Long-term studies on the evolution of resistance of Myzus persicae (Hemiptera: Aphididae) to insecticides in Greece.

The aphid Myzus persicae s.l. (Hemiptera: Aphididae) is an important pest of many crops worldwide with a complex life cycle, intensely controlled by chemical pesticides, and has developed resistance to almost all used insecticides. In Greece, the aphid exhibits high genetic variation and adaptability and it is a classic example of evolution in the making. We have been studying M. persicae for over 20 years, on different host plants and varying geographical areas, analyzing its bio-ecology and the ability to develop resistance to insecticides. In this review, we present new and historical data on the effectiveness of insecticides from seven chemical groups used to control the aphid in Greece and the incidence of seven resistance mechanisms, including the new fast-spreading R81T point mutation of the postsynaptic nicotinic acetylcholine receptor. Thousands of samples were tested by biological, biochemical and molecular assays. The aphid populations were found to have developed and maintain resistance at medium to high levels to organophosphates, carbamates, pyrethroids and neonicotinoids for decades. In the latter group, a marked increase is recorded during an ~10-year period. The data analyzed and the extensive bibliography, advocate the difficulty to control the aphid making the design and application of IPM/IRM programs a challenge. We discuss principles and recommendations for the management of resistance, including the use of compounds such as flonicamid, spirotetramat, flupyradifurone and sulfoxaflor. We emphasize that resistance is a dynamic phenomenon, changing in time and space, requiring, therefore, continuous monitoring.

Toxicity and Influence of Sublethal Exposure to Sulfoxaflor on the Aphidophagous Predator Hippodamia variegata (Coleoptera: Coccinellidae).

Hippodamia variegata (Goeze), the variegated ladybug, is a predator of many insect pests, especially aphids. Sulfoxaflor is a chemical insecticide that can be used to control many sap-feeding insect pests, for instance, plant bugs and aphids, as an alternative to neonicotinoids in different crops. To improve the combination of the H. variegata and sulfoxaflor in an IPM (integrated pest management) program, we studied the ecological toxicity of the insecticide to the coccinellid predator at sublethal and lethal doses. We examined the influence of sulfoxaflor on larvae of H. variegata using exposure doses of 3, 6, 12, 24, 48 (maximum recommended field rate (MRFR)), and 96 ng a.i. per insect. In a 15-day toxicity test, we observed decreased adult emergence percentage and survival, as well as an increased hazard quotient. The LD50 (dose causing 50% mortality) of H. variegata due to sulfoxaflor decreased from 97.03 to 35.97 ng a.i. per insect. The total effect assessment indicated that sulfoxaflor could be grouped as slightly harmful for H. variegata. Additionally, most of the life table parameters were significantly decreased after exposure to sulfoxaflor. Overall, the results present a negative influence of sulfoxaflor on H. variegata when applied at the recommended field dose for controlling aphids in Greece, which demonstrates that this insecticide may only be employed with care when used in IPM programs.

Toxicity and Lethal Effect of Greenhouse Insecticides on Coccinella septempunctata (Coleoptera: Coccinellidae) as Biological Control Agent of Myzus persicae (Hemiptera: Aphididae).

Deltamethrin and imidacloprid are commonly used insecticides for controlling sub-sucking insects in greenhouses. However, their application may cause sublethal effects on the aphid coccinellid predator Coccinella septempunctata (Coleoptera: Coccinellidae). Here, we study (i) the toxicity and the effect of two sublethal doses (LD10 and LD30) of deltamethrin and imidacloprid on C. septempunctata in a laboratory microcosm and (ii) the residual toxicity of the two insecticides in a greenhouse. The results showed that both insecticides reduced fecundity, longevity, the intrinsic rate of increase, the finite rate of increase and the net reproductive rate. However, the developmental time of the fourth instar larvae was prolonged by both insecticides at LD10 and LD30. Deltamethrin residues were toxic 21 DAT (days after treatment) to C. septempunctata fourth instar larvae. In contrast, imidacloprid began in the slightly harmful category (75%) 1 DAT and declined to the harmless category (18.33%) 21 DAT. These results indicate that deltamethrin and imidacloprid have potential risks to C. septempunctata. This study provides information to guide the development of integrated pest management (IPM) strategies in greenhouses.

Toxicity, Sublethal and Low Dose Effects of Imidacloprid and Deltamethrin on the Aphidophagous Predator Ceratomegilla undecimnotata (Coleoptera: Coccinellidae).

Ceratomegilla undecimnotata (Coleoptera: Coccinellidae) is a common aphidophagous coccinellid predator used in biological control against aphid pests. Knowing toxicity, lethal, and sublethal effects of insecticides on natural enemies is essential in order to incorporate them into Integrated Pest Management (IPM). In the present study, the lethal and sublethal effects of imidacloprid and deltamethrin were evaluated on the fourth instar larvae of C. undecimnotata and subsequently on the full life cycle. Our results strongly suggest that sublethal and low doses of imidacloprid and deltamethrin at LD10 and LD30 affected fourth instar larvae duration time, adult preoviposition period, total preoviposition period, and fecundity. Moreover, the intrinsic (r) and finite (λ) rate of increase and the net reproduction rate (R0) significantly decreased in populations treated with imidacloprid compared to the control population. The data clearly suggest that imidacloprid and deltamethrin have a negative influence on population growth parameters of C. undecimnotata at sublethal and low doses and, therefore, these insecticides should be used with caution within the context of IPM.

Global patterns in genomic diversity underpinning the evolution of insecticide resistance in the aphid crop pest Myzus persicae.

The aphid Myzus persicae is a destructive agricultural pest that displays an exceptional ability to develop resistance to both natural and synthetic insecticides. To investigate the evolution of resistance in this species we generated a chromosome-scale genome assembly and living panel of >110 fully sequenced globally sampled clonal lines. Our analyses reveal a remarkable diversity of resistance mutations segregating in global populations of M. persicae. We show that the emergence and spread of these mechanisms is influenced by host-plant associations, uncovering the widespread co-option of a host-plant adaptation that also offers resistance against synthetic insecticides. We identify both the repeated evolution of independent resistance mutations at the same locus, and multiple instances of the evolution of novel resistance mechanisms against key insecticides. Our findings provide fundamental insights into the genomic responses of global insect populations to strong selective forces, and hold practical relevance for the control of pests and parasites.

Tracking the global dispersal of a cosmopolitan insect pest, the peach potato aphid.

BACKGROUND: Global commerce and human transportation are responsible for the range expansion of various insect pests such as the plant sucking aphids. High resolution DNA markers provide the opportunity to examine the genetic structure of aphid populations, identify aphid genotypes and infer their evolutionary history and routes of expansion which is of value in developing management strategies. One of the most widespread aphid species is the peach-potato aphid Myzus persicae, which is considered as a serious pest on various crops in many parts of the world. The present study examined the genetic variation of this aphid at a world scale and then related this to distribution patterns. In particular, 197 aphid parthenogenetic lineages from around the world were analysed with six microsatellite loci. RESULTS: Bayesian clustering and admixture analysis split the aphid genotypes into three genetic clusters: European M. persicae persicae, New Zealand M. persicae persicae and Global M. persicae nicotianae. This partition was supported by FST and genetic distance analyses. The results showed two further points, a possible connection between genotypes found in the UK and New Zealand and globalization of nicotianae associated with colonisation of regions where tobacco is not cultivated. In addition, we report the presence of geographically widespread clones and for the first time the presence of a nicotianae genotype in the Old and New World. Lastly, heterozygote deficiency was detected in some sexual and asexual populations. CONCLUSION: The study revealed important genetic variation among the aphid populations we examined and this was partitioned according to region and host-plant. Clonal selection and gene flow between sexual and asexual lineages are important factors shaping the genetic structure of the aphid populations. In addition, the results reflected the globalization of two subspecies of M. persicae with successful clones being spread at various scales throughout the world. A subspecies appears to result from direct selection on tobacco plants. This information highlights the ultimate ability of a polyphagous aphid species to generate and maintain ecologically successful gene combinations through clonal propagation and the role of human transportation and global commerce for expanding their range.

Lethal and sub-lethal effects of imidacloprid on the aphidophagous coccinellid hippodamia variegata.

Hippodamia variegata is one of the most abundant ladybird species in Greece, preying on several aphid species and other arthropods, of which many are pests of cultivated plants. Imidacloprid, a neonicotinoid insecticide, is commonly used for controlling sucking insects; at the same time, however, it can cause various sub-lethal effects on non-target organisms. The development of IPM programs against pests requires an evaluation of the side effects of insecticides on natural enemies. We evaluated the sub-lethal effects of imidacloprid on H. variegata. Our results demonstrate that imidacloprid at a LC10 (3.92 mg (a.i.) L-1 and LC30 (8.69 mg (a.i.) L-1) decreased adult longevity and survival rate. In addition, demographic parameters, such as the intrinsic rate of increase (r), finite rate of increase (λ) and net reproductive rate (R0), were reduced when exposed to sub-lethal concentrations of imidacloprid. These results demonstrate a negative influence of imidacloprid at sub-lethal concentrations on H. variegata which could reduce biological control services provided by this predator.

Efficacy of the pyrethroid alpha-cypermethrin against Bactrocera oleae populations from Greece, and improved diagnostic for an iAChE mutation.

BACKGROUND: The most important pest of olive orchards worldwide is the olive fruit fly Bactrocera oleae (Gmelin). Its control in Greece has been based on organophosphates (OPs), but their intense use has led to the development of resistance. A test previously developed to monitor the trait may not be as robust as originally thought. The pyrethroid alpha-cypermethrin has recently been registered for bait sprays, as an alternative to OPs. RESULTS: The susceptibility of 20 B. oleae populations to alpha-cypermethrin was examined. Variation was observed in their response, with LD(50) ranging from 0.14 to 3.28 ng insect(-1) and resistance factors from 2.3 to 54.7. Resistance mechanisms were investigated. Cytochrome P450 monoxygenase activities showed an association with resistance. Sequences in the domain IIS4-IIS6 of the B. oleae para-type sodium channel were also analysed, but no resistance-associated mutations were identified. Finally, a novel diagnostic assay able to reliably monitor the frequency of the iAChE G488S resistance mutation was developed. CONCLUSION: This is the first attempt to evaluate the efficacy of alpha-cypermethrin against B. oleae from Greece. Data showed that it can be used effectively, but also highlighted the importance of continuous monitoring. The IIS4-IIS6 sodium channel region is the default area in which to look for resistance mutations if target-site resistance to pyrethroids arises. The application of the novel iAChE molecular diagnostic may facilitate the introduction of pyrethroids alongside OPs currently in use.

Insecticide resistance status of Myzus persicae (Hemiptera: Aphididae) populations from peach and tobacco in mainland Greece.

The susceptibility of 88 and 38 field samples of Myzus persicae (Sulzer) to imidacloprid and deltamethrin respectively was examined using the FAO dip test bioassay. The field samples were collected from tobacco and peach from various regions of Greece in the period from 2004 to 2006. In addition, 497, 349 and 370 clones originating from peach and tobacco were screened for the three known resistance mechanisms, elevated esterases, modified acetylcholinesterase (MACE) and knockdown resistance (kdr) respectively, using biochemical and DNA diagnostics. Most of the samples assayed with imidacloprid showed low resistance factors (RFs)-39% below 5 and 21% between 5 and 10. However, 9% of the samples (all from tobacco) showed relatively high RF values (24-73). Differences were found between crops, with higher RF values recorded in samples from tobacco than in those from peach. Bioassays with deltamethrin revealed the development of strong resistance in the populations examined. The RFs were mostly higher than 23, and in 29% of the samples they were extremely high (152-436). Finally, the three known resistance mechanisms were found in high frequencies in the populations examined, although some differences between crops and years were detected. The implications of the study for management schemes against M. persicae are discussed.

Organophosphate resistance in olive fruit fly, Bactrocera oleae, populations in Greece and Cyprus.

The olive fruit fly Bactrocera oleae (Gmelin) (Diptera: Tephritidae) is the most important pest of olives in countries around the Mediterranean basin. Its control has been based mostly on bait sprays with organophosphate insecticides (usually dimethoate or fenthion) for about 40 years. In the present study, the resistance status of olive fruit fly populations to dimethoate was examined in Greece and Cyprus over 2 years. Thirty-one populations from various regions of Greece, nine from Cyprus and one laboratory susceptible strain, which served as a control, were assayed by topical application of dimethoate. Considerable variation in the resistance levels to dimethoate was recorded in the populations of B. oleae, with resistance ratios ranging from 6.3 to 64.4 (ED(50) values 12.5-128.7 ng dimethoate per insect). The highest resistance ratios were found in populations from Crete, and the lowest in those from Cyprus. This variation could be attributed to different selection pressures from insecticidal applications among populations from the various regions. Migration of resistant genotypes, either autonomous or via commerce, may also be involved.

Evolution of imidacloprid resistance in Myzus persicae in Greece and susceptibility data for spirotetramat.

BACKGROUND: Myzus persicae s.l. is a major crop pest globally and has evolved resistance to a range of insecticide classes making it increasingly difficult to control in some areas. Here we compare bioassay monitoring data for two important compounds, imidacloprid and spirotetramat, on field samples/clones collected in Greece. RESULTS: A total of 122 aphid samples/clones from central and northern Greece were examined in dose-response bioassays with imidacloprid. There was an overall increase in the level of resistance (resistance factor = 15-40) within tobacco-collected samples from 78.7% in 2007 to 86.7% in 2015. The corresponding frequencies for peach samples were 13.3% and 6.7%. These results were confounded however by the first identification of the R81T target mutation in Greece during 2015 (4.3% as heterozygotes in peach) and 2016 (21.3% as heterozygotes in peach). No resistance to spirotetramat was found at the 60 clones collected in 2015. CONCLUSION: Resistance to imidacloprid is continuing to increase within Greek M. persicae s.l. populations and the situation is likely to deteriorate further with the recent identification of the R81T resistance mutation. Resistance to spirotetramat has not been found and is therefore a good alternative to neonicotinoids for resistance management. © 2017 Society of Chemical Industry.

Transmission of Potato virus Y in Tobacco Plants by Myzus persicae nicotianae and M. persicae s.str.

The transmission efficiency of Potato virus Y N (PVYN) from and to tobacco plants by Myzus persicae nicotianae, a tobacco specialist, and M. persicae s.str. (Hemiptera: Aphididae), a generalist, was evaluated. In direct transmission tests, 1,004 spring migrants collected as nymphs from peach and 1,434 wingless females from 12 clonal lineages of both taxa were examined. Both the winged and wingless females were starved before being transferred individually for a 3-min acquisition period on infected plants and then moved to healthy plants for a 10-min inoculation access period. Little variation in transmission efficiency was observed. Although not statistically significant, M. persicae nicotianae was slightly more effective. This may suggest similar genetic properties (e.g., virion receptors in mouthparts) between the taxa in relation to virus transmission. M. persicae s.str. transmitted the virus less efficiently when a shorter inoculation period was given. This may indicate differences in probing behavior during the early phase of host selection. Nevertheless, behavioral differences related to host selection have been found using arena tests with infected and healthy tobacco plants where 180 winged females from each taxon were tested. M. persicae s.str. had significantly higher propensity to transmit PVYN than M. persicae nicotianae (31.9 versus 15.3%).

Insecticide resistance status of Myzus persicae in Greece: long-term surveys and new diagnostics for resistance mechanisms.

BACKGROUND: Myzus persicae nicotianae is an important pest in Greece, controlled mainly by neonicotinoids. Monitoring of the aphid populations for resistance mechanisms is essential for effective control. RESULTS: Two new RFLP-based diagnostics for the detection of the M918T (super-kdr pyrethroid resistance) and nAChR R81T (neonicotinoid resistance) mutations were applied, along with other established assays, on 131 nicotianae multilocus genotypes (MLGs) collected from tobacco and peach in Greece in 2012-2013. Furthermore, we present resistance data from aphid clones (>500, mainly nicotianae) collected in 2006-2007. About half of the clones tested with a diagnostic dose of imidacloprid were tolerant. The R81T mutation was not found in the 131 MLGs and 152 clones examined. Over half (58.6%) of a subset of 29 clones showed a 9-36-fold overexpression of CYP6CY3. M918T was found at low to moderate frequencies. The kdr and MACE mechanisms and carboxylesterase-based resistance were found at high frequency in all years. CONCLUSION: The aphid retains costly resistance mechanisms even in the absence of pressure from certain insecticides, which could be attributed to factors related to climate and genetic properties of the populations. The indication of build-up of resistance/tolerance to neonicotinoids, related to CYP6CY3 overexpression, is a matter of concern. © 2015 Society of Chemical Industry.