamethrin standard impregnated papers from WHO. Athens’ population were further selected in the laboratory for 10-generation and the molecular mechanism of resistance to temephos, was examined. Elevated esterases were implicated in the temephos resistance phenotype of Ae. albopictus. Finally, we have genetically characterized the composition of Cx. pipiens (pipiens and molestus). Samples were collected during the 2010 outbreak from the region of Thessaloniki, where WNV cases occurred, and from the region Schinias-Marathonas, with no reported cases at the time. Information on bird fauna was also obtained for the two regions. Application of genetic diagnostic tools revealed the presence of hybrids in Thessaloniki and suggested a great receptivity for the establishment of WNV zoonotic cycles

Bioassay and molecular monitoring of insecticide resistance status in Aedes albopictus populations from Greece, to support evidence-based vector control

Background Aedes albopictus has a well-established presence in southern European countries, associated with recent disease outbreaks (e.g. chikungunya). Development of insecticide resistance in the vector is a major concern as its control mainly relies on the use of biocides. Data on the species’ resistance status are essential for efficient and sustainable control. To date the insecticide resistance status of Ae. albopictus populations from Greece against major insecticides used in vector control remains largely unknown. Methods We investigated the insecticide resistance status of 19 Ae. albopictus populations from 11 regions of Greece. Bioassays were performed against diflubenzuron (DFB), Bacillus thuringiensis var. israelensis (Bti), deltamethrin and malathion. Known insecticide resistance loci were molecularly analysed, i.e. voltage-gated sodium channel (VGSC) mutations associated with pyrethroid resistance; presence and frequency of carboxylesterases 3 (CCEae3a) and 6 (CCEae6a) gene amplification associated with organophosphate (OP) resistance and; chitin synthase-1 (CHS-1) for the possible presence of DFB resistance mutations. Results Bioassays showed full susceptibility to DFB, Bti and deltamethrin, but resistance against the OP malathion (range of mortality: 55.30–91.40%). VGSC analysis revealed a widespread distribution of the mutations F1534C (in all populations, with allelic frequencies between 6.6–68.3%), and I1532T (in 6 populations; allelic frequencies below 22.70%), but absence of V1016G. CCE gene amplifications were recorded in 8 out of 11 populations (overall frequency: 33%). Co-presence of the F1534C mutation and CCEae3a amplification was reported in 39 of the 156 samples analysed by both assays. No mutations at the CHS-1 I1043 locus were detected. Conclusions The results indicate: (i) the suitability of larvicides DFB and Bti for Ae. albopictus control in Greece; (ii) possible incipient pyrethroid resistance due to the presence of kdr mutations; and (iii) possible reduced efficacy of OPs, in a scenario of re-introducing them for vector control. The study highlights the need for systematic resistance monitoring for developing and implementing appropriate evidence-based control programmes

Transcriptome Profiling and Genetic Study Reveal Amplified Carboxylesterase Genes Implicated in Temephos Resistance, in the Asian Tiger Mosquito Aedes albopictus

International audienceBackground: The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chikungunya, has been largely reliant on the use of the larvicide temephos for many decades. This insecticide remains a primary control tool for several countries and it is a potential reliable reserve, for emergency epidemics or new invasion cases, in regions such as Europe which have banned its use. Resistance to temephos has been detected in some regions, but the mechanism responsible for the trait has not been investigated.Principal findings: Temephos resistance was identified in an Aedes albopictus population isolated from Greece, and subsequently selected in the laboratory for a few generations. Biochemical assays suggested the association of elevated carboxylesterases (CCE), but not target site resistance (altered AChE), with this phenotype. Illumina transcriptomic analysis revealed the up-regulation of three transcripts encoding CCE genes in the temephos resistant strain. CCEae3a and CCEae6a showed the most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and temephos resistance, by demonstrating a strong association between survival to temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins, were upregulated in resistant mosquitoes, indicating that the co-evolution of multiple mechanisms might contribute to resistance.Significance: The identification of specific genes associated with insecticide resistance in Ae. albopictus for the first time is an important pre-requirement for insecticide resistance management. The genomic resources that were produced will be useful to the community, to study relevant aspects of Ae. albopictus biology

Annotated <i>Aedes albopictus</i> genes encoding for detoxification and/or redox genes and insecticide target subunits.

<p>Annotated <i>Aedes albopictus</i> genes encoding for detoxification and/or redox genes and insecticide target subunits.</p

Temephos toxicity in the temephos–selected resistant strain (Tem-GR) as compared to its parental (Par-GR), a reference laboratory strain (Lab) and a susceptible field population from Italy (Field-S-IT) [7].

<p>LC₅₀ values are in ppm (μgr/L).</p><p>¹Resistant ratios calculated over the Lab-S strain</p><p>²Resistant ratios calculated over the Field-S-IT field population [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003771#pntd.0003771.ref006" target="_blank">6</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003771#pntd.0003771.ref007" target="_blank">7</a>], collected in 2002 from neighboring region (Genoa, Italy).</p><p>Temephos toxicity in the temephos–selected resistant strain (Tem-GR) as compared to its parental (Par-GR), a reference laboratory strain (Lab) and a susceptible field population from Italy (Field-S-IT) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003771#pntd.0003771.ref007" target="_blank">7</a>].</p

Gene copy number analysis of <i>CCEae3a</i>, <i>CCEae6a</i> and AAEL015578 CCEs.

<p>Error bars represent the standard error of the calculated mean based on three biological replicates and a star indicates statistical significance (p value<0.05).</p

Genetic association of <i>CCEae3α</i> and <i>CCEae6α</i> copy numbers with resistance to temephos.

<p>Genomic DNA from surviving and dead F2 individuals was analyzed by qPCR. Histone 3 is used as a reference gene and values are expressed as the reverse ratio of the esterase Ct over the histone 3 Ct. A. Difference in <i>CCEae3α</i> copy numbers between survivors and dead individuals are statistically significant based on a Welsh test (p-value<0.05). B. Differences in <i>CCEae6α</i> (B) copy numbers between survivors and dead individuals are statistically significant based on a Welsh test (p-value<0.05).</p