Phenotypic and molecular analysis of insecticide resistance in Aedes albopictus populations from Greece

doi:10.21203/rs.3.rs-21133/v1

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Phenotypic and molecular analysis of insecticide resistance in Aedes albopictus populations from Greece

https://doi.org/10.21203/rs.3.rs-21133/v1

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published 29 Jun, 2020

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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 it’s control mainly relies on the use of biocides. Data on the specie’s resistance status is essential for efficient and sustainable control.

Methods: We investigated the insecticide resistance status of several Ae. albopictus populations from Greece. Bioassays were performed against diflubenzuron (DFB), B. thuringiensis var. israelensis (Bti), deltamethrin and malathion. Molecular analysis of known insecticide resistance loci was performed, 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. VGSC analysis revealed a widespread distribution of mutations F1534C (in all populations, with allelic frequencies between 6.6% - 68.3%), and I1532T (in 6 populations), but absence of V1016G. CCE gene amplifications were recorded in 8 out of 11 populations. Co-presence of mutation F1534C and CCEae3a amplification was reported in a subgroup of samples. No mutations at the CHS locus I1043 were detected.

Conclusions: The results indicate: (i) the suitability of larvicides DFB and Bti for Ae. albopictus control in Greece, (ii) a possible incipient pyrethroid resistance due to the presence of kdr mutations and (iii) a 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 programs. Key words: diagnostic, arbovirus, mosquito tiger, insecticide resistance, vector control, Europe

Parasitology

diagnostic

arbovirus

mosquito tiger

insecticide resistance

vector control

Europe

In the last two decades southern European countries have suffered from a number of vector-borne diseases (VBDs), such as Chikungunya (CHIKV), Dengue (DENV) and West Nile virus (WNV) outbreaks [1, 2]. This has been attributed to several factors including the introduction and establishment of invasive vector species, associated with the ongoing environmental and climate change and the globalization of human activities [3].

Aedes albopictus is an important vector and invasive mosquito species. Originating from the tropical and subtropical regions of southern Asia, it currently displays a worldwide distribution [4] facilitated by its ecological plasticity, competitive behavior and egg diapause during the winter period [5]. In Europe, Ae. albopictus was first recorded in Albania in 1979 [6] and to date the species is well-established in the Mediterranean region and also reported in northern European countries [7]. Being a competent vector of more than 22 arboviruses, including CHIKV and DENV [8], Ae. albopictus constitutes a major public health threat. Its presence in Europe has been associated with the local transmission of CHIKV in Italy (2007, 2017) and southeastern France (2010, 2014) and of DENV in France (2010, 2013 -2015) and Croatia (2010) [1]. Furthermore, since 2016 more than 2,400 imported Zika cases have been reported in Europe [9, 10], which in conjunction with Ae. albopictus known distribution and Zika vectorial capacity raises concerns for potential autochthonous Zika transmission [11].

Ae. albopictus has also been reported in Greece [12], a country witnessing consecutive WNV outbreaks since 2010, with the largest outbreak in 2018 resulting in a total of 316 human cases and 50 fatalities [13]. Although Culex pipiens has been implicated as the primary WNV vector in Greece [14], Ae. albopictus could potentially have a role in disease transmission, as a competent vector [15]. Apart from a major public health concern, Ae. albopictus causes a significant biting nuisance problem, negatively affecting the quality of life and potentially acting as a discouraging factor for tourist economy [16].

The prevention of mosquito-borne diseases (MBDs) relies to a great extent on vector control [17], mainly through the utilization of synthetic insecticides [18]. In Europe, pyrethroids (e.g. permethrin, deltamethrin, alpha-cypermethrin) and the larvicides diflubenzuron (DFB) and pyriproxyfen are the main insecticides used to control Aedes adults and immature stages, respectively [18, 19]. In Greece, applications of B thuringiensis israelensis (Bti) and of the insect growth regulator (IGR) DFB compose the majority of the vector control interventions implemented in large-scale (regional level) control programs. Pyrethroid insecticides, both type II (such as deltamethirn) and type I (such as permethrin and alpha-cypermethrin) formulations are registered and used in professional vector control programs and for personal protection/ household level applications [20]. A major issue associated with the intensive use of a limited number of insecticides in mosquito and agricultural pest control is the development of insecticide resistance [18]. Insecticide resistance has been reported in Ae. albopictus, yet a big knowledge gap remains in regards to the susceptibility status, geographical distribution, frequency and co-occurrence of resistance traits and underlying mechanisms in the vector populations. Bioassay experiments have recorded resistance to several insecticides including pyrethroids, DDT, temephos, malathion, etc. mainly in rural and urban central and southern Asian populations (e.g. from China, Thailand, Singapore, India), while only sporadical cases of resistance have been reported in Europe, America and western Africa [18].

The two insecticide resistance mechanisms reported so far in Aedes mosquitoes are a) target-site resistance, involving mutations at the insecticide’s target site of action and b) metabolic detoxification, through overexpression or conformational changes of enzymes involved in the metabolism of the insecticide [18, 21]. To date, 5 mutations in 2 loci of the voltage-gated sodium channel (VGSC) gene; V1016G (valine to glycine), I1532T (isoleucine to threonine) and F1534C/L/S (phenylalanine to cysteine/ leucine/ serine), have been reported in Ae. albopictus populations from central and southeastern Asia, European Mediterranean countries, the USA and Brazil [18].

The involvement of all these five mutations in insecticide resistance has been further examined by expressing the mutated VGSC channels in Xenopus eggs and investigating how their electrophysiological properties are affected in the presence of insecticides. All three mutations at position F1534 (F1534C/L and S), as well as the I1532T mutation have been shown to significantly reduce significantly the channel’s sensitivity to type I pyrethroids, but not to type II pyrethroids [22, 23]. Mutation V1016G mutation also reduces the channel’s sensitivity mainly to permethrin and slightly to deltamethrin [22]. However, a synergistic effect has been reported in the presence of the triple mutant V1016G + F1534C+ S989P (a third mutation identified in Ae. aegypti populations), which substantially reduced sensitivity to both permethrin and deltamethrin [24].

Pyrethroid resistance has also been correlated to CYP6P12 over-expression conferring increased metabolic detoxification [25]. Metabolic resistance to the larvicide temephos (OP) has been functionally associated with the up-regulation of carboxylesterases CCEae3a and CCEae6a, due to gene amplification [26]. Ae. albopictus populations from Greece and Florida (USA) have been found to carry this CCEae3a gene amplification or the CCEae3a-CCEae6a co-amplification [27]. Concerning alternative insecticides, such as IGRs or Bti, no genotypic resistance data have been reported for Aedes mosquitoes, but three point mutations I1043M/L/F in the chitin synthase gene 1 (CHS-1) were identified in Cx. pipiens mosquitoes, conferring very high levels of resistance to the larvicide DFB [28- 30].

The re-appearance of VBDs in Europe, the widespread distribution of Ae. albopictus in southern Europe, the sporadic information on the vectors’ insecticide resistance status and the need for evidence-based mosquito control programs acting in advance of new outbreaks, make the monitoring and analysis of the Ae. albopictus insecticide resistance traits a necessity [31]. Here, we analysed the insecticide resistance status in a number of Ae. albopictus populations from Greece, using bioassays and molecular genotyping assays targeting known resistance markers.

1. Study sites, sample collections and mosquito handling

Adults and immature stages of Ae. albopictus were collected during the summer of 2017, 2018 and 2019, in a total of 19 urban and peri-urban localities in Greece, in the regions of Thessaloniki [32] and Rodopi (Northern Greece), Attica and Argolida (Central Greece), the Island of Chios (North Eastern Aegean Islands complex) [32], Patra and Kalamata (Western Greece), the Island of Kefalonia (Ionian Islands complex) and Crete (Chania, Rethymno and Heraklion-Southern Greece) (Table 1).

Adult specimens were collected with aspiration catches and CDC-light traps baited with dry ice. Larvae were sampled with dipping collections and eggs were collected with oviposition traps (black plastic cup with 2 wooden tongue depressors as ovipositional substrate). Both larvae and eggs were collected from at least five different sites within each locality in order to minimize the probability of including isofemale mosquitoes in the molecular analyses.

Following ovitrap collections, eggs were reared to adults in standard insectary conditions (temperature 27± 2^oC and relative humidity 70-80%), identified morphologically to species [33] and stored in ethanol at -4^oC for the subsequent molecular analysis. A subgroup of eggs from Aghios Stefanos (region of Attica), Kefalonia, Patras and Heraklion were reared to larvae or adults to use for susceptibility bioassays, as described below.

2. Genomic DNA extraction and molecular identification of mosquito species

Genomic DNA (gDNA) was extracted from individual larvae or adult mosquitoes and from pools of eggs (10 eggs per pool per locality; 3 localities), using DNazol reagent according to manufacturer’s instructions (Invitrogen, Carlsbad, CA).

Species identification was based on the PCR amplification (KAPA Taq PCR Kit) of the nuclear ribosomal spacer gene ITS2 [34] (primers 5.8S, 28S; Additional file 1: Table S1), following an assay that discriminates between Ae. albopictus, Ae. cretinus and Ae. aegypti, by generating PCR products of 509 bp, 385 bp and 324 bp in length, respectively. The applied thermal protocol was the following: initial denaturation at 94^oC for 10 min, 40 cycles x [denaturation at 94^oC for 1 min, primer annealing at 52^oC for 1 min, primer extension at 72^oC for 1 min] and final extension at 72^oC for 10 min. The PCR products were electrophoresed on a 1.5% w/v agarose gel containing ethidium bromide.

3. Insecticide susceptibility bioassays

a) Larval bioassays

Following the WHO guidelines for laboratory and field testing of mosquito larvicides [35], we examined the susceptibility of Ae. albopictus populations to two larvicides; the bacterial larvicide Bti (VectoBac 1200 ITU (International Toxin Units)/ mg; 11,61% w/v) and the insect growth regulator DFB (DU-DIM 15SC; 15% w/v). Both insecticides were diluted in distilled water. Bioassays were performed using Ae. albopictus third-early fourth instar larvae (F_0-F₁ generation), reared under standard insectary conditions (temperature 27±2^oC and relative humidity 70-80%). Fifteen to twenty larvae were placed in 99 ml water, to which 1 ml of the insecticide solution was added. A range of five to nine concentrations was tested for each insecticide (Bti: 0,008 - 0 ,50 mg/L; DFB: 0,0004 – 0,02 mg/L) in order to define a mortality range between 10 and 95% and determine the LC₅₀and LC₉₅ values. At least three replicates were prepared for each concentration. Larval mortality was recorded after the WHO recommended exposure time for each insecticide. Moribund larvae were counted as dead [35]. Results were compared to the LC₅₀and LC₉₅ values reported for susceptible-laboratory Ae. albopictus strains reported in other studies [36, 37, 38].

b) Adult bioassays

Three to five days old, non-blood fed female mosquitoes were subjected to insecticide susceptibility tests against deltamethrin and malathion, following the CDC Bottle Bioassay Guidelines [39]. A standard Ae. albopictus susceptible laboratory strain was included [40]. Insecticide stock solutions in acetone were prepared and Wheaton bottles were cleaned and coated as described in the CDC guidelines. The diagnostic dose of the insecticide under evaluation was used: Deltamethrin 10 ug/bottle and Malathion 50 ug/bottle. Tests were performed using 20-25 mosquitoes per bottle. Four insecticide treated replicate bottles and at least one control bottle (coated with acetone only) were used in each experiment set. The diagnostic time for both insecticides tested is 30 minutes [39]. Alive and dead mosquitoes in each bottle were recorded at time intervals of 5-15 minutes. Mortality rate at the diagnostic time was calculated to determine the insecticide susceptibility status, according to CDC recommendations.

4. Genotyping of target site resistance mutations

a) Detection of knock-down resistance (kdr) mutations in the VGSC gene

The VGSC domain I was investigated for the presence of the V1016G mutation and domain III for mutations I1532T and F1534L/S/C via PCR and product sequencing.

The PCR (KAPA Taq PCR Kit) for domain II was carried out in 25 ul containing: 1.5 ul of mixed gDNA extracted from 5-8 Ae. albopictus individuals of the same locality, 10X DNA polymerase Buffer, 0.4 uM of each primer (primers kdr2F, kdr2R; Additional file 1: Table S1), 0.4 uM of dNTPs and 1.5 U of Taq polymerase. The PCR thermal conditions were: initial denaturation at 95^oC for 3 min, 40 cycles x [denaturation at 94^oC for 30 sec, primer annealing at 55^oC for 30 sec, primer extension at 72^oC for 1 min] and final extension at 72^oC for 5 min. A small amount of the PCR products (5 ul) was electrophoresed on a 1% w/v agarose gel to verify the presence of the correct size amplicon (500bp), and the remaining amount was purified using the Nucleospin PCR & Gel Clean-Up Kit and sequenced using primer kdr2F.

The PCR for domain III was carried out in 25 ul containing: 1.5 ul of gDNA from Ae. albopictus individuals, 10X DNA polymerase Buffer, 2 mM MgCl₂, 0.3 uM of each primer (primers kdr3F, kdr3R; Additional file 1: Table S1), 0.4 uM of dNTPs and 1.5 U of Taq polymerase. The thermal conditions of the PCR were: initial denaturation at 95^oC for 3 min, 40 cycles x [denaturation at 94^oC for 30 sec, primer annealing at 57^oC for 30 sec, primer extension at 72^oC for 1 min] and final extension at 72^oC for 5 min. The products were electrophoresed on a 1% w/v agarose gel and the specific 740 bp band was gel extracted and purified using the Nucleospin PCR & Gel Clean-Up Kit and sequenced with primer kdr3Rin.

b) Analysis of the CHS locus 1043

Analysis of the CHS1 locus I1043, to identify possible conserved DFB resistance mutations that have been found in other species [28, 30] was performed in pools of mixed gDNA extracted from 5 to 8 Ae. albopictus individuals of the same locality. Available Ae. albopictus DNA samples from other countries [27] were included in the analysis and genotyped individually. A 350bp fragment of the Ae. albopictus chitin synthase gene, spanning the locus 1043 (numbering based on Musca domestica genomic sequence) was amplified in a 25 ul PCR (KAPA Taq PCR Kit) containing 1.5 ul DNA, 10X DNA polymerase Buffer, 0.4 uM of each primer (primers kkv F3, kkv R3; Additional file 1: Table S1), 0.4 uM of dNTPs and 1.5 U of Taq polymerase. The thermal conditions were: initial denaturation at 95^oC for 5 min, 40 cycles x [denaturation at 94^oC for 30 sec, primer annealing at 55^oC for 30 sec, primer extension at 72^oC for 1 min] and final extension at 72^oC for 10 min. A small amount of the PCR products was electrophoresed on a 1.5% w/v agarose gel containing ethidium bromide to verify amplification. The remaining amount of the PCR products was purified using the Nucleospin PCR & Gel Clean-Up Kit (Macherey Nagel, Dueren, Germany) and sequenced using the kkv F3 primer.

5. Metabolic resistance: detection of esterase gene amplification

CCEae3a and CCEae6a gene copy numbers were determined using quantitative real-time PCR on individual Ae. albopictus specimens. Amplification reactions at a 10ul final volume were performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) containing 0.5 ul of gDNA, 0.2 uM of each primer (CCEae3aF, CCEae3aR and CCEae6aR, CCEEae6aF; Grigoraki et al., 2017; Additional file 1: Table S1) and SYBR Select Master Mix (Applied Biosystems, Thermo Fischer Scientific). Histone 3 (NCBI: XM_019696438.1) was used as a reference gene for normalization (primers His3 TaqF, His3 TaqR; Additional file 1: Table S1). The thermal parameters were: 50^oC for 2 min, 95^oC for 2 min and 40 cycles x [95^oC for 3 sec, 60^oC for 30 sec]. Melting curves were performed for reference and target genes to verify the presence of a unique specific PCR product, which was also checked on a 1% w/v agarose gel. A no-template control was included to detect possible contamination. Two replicates per sample were included. CCEae3a and CCEae6a gene copy numbers were estimated relatively to a temephos susceptible Αe. albopictus lab-strain from Greece.

Molecular identification of mosquito species

A total of 482 individual mosquitoes (larvae and adults) and 30 eggs (in pools) were identified to species by PCR discrimination; of the ITS2 genomic sequence length; 97.6% of the samples were identified as Ae. albopictus, while only 12 specimens, all from Chania-Crete, were identified as Ae. cretinus (corresponding to 17.4% of the Chania population) (Table 1). No Ae. aegypti mosquitoes were recorded.

Insecticide susceptibility bioassays

a) Larval bioassays

Ae. albopictus populations from Aghios Stefanos- Attica (ATT), Patras and Heraklion, were tested for Bti resistance and found to be susceptible (Figure 1). The calculated LC₅₀values were below 0.20 mg/L (corresponding to less than 0.240 ITU/mL) for all populations (Table 2), which is less than the LC₅₀ values reported for susceptible Ae. albopictus laboratory strains in other studies. Indicatively, in Li et al., 2018 [37] and Su et al., 2019 [38] the reported LC₅₀ values of the control susceptible strains were 0.036 mg/L and 0.044 mg/L, respectively (both corresponding to more than 0.240 ITU/ mL).

All three populations were also tested for diflubenzuron resistance and showed mortality of 100% in DFB doses below 0.02 mg/L. This is significantly lower than the recommended field doses (DU-DIM 15SC: 0.32 - 0.63 mg/L), the recommended WHO dosage of DFB in potable water containers (0.25 mg/L) [41], as well as the emergence inhibition dose (EI₅₀ of 0.376 mg/L) previously reported for susceptible Ae. albopictus field strains [36].

b) Adult bioassays

Ae. albopictus populations from Aghios Stefanos- Attica (ATT), Kefalonia, Patras and Heraklion were susceptible to deltamethrin (Figure 1), as the mortality recorded at the 30 min diagnostic time was 100% (Table 3, Additional file 2: Figure S1). On the other hand, populations from Aghios Stefanos-Attica (ATT), Kefalonia and Heraklion, were resistant to malathion, displaying approximately 55% mortality at the diagnostic time (30 min), while the Patras population had a mortality of 91.4%, indicating possible resistance (Table 3, Additional file 2: Figure S1).

Genotyping of target site resistance mutations

a) VGSC: kdr mutations at positions V1016, I1532 and F1534.

Genotyping of the VGSC domain II was performed for a total of 323 larvae/adult mosquitoes (in pools of 5-7 individuals) and 20 eggs (in pools of 10). The wild type allele V1016 (codon GTA) was recorded in all cases (Figure 1, Table 4).

For the detection of mutations I1532T and F1534C/L/S in the VGSC domain III, 319 individuals were genotyped. For the first time in Greece, mutation I1532T was detected in 6 out of the 11 surveyed regions. Particularly, 23 genotyped specimens from Rodopi, Thessaloniki, Patras, Argolida, Rethymno and Chania were found to have this substitution, mostly in heterozygosis (genotype 1532I/ 1532T). In the majority of the above-mentioned regions, the mutant allele 1532T frequency was low, varying from 1.7 to 6.5% (Figure 1). The highest frequency was observed in Patra (where the only 2 homozygotes 1532T/1532T were reported) (Table 4).

Mutation F1534C was found in all regions. The regions with the highest 1534C allele frequency were: Attica (overall allele frequency: 68.3%), Argolida (45.2%), Rethymno (48.3%), Heraklion (44.3%) and Chania (29%), all located in Central and Southern Greece (we excluded Kalamata results due to the small number of specimens collected and analyzed). In contrast, regions from Northern Greece (Rodopi and Thessaloniki) and Western Greece (Patra and Kefalonia) displayed lower 1534C allele frequencies, ranging between 6.6 and 16.7% (Figure 1). The F1534C mutation appeared mainly in heterozygosis, with the exception of Athens, where more than half of the genotyped specimens (51.6%) were homozygous for the mutation (genotype 1543C/1534C) (Table 4). Only two individuals (sampled from Argolida and Patra) harbouring both mutations, I1532T and F1534C in heterozygosis (genotype 1532I/1532T - 1534F/1534C), were recorded.

b) Chitin Synthase (CHS-1): mutations at position I1043

The CHS-1 genomic sequence of 325 Ae. albopictus mosquitoes (larvae and adults in pools of 5-7 individuals) and a total of 20 eggs (in pools of 10) were analyzed for the presence of either of the three mutations I1043L/M/F, previously linked to DFB resistance. No mutations were detected (Figure 1, Table S2). Likewise no mutations were recorded in the 178 genotyped samples from USA, Brazil, Belize, Gabon, Switzerland, Taiwan, France, Mexico, China, Sri Lanka, Australia, Japan, Lebanon and Bangladesh (Table S3).

Esterase gene amplification

CCEae3a and CCEae6a amplification associated with temephos resistance was recorded in 8 out of the 11 surveyed regions in Greece. Two types of amplification were found: a CCEae3a amplicon and a CCEae3a - CCEae6a co-amplicon.

Amplified esterase genes were detected in specimens from Chios, Argolida, Patra, Kalamata, Attica, Chania, Rethymno and Heraklion. The CCeae3a amplicon frequency reported in the eight locations (i.e frequency of specimens harbouring the amplicon), ranged from 16.6 to 84 % and that of CCEae3a-CCEae6a amplicon from 5 to 80 %. The majority of samples with amplified esterases harboured between 2-10 gene copies. Individuals with more than 10 (11-20) and more than 20 gene copies (in the case of one individual from Rethymno) were also recorded. Attica was the region with the highest percentage of individuals carrying ≥2 copies of either CCEae3a or CCEae6a gene (84% and 80%, respectively) and Chios of individuals with >10 copies (25% and 33.3%, respectively) (Figure 2).

No carboxylesterase gene amplification was detected in Rodopi, Thessaloniki and Kefalonia populations (Figure 1).

This study represents an extended investigation of the insecticide resistance status of Aedes albopictus in Greece. Entomological monitoring revealed the dominant presence of Ae. albopictus over other Aedes container-breeding species and confirmed the species’ widespread distribution in the country [12, 42]. The discrimination of Ae. albopictus from morphologically similar Stegomyia species using the nuclear ITS2 as a molecular marker [34] is of high importance in order to distinguish this species from the primary arbovirus vector Aedes aegypti [43] and the non-vector Aedes cretinus [44]. The latter, although considered a native mosquito species in Greece [45], that had been extensively reported in the past in Attica and Crete [42], was detected in our study only in Chania, Crete corresponding to an overall frequency of 2.4%. The absence/very low Ae. cretinus frequency in urban and peri- urban settings of the country may be correlated to the competitive and highly adaptable behavior displayed by Ae. albopictus [46].

The Ae. albopictus WHO and CDC bioassays showed full susceptibility of the tested populations against deltamethrin and the larvicides DFB and Bti, suggesting their current suitability for the vector control programs in Greece. On the contrary, mortality levels < 90 % were recorded in the malathion assays indicating resistance against this OP insecticide. Although malathion is not currently used in Greece, the observed resistance raises concerns, regarding the potential need for re-introducing OP insecticides used in the past.

Genotyping of the VGSC gene for the detection of mutations associated with pyrethroid resistance revealed a widespread distribution of the kdr mutation F1534C across the country, following initial reports of the mutation in Greece [47, 48]. The recorded 1534C allele frequencies raised above 40% in populations from southern and central Greece, peaking in Attica (68.3%). Previous studies also reported relatively high mutation frequencies (reaching 66%) in populations sampled from the Attica region [47, 48]. To date, kdr F1534C has also been recorded in Brazil [49], Singapore, Vietnam [50] and China [51], while other substitutions in the same genomic locus, F1534L and F1534S, also associated with pyrethroid resistance, have been identified in the USA, China, Vietnam and Italy [47, 50- 52].

The involvement of both F1534L and F1534S mutations in resistance to the type I pyrethoids was recently further supported by in vitro functional characterization data [23]. The F1534L substitution was shown to confer similar levels of insensitivity to the previously characterized F1534C, while F1534S seemed to have an even bigger effect. Thus, monitoring the presence of these mutations in countries, like Greece, were type I pyrethroids are used for vector control is of great importance.

Mutation F1534C in the homozygous state has been associated with resistance to permethrin [22] in Aedes mosquitoes. In the analyzed populations, F1534C was observed mainly in heterozygosis, potentially accounting for low resistance levels, due to the recessive nature Nevertheless, the recorded mutation frequencies should not be undermined, as a notable raise of insecticidal pressure may lead to rapid increase in mutation selection potentially hampering the effectiveness of permethrin- based applications for Ae. albopictus control. Additionally, the investigation of P450s-mediated pyrethroid detoxification, which in combination with target-site mutations has been shown to confer operationally significant resistance levels [54], would be critical complementary evidence facilitating the development of efficient control programs.

Another mutation in the VGSC gene, I1532T, was found in Greece at low frequencies (< 10%) in several surveyed regions with the exception of Patras (western Greece) where the mutant allele reached a frequency of 22.7%. This mutation has been also monitored and reported at considerable frequencies in Ae. albopictus populations from Italy [47, 48], Albania [48] and China [51]. Although Kasai et al., 2019 [50] demonstrated a lack of association between this mutation and resistance to pyrethroids, a recent study revealed that VGSC channels carrying this mutation have reduced sensitivity to type I pyrethoids, notably at similar levels to channels harbouring the mutation F1534S [23]. Therefore, it is important to monitor the presence and distribution of this mutation in field populations. We also identified in our samples a small number of individuals carrying both the I1532T and F1534C mutations. In some cases, combination of mutations can have an additive or even synergistic effect resulting in very high levels of resistance [21]. Although the combination of I1532T+F1534C has not been functionally characterized yet, the combinations of I1532T with F1534S and L have been recently shown to have a similar effect to the single mutants in conferring insensitivity to type I pyrethroids [23].

The kdr mutation V1016G, correlated with “stronger” resistance (than F1534C) to type I and type II pyrethroids [22, 50] was not detected in any of the analyzed samples from Greece. However, it was recently recorded in Ae. albopictus populations from Hanoi-Vietnam, in Beijing-China and across Italy [48, 50, 55]. The systematic monitoring of this mutation in Ae. albopictus populations from Greece and elsewhere is strongly recommended, given the primary role of pyrethroids in Aedes mosquito control worldwide [18] and the possibility of low mutation frequencies and/or focal mutation distribution going undetected.

Resistance to DFB was assessed molecularly in specimens from all 11 surveyed regions in Greece through monitoring of the substitutions I1043L/M/F in the CHS gene, associated with high resistance levels in Cx. pipiens mosquitoes [28- 30]. No CHS mutations were recorded, a result in line with the respective bioassay outcome, indicating the effectiveness of DFB for Ae. albopictus control in the study regions. Likewise all samples analyzed from France, Switzerland, Lebanon, Belize, Gabon, Taiwan, Sri Lanka, China, Japan, Australia, Bangladesh, USA, Brazil and Mexico were homozygous for the wild type (susceptible) allele I1043. Taking into account the highly focal pattern of DFB resistance reported in Cx. pipiens [29], the non detection of resistant alleles in the sampled Aedes populations does not imply their absence from other populations in the region, emphasizing the need for incorporating systematic monitoring of DFB resistant mutations in integrated Ae. albopictus surveillance programs.

Carboxylesterase CCEae3a and CCEae6a gene amplifications linked to temephos resistance [26, 27] were detected in 8 out of 11 surveyed regions. Almost 35% of the total Ae. albopictus specimens analyzed, the majority of which were sampled from central and southern Greece, carried more than 1 copy of CCEae3a or both CCEae3a – CCEae6a genes indicating elevated temephos detoxification.

Interestingly out of the 156 Ae. albopictus samples commonly analyzed for the detection of the kdr mutation F1534C and CCEae3a amplification, 25% were found to harbor both mutations, denoting a potential risk for multi-resistance against pyrethroid and organophosphate insecticides.

The observed resistance against malathion may be associated with the high occurrence of amplified CCEs in the analyzed populations. CCEae3a and CCEae6a gene amplification was originally associated with temephos resistance in Ae. albopictus mosquitoes [26], yet Marcombe et al., 2019 [56] also correlated the presence of multiple CCEae3a gene copies with malathion resistance in Ae. aegypti mosquitoes. Thus, it is possible that the detected CCEs amplifications in the Greek Ae. albopictus populations are responsible for the resistance levels recorded in the malathion bioassays, by conferring cross -resistance across additional OPs.

Evident differences were noted in the frequencies of the kdr mutations F1534C, between populations from Thessaloniki (northern Greece; mutant allele F1534C frequency <7%) and sampling locations in central and southern Greece (mutant allele F1534C frequencies > 29%) which may partially reflect the imposed insecticidal pressures. Similarly, in Thessaloniki CCEs gene amplification was not detected in any sample, while 26-84% of the Attica, Argolida, Chania, Rethymno and Heraklion populations has more than 1 CCEae3a gene copy. The latter regions of central and southern Greece have intense agricultural activities, where pyrethroids and OPs have been used extensively for pest control over the years, possibly contaminating nearby Ae. albopictus breeding sites and leading to the selection of the respective resistance mutations [57]. On the contrary, in Thessaloniki the majority of the specimens were sampled from an organic farm (Litsa-Thermi) where no insecticides have been applied over the last 10 years possibly explaining the low kdr mutation frequency and the absence of CCEs amplification.

The limited number of approved insecticides for public health purposes and the worldwide data on insecticide resistance generated to date raise concerns regarding the effectiveness and suitability of the current vector control interventions. Our findings significantly enrich the available information on Ae. albopictus insecticide resistance status. In a context of evidence-based vector control programs, regular investigation of the species composition, population dynamics and systematic resistance monitoring encompassing both bioassays and molecular diagnostics (highly complementary yet not mutually exclusive tools) are critical components for the development of resistance management strategies.

This study delineates the susceptibility profile of Aedes albopictus populations from Greece against commonly used insecticides. Our findings suggest the suitability of the larvicides DFB and Bti and the current effectiveness of pyrethroids for Ae. albopictus control. However, the presence of high kdr mutation frequencies raises concerns, given the dominant role of pyrethroids in mosquito control and the few alternative synthetic compounds available for public health purposes. The OPs malathion and temephos appear unreliable alternative insecticides for possible future re-introduction in mosquito control, as resistance against malathion was detected in the country. Systematic insecticide resistance monitoring is imperative for the development of insecticide resistance management programs ensuring the sustainability of the current chemical control tools.

Bti: Bacillus thuringiensis var. israelensis; CCE: Carxoxylesterase; (E)CDC: (European) Centre for Disease Control; CHIKV: Chikungunya virus; CHS-1: Chitin synthase 1; CI: Confidence intervals; CYP: Cytochrome P450 monooxygenase; DDT: Dichlorodiphenyl- trichloroethane; DENV: Dengue virus; df: degrees of freedom; DFB: Diflubenzuron; EI: Emergence inhibition; GCN: Gene copy number; gDNA: Genomic DNA; IGR: Insect growth regulator; ITS2: Internal transcribed spacer 2; ITU: International toxic units; LC: Lethal concentration; MBDs: Mosquito-borne diseases; OP: organophosphate; PCR: Polymerase chain reaction; SD: Standard deviation; VBDs: Vector-borne diseases; VGSC: Voltage-gated sodium channel; WHO: World Health Organization; WNV: West Nile virus

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare that they have no competing interests

Funding

This work was partly supported by the General Secretariat for Research and Technology (GSRT) and the Hellenic Foundation for Research and Innovation (HFRI) in the context of the action “1st Proclamation of Scholarships from ELIDEK for PhD Candidates”.

Authors' contributions

SB conducted the majority of molecular and bioassay analyses and made substantial contributions to the conception and design of the work, analysis and interpretation of the data and manuscript preparation. E.A.F conducted molecular and bioassay analyses and contributed to the design of the work, interpretation of the data generated and manuscript preparation. IK made substantial contributions in the sample collections and bioassay experiments. LG contributed in the molecular analyses and manuscript preparation. SM provided samples for analysis. AC provided samples for analysis and contributed in data interpretation and manuscript revisions. JV made substantial contributions to the design of the work, interpretation of data and substantively revised the work/manuscript. All authors have approved the submitted version

Acknowledgements

Not applicable

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Table 1: Aedes species composition per study site.

Collection date

Species identification

Region

Study sites/Localities

Ae. albopictus

Ae. cretinus

Rodopi

Iasmos

August 2018

Thessaloniki

Thermi- Litsa^

Refugee Camp Diavata^¥

Refugee Camp Lagkadikia^¥

September 2018

June 2017

10*

18*

10*

18*

Chios

Refugee Camp Souda^¥

June 2017

23*

Attica

Aigaleo

Filothei

Aghios Stefanos

Aghios Eleftherios

August-September 2018

October 2018

Kefalonia

Paliki

November 2019

Patras

Rio

September 2019

Argolida

Aghia Triada

Kilada

August-September 2018

September 2018

Kalamata

Town

August 2018

Chania

Town

Souda

September- October 2018

August- October 2018

Rethymno

Town

Panormos

August 2018

August- September 2018

Heraklion

Giofyro

June-July 2019

N: total number of specimens analyzed per sampling region for species identification. *: includes 10 eggs analyzed as a pool. ^Thermi –Litsa is an organic farming locality. ^¥Samples from Diavata, Lagkadikia and Souda refugee camps were collected and analysed in Fotakis et al., 2020 [32].

Table 2: WHO bioassay mortalities for Ae. albopictus populations tested against Bti.

	Population	N	LC₅₀ (95% CI)			LC₉₅ (95% CI)		Slope ± SE	χ²	df
*Bti*	Population	N	mg/ L		ITU/ mL	mg/ L	ITU/ mL	Slope ± SE	χ²	df
1200 ITU/ mg	Patras	250	0.130 (0.08-0.171)	0.156 (0.096-0.205)		0.356 (0.250-0.994)	0.427 (0.300-1.193)	3.76 ± 0.45	113.3	23
	A. Stefanos- ATT	301	0.195 (0.156-0.235)	0.234 (0.187-0.282)		0.465 (0.360-0.736)	0.547 (0.432-0.883)	4.39 ± 0.41	88.59	28
	Heraklion	243	0.145 (0.113-0.179)	0.174 (0.136-0.215)		0.383 (0.276-0.874)	0.459 (0.331-1.049)	3.90 ± 0.49	75.46	23
7000 ITU/ mg 7000 ITU/ mg	Susceptible lab strains
	Li et al., 2018		0.036 (0.028-0.047)	0.252 (0.196-0.329)
	Su et al., 2019		0.044 (0.040-0.050)	0.308 (0.280-0.350)

Log-dose probit-mortality data for larvicides tested against Ae. albopictus larvae. The results are compared to the susceptible laboratory Ae. albopictus control strains of other studies [37, 38]. N: total number of larvae tested to a range of insecticide concentrations (4 replicates per concentration). LC₅₀, LC₉₅: lethal concentration (mg/ L) that kills 50% and 95% of the population, respectively. CI: Confidence Intervals. ITU: International Toxic Units; χ²: Chi-square testing linearity of dose-mortality response with degrees of freedom (df).

Table 3: CDC bottle bioassay mortalities for Ae. albopictus populations tested against deltamethrin (pyrethroid) and malathion (OP).

	Deltamethrin 10 ug/ml			Malathion 50 ug/ ml
Population	N₁	Mortality (%) ± SD	Status	N₂	Mortality (%) ± SD	Status
A. Stefanos- ATT	75	100	S	79	55.7 ± 25.8	R
Patras	70	100	S	83	91.4 ± 5.9	R*
Kefalonia	88	100	S	83	55.6 ± 40.5	R
Heraklion	78	100	S	78	55.3 ± 20.6	R
Susceptible lab strain	70	100	S	76	100	S

Mortality percentages ± SD (Standard Deviation) correspond to the discriminating exposure time DT=30 min for both insecticides [39]. N₁ and N₂ refer to the total number of female mosquitoes tested against deltamethrin and malathion, respectively. R: Resistance (*: possibility of resistance), S: susceptibility. According to WHO recommendations for CDC bottle bioassays, 98-100% mortality at the diagnostic time indicates susceptibility, 80-97% suggests the possibility of resistance that requires further confirmation and mortality <80% denotes resistance.

Table 4: Genotype and allele frequencies (%) of VGSC domain II locus V1016 and domain III loci I1532 and F1534.

		V1016G			I1532T						F1534C
	Collection Year		Genotype			Genotype			% allele freq		Genotype			% allele freq
Region	Collection Year	N₁	VV	N₂	II		IT	TT	(I)	(T)	FF	FC	CC	(F)	(C)
Rodopi	2018	13	13	12	11		1	0	95.8	4.2	8	4	0	83.3	16.7
Thessaloniki: Diavata, Lagkadikia^¥ Thermi- Litsa	2017 2018	18* 30	18* 30	8 30	8 28		0 2	0 0	100 96.7	0 3.3	7 26	1 4	0 0	93.8 93.3	6.2 6.7
Chios^¥	2017	22*	22*	11	11		0	0	100	0	6	4	1	72.7	27.3
Kefalonia	2019	45	45	41	41		0	0	100	0	37	1	3	91.5	8.5
Patras	2019	33	33	33	20		11	2	77.3	22.7	28	5	0	92.4	7.6
Argolida	2018	21	21	21	19		2	0	95.2	4.8	10	3	8	54.8	45.2
Kalamata	2018	7	7	6	6		0	0	100	0	2	3	1	58.3	41.7
Rethymno	2018	20	20	30	29		1	0	98.3	1.7	10	11	9	51.7	48.3
Chania	2018	31	31	31	27		4	0	93.5	6.5	15	14	2	71	29
Heraklion	2019	44	44	44	44		0	0	100	0	15	19	10	55.7	44.3
Attica:
Ag. Stefanos	2018	10	10	9	9		0	0	100	0	9	0	0	100	0
Ag. Eleftherios	2018	20	20	15	15		0	0	100	0	0	4	11	13.3	86.7
Aigaleo	2018	20	20	20	20		0	0	100	0	3	5	12	27.5	72.5
Filothei	2018	9	9	8	8		0	0	100	0	8	0	0	100	0
Overall^		59	59	52	52		0	0	100	0	12	9	31	31.7	68.3

N₁, N₂: total number of genotyped specimens for VGSC domain II and domain III respectively, per sampling region. *: Includes 10 eggs analysed in a pool. Results are presented cumulatively for the different sampling points in each region, except for Thessaloniki (¥: Samples from Diavata and Lagkadikia refugee camps and Chios were analysed in Fotakis et al., 2020 [32]) and Attica (^: overall results also given). VV: 1016V/1016V homozygous susceptible. Allele frequencies for VGSC locus 1016 are not given as the wild-type allele V1016 was detected in all samples. I: 1532I susceptible allele, T: 1532T mutant allele; II: 1532I/1532I homozygous, IT: 1532I/1532T heterozygote, TT: 1532T/1532T homozygous mutant. F: 1534F susceptible allele, C: 1534C mutant allele; FF: 1534F/1534F homozygous, FC: 1534F/1534C heterozygote, CC: 1534C/1534C homozygous mutant.

Additional file 1: Table S1. Primers used in this study for regular and real-time PCR. Table S2. Genotype and allele frequencies of the CHS-1 locus 1043, Greece. Table S3. Genotype and allele frequencies of the CHS-1 locus 1043, other countries. (DOCX 22kb)

Additional file 2: Figure S1. CDC bioassay mortality percentages corresponding to exposure time against malathion and deltamethrin for Aedes albopictus populations from Greece. (DOCX 22kb)

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Phenotypic and molecular analysis of insecticide resistance in Aedes albopictus populations from Greece

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