Panagiotis J. Skouras

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.

A small deletion in the olive fly acetylcholinesterase gene associated with high levels of organophosphate resistance

Organophosphate resistance in the olive fly was previously shown to associate with two point mutations in the ace gene. The frequency of these mutations was monitored in Bactrocera oleae individuals of increasing resistance. In spite of the difference in resistance among the individuals, there was no correlation between mutation frequencies and resistance level, indicating that other factors may contribute to this variation. The search for additional mutations in the ace gene of highly resistant insects revealed a small deletion at the carboxyl terminal of the protein (termed Delta3Q). Significant correlation was shown between the mutation frequency and resistance level in natural populations. In addition, remaining activity of acetylcholinesterase enzyme (AChE) after dimethoate inhibition was higher in genotypes carrying the mutation. These results strongly suggest a role of Delta3Q in high levels of organophosphate (OP) resistance. Interestingly, the carboxyl terminal of AChE is normally cleaved and substituted by a glycosylphosphatidylinositol (GPI) anchor. We hypothesize that Delta3Q may improve GPI anchoring, thus increasing the amount of AChE that reaches the synaptic cleft. In this way, despite the presence of insecticide, enough enzyme would remain in the cleft for its normal role of acetylcholine hydrolysis, allowing the insect to survive. This provides a previously un-described mechanism of resistance.

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.

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.

Susceptibility of Ceratitis capitata to deltamethrin and spinosad in Greece

Ceratitis capitata is an important pest of citrus in Greece, also causing severe damage to other fruits. Its control has been based on organophosphates for many decades, but recently there has been a switch to pyrethroids. In the present study, the resistance status to deltamethrin and spinosad in 27 C. capitata samples from various hosts and regions of Greece was examined with toxicity bioassays. The bioassays did not reveal significant levels of resistance in either of the two insecticides. All but one of the samples tested with deltamethrin showed a significantly higher LD50 than the susceptible strain (LS) but the resistance factor (RF) values were low (1.2–4.6). In support of this, no significant overexpression of the CYP6A51 gene (associated with pyrethroid resistance in other studies) was detected in the samples examined compared to the LS, indicating the absence of incipient pyrethroid resistance. Lower between-sample variation was observed in the response to spinosad compared to deltametrin; CV for LD50 values: 19.4 vs. 40.0%, respectively, RF values: 1.1–2.4 and 23 out of the 27 field samples had LD50 significantly higher than the LS. This might suggest lower genetic variation related to resistance where selection can act. Our results suggest that deltamethrin can effectively control C. capitata in Greece and spinosad may be a valuable alternative, once registered for the control of this pest in Greece. However, continuous monitoring is important given that P450-mediated metabolic resistance has been demonstrated for C. capitata populations in the Mediterranean basin.

Effect of synthetic insecticides on the larvae of Coccinella septempunctata from Greek populations

Coccinella septempunctata L. 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. These pests are usually controlled with chemical insecticides. During this process, however, beneficials are also exposed to pesticides. The development of Integrated Pest Management (IPM) programs against aphids requires the evaluation of the effects of insecticides on beneficial insects. We evaluated the LD50 of imidacloprid, acetamiprid, bifenthrin and deltamethrin on first, second, third and fourth instar larvae of C. septempunctata by topical application. Moreover, we studied their sublethal effects (LD10) on the development, weight and prey consumption of fourth instar larvae. The topical application bioassays showed that deltamethrin and bifenthrin were highly toxic to all larval instars, whereas imidacloprid and acetamiprid were less toxic to fourth instar larvae. The LD10 dose significantly affected, developmental time, adult weight and daily predation. These results show the importance of assessing potential effect of insecticides on C. septempunctata for developing effective IPM programs of aphids in Greece.

Data on the ecology of Kermes echinatus Balachowsky (Hemiptera, Coccomorpha, Kermesidae) on Quercus ilex (Fagaceae) in Greece

The Phenology and natural enemies of the scale insect Kermes echinatus Balachowsky (Hemiptera: Kermesidae), which infests the holly oak Quercus ilex (Fagaceae), were studied between November 2015 and November 2017, in southern Greece (Kalamata, Peloponnese). Kermes echinatus is a univoltine, oviparous and biparental species. It overwinters as 1st instar nymphs, settling on the branches of the trees. The second instar nymphs appear by the middle of April, followed by 3rd instar nymphs until the end of May. The male larvae and pupae were recorded from the end of April to the end of May. Mature females, pre-ovipositing and ovipositing adults, were recorded during May and June. The newly hatched crawlers appeared by the end of June, which remained at this stage, settled on branches during the rest of summer and winter periods till next April. The parasitoid species Metaphycus gennaroi Guerrieri & Noyes and the hyperparasite Cheiloneurus claviger Thomson (Hymenoptera: Encyrtidae) were recorded on the colonies of the scale. The parasitism level of the scale reached to 21%. The predator Chilocorus bipustulatus L. (Coleoptera: Coccinellidae) was also recorded on the colonies of the scale.