Transcriptomic Analysis of a Susceptible and a
 Resistant Strain of the Lesser Grain Borer, Rhyzopertha
 dominica, and Laboratory Evaluation of s-metheprene
    and the Synergist Piperonyl Butoxide - Abstract

      Maria K. Sakka1, Maria Riga2, Panagiotis Ioannidis2, Georgia V. Baliota1,
Rajeswaran Jagadeesan3, Manoj K. Nayak3, John Vontas2,4, Christos G. Athanassiou1
     1
        Laboratory of Entomology and Agricultural Zoology, Department of Agriculture Crop
 Production and Rural Environment, University of Thessaly, Phytokou str., 38446, Nea Ionia,
                         Magnesia, Greece; e-mail: mpaliota@agr.uth.gr
    2
     Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology
               Hellas, 100 N. Plastira Street, GR-70013, Heraklion, Crete, Greece.
      3
       Department of Agriculture and Fisheries, Queensland, Ecosciences Precinct, GPO Box
                              267, Brisbane, QLD 4001, Australia
    4
      Laboratory of Pesticide Science, Department of Crop Science, Agricultural University of
                     Athens, 75 Iera Odos Street, GR-11855 Athens, Greece



Summary

   One of the newer active ingredients (a.i.) that have been registered in many
countries for the control of Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) is
the juvenile hormone analogue (JHA) s-methoprene. Piperonyl butoxide (PBO) is
well-known as a synergist in combination with a specific group of a.i., such as
pyrethroids, that exhibits toxicity through mixed function oxidases. The mechanism
through which PBO inhibits P450s is mostly unknown. Moreover, resistance to s-
methoprene has not been analyzed yet in R. dominica, largely due to the lack of
genomic resources for this species. In the current study we investigate, for the first
time, the mechanisms underlying s-methoprene resistance in R. dominica using
transcriptome analysis to identify the potential role of a set of differentially expressed
genes related to cytochrome P450s in resistance to s-methoprene against two strains,
a resistant and a susceptible of R. dominica. Moreover, laboratory bioassays were
performed in order to evaluate the efficacy of s-methoprene alone and in combination
with PBO.
   For the transcriptome analysis we sequenced the transcriptomes of both strains and
identified the Cytochrome P450 (CYP) genes. Larvae of R. dominica (both strains)
were pooled respectively and preserved in RNA later, and total RNAs of each was
extracted using the GeneJet RNA Purification kit (ThermoScientific), according to the
manufacturer’s protocol. For the bioassays, treated wheat grains with s-methoprene, in
presence and absence of PBO, were used for laboratory bioassays. S-methoprene
concentrations of 0, 0.01, 0.03, 0.1 and 0.3 mg kg− 1 for susceptible and 0, 1, 3, 10 and
30 mg kg− 1 for resistant strain, were used. For PBO, the recommended label rate for
combinations, 0.013 lt per 45.3 kg wheat, was used.




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   The results of the present study indicate that resistance to s-methoprene is
potentially mediated by cytochrome CYPs. MET gene sequence analysis on the
resistant and the susceptible strains clearly identified P489L substitution in the open
reading frame (ORF) of resistant strain, conferring resistance to s-methoprene. For the
bioassays the progeny production varied. In the case of the susceptible strain, similar
results had the treated wheat with s-methoprene alone or in combination with PBO.
Moreover, progeny production was higher in the lowest dose of s-methoprene,
regardless the existence of PBO. In contrast, for the resistant population, when s-
methoprene was applied alone, progeny production was significantly lower than that
in the control vials. However, there was a considerably high offspring emergence,
regardless of the concentration. Similarly, when s-methoprene was applied with PBO,
the increase in the concentrations reduced progeny production. Furthermore, for the
two lowest s-methoprene concentrations, progeny production was not affected,
regardless of the presence of PBO. Nevertheless, for the two higher concentrations,
progeny production was considerably lower when s-methoprene was applied in
combination with PBO, than for the application of s-methoprene alone.
   Subsequently, we sequenced the transcriptomes of s-methoprene-resistant and
susceptible strains and identified the CYP genes. Interestingly, their analysis revealed
that a number of them were significantly upregulated in the s-methoprene-resistant
strain and are thus worth of further investigation to determine their role in insecticide
resistance.

       Keywords: piperonyl butoxide; s-methoprene; Rhyzopertha dominica;
       transcriptome analysis.


Acknowledgment. This research has been co-financed by the European Union and
Greek national funds through the Operational Program Competitiveness,
Entrepreneurship and Innovation, under the call RESEARCH – CREATE –
INNOVATE (project code: T1EDK-01491).




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