The use of chemical insecticides for the control of destructive insects is one of the most efficient and
cost-effective methods employed for their control. However, their constant use in vector control and
agriculture over the years has led to the establishment of resistant insect populations, therefore
creating an ever growing need to understand the molecular mechanisms that govern insecticide
resistance. One of the most widely used insecticide classes in the control of vectors of diseases are
pyrethroids, which bind on the voltage gated sodium channel altering its kinetics, whereas
neonicotinoids, selective agonists of the acetylcholine receptor, are traditionally used in agriculture,
although they have recently started being used in vector control as well. Resistance against pyrethroids
is very prevalent in the major malaria vector mosquito Anopheles gambiae. Specific neonicotinoid
resistance mechanisms haven’t been identified in mosquitoes yet, although they are common in
agricultural pests. In the current bibliography, most studies approach insecticide resistance by
examining individual mechanisms whereas here, various resistance mechanisms from medically and
agriculturally important insects and their combinations are analyzed. Firstly, pyrethroid insecticide
resistance mechanisms utilized by An. gambiae are functionally analyzed using laboratory An. gambiae
strains. Specifically, the resistance effect of the L1014F kdr target-site mutation, that is heavily
associated with pyrethroid insecticide resistance, in combination with the overexpression of known
pyrethroid metabolizing P450 enzyme Cyp6M2 is studied. Mosquitoes harboring the L1014F mutation
and overexpressing Cyp6M2 show a synergistic effect between the mechanisms, displaying significantly
higher pyrethroid resistance than the sum of the isolated mechanisms. Another object of this study is
the determination of the resistance effect conferred by the combination of neonicotinoid resistance
mechanisms from the agriculturally important insects Myzus persicae, Aphis gossypi and Bemisia tabaci
when introduced in the model organism Drosophila melanogaster. More specifically, the R81T targetsite mutation, which is known to confer neonicotinoid resistance, is studied in transgenic flies also
overexpressing known well-characterized neonicotinoid metabolizing enzymes Cyp6M1 and Cyp6CY3.
R81T and Cyp6CM1 appear to work in great synergism, while R81T and Cyp6CM1 display a higher
resistance phenotype than the addition of the effects of each individual mechanism, but the effect- even
though synergistic is moderate. Finally, the creation of transgenic Anopheles gambiae strains is
attempted, utilizing a CrispR/Cas9 transgenesis approach. The reversion of the L1014F mutation to its
wild type state is attempted in a multi-insecticide resistant mosquito strain in order to measure its
contribution to the pyrethroid resistance phenotype. The introduction of the R81T mutation in an
insecticide susceptible An. gambiae strain is also attempted in order to measure its potential resistance
effect and validate the possibility of this mutation being viable and developing in mosquitoes, due to
increasing neonicotinoid use in vector control. No transgenic strains are reported as of yet, although
experimental planning, creation of the necessary constructs and some early embryo microinjection
attempts in order to generate these strains have been performed.
(EL)
The use of chemical insecticides for the control of destructive insects is one of the most efficient and
cost-effective methods employed for their control. However, their constant use in vector control and
agriculture over the years has led to the establishment of resistant insect populations, therefore
creating an ever growing need to understand the molecular mechanisms that govern insecticide
resistance. One of the most widely used insecticide classes in the control of vectors of diseases are
pyrethroids, which bind on the voltage gated sodium channel altering its kinetics, whereas
neonicotinoids, selective agonists of the acetylcholine receptor, are traditionally used in agriculture,
although they have recently started being used in vector control as well. Resistance against pyrethroids
is very prevalent in the major malaria vector mosquito Anopheles gambiae. Specific neonicotinoid
resistance mechanisms haven’t been identified in mosquitoes yet, although they are common in
agricultural pests. In the current bibliography, most studies approach insecticide resistance by
examining individual mechanisms whereas here, various resistance mechanisms from medically and
agriculturally important insects and their combinations are analyzed. Firstly, pyrethroid insecticide
resistance mechanisms utilized by An. gambiae are functionally analyzed using laboratory An. gambiae
strains. Specifically, the resistance effect of the L1014F kdr target-site mutation, that is heavily
associated with pyrethroid insecticide resistance, in combination with the overexpression of known
pyrethroid metabolizing P450 enzyme Cyp6M2 is studied. Mosquitoes harboring the L1014F mutation
and overexpressing Cyp6M2 show a synergistic effect between the mechanisms, displaying significantly
higher pyrethroid resistance than the sum of the isolated mechanisms. Another object of this study is
the determination of the resistance effect conferred by the combination of neonicotinoid resistance
mechanisms from the agriculturally important insects Myzus persicae, Aphis gossypi and Bemisia tabaci
when introduced in the model organism Drosophila melanogaster. More specifically, the R81T targetsite mutation, which is known to confer neonicotinoid resistance, is studied in transgenic flies also
overexpressing known well-characterized neonicotinoid metabolizing enzymes Cyp6M1 and Cyp6CY3.
R81T and Cyp6CM1 appear to work in great synergism, while R81T and Cyp6CM1 display a higher
resistance phenotype than the addition of the effects of each individual mechanism, but the effect- even
though synergistic is moderate. Finally, the creation of transgenic Anopheles gambiae strains is
attempted, utilizing a CrispR/Cas9 transgenesis approach. The reversion of the L1014F mutation to its
wild type state is attempted in a multi-insecticide resistant mosquito strain in order to measure its
contribution to the pyrethroid resistance phenotype. The introduction of the R81T mutation in an
insecticide susceptible An. gambiae strain is also attempted in order to measure its potential resistance
effect and validate the possibility of this mutation being viable and developing in mosquitoes, due to
increasing neonicotinoid use in vector control. No transgenic strains are reported as of yet, although
experimental planning, creation of the necessary constructs and some early embryo microinjection
attempts in order to generate these strains have been performed.
(EN)
Πανεπιστήμιο Κρήτης
