Immediate and Delayed Mortality of Tribolium confusum Adults and Larvae on Concrete Surfaces Treated with Chlorantraniliprole

: Adults and larvae of Tribolium confusum were exposed for 1, 2, 3, 4 and 5d on concrete treated with chlorantraniliprole SC at four doses (0.01, 0.05, 0.1 and 0.5 mg active ingredient/cm 2 ). Then, the adults and larvae alive were transferred on untreated concrete and delayed mortality was evaluated after 7d. Larval mortality ranged from 61.1 to 78.9% while adult mortality ranged between 40.0 and 70.0%, after 5d. Delayed mortality was high for both life stages, reaching 97.2 and 100.0% for adults and larvae, respectively. To conclude with, chlorantraniliprole is an effective management tool against T. confusum on concrete surfaces.


Introduction
The confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), is considered one of the most serious pest of stored and processed grains worldwide [1,2], infecting 138 different types of commodities (e.g., flours, bran, cereal and oil seeds, spices, and a broad spectrum of dry plant materials) [3].It is also a serious insect pest in mills, as it can easily find refuges and food in cracks and crevices in milling facilities, as well as the ideal conditions for its growth and development [5].However, management of this species is an important issue since it is tolerant to several insecticides, such as the pyrrole insecticide chlorfenapyr [6], or the members of spinosyn group, spinosad and spinetoram [7].Moreover, T. confusum was found to be the least susceptible among the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), and the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), to three different formulations of diatomaceous earths [8].
Chlorantraniliprole, is a novel insecticide that belongs to the chemical group of anthranilic diamides [9,10], which has low mammalian toxicity and can kill a wide range of insect pests without harming beneficial arthropods [11][12][13][14].For instance, it is very effective for species of agricultural importance belonging to different orders, e.g.Coleoptera, Lepidoptera, Hemiptera, Diptera, Isoptera and Thysanoptera [15][16][17][18][19][20][21].Furthermore, recent studies have shown the insecticidal activity of chlorantraniliprole for stored-product protection, either as grain protectant [22,23] or as surface treatment [24].Thus, the objective of the present work was to examine the immediate and delayed mortality of chlorantraniliprole SC on concrete surface, against adults and larvae of T. confusum.

Insect, commodity and Insecticidal Formulation
The adults and larvae of T. confusum used in the bioassays are kept in the Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, since 2014.The insects were initially collected from Greek storage facilities in 2003 and reared in wheat flour including 5% brewer's yeast (w/w) at 27°C and 60% relative humidity, in continuous darkness.Moreover, all adults of the tested species were 7 days old and larvae were 3rd -4th instar [25].Clean, without infestation, and pesticide-free white soft wheat flour (a variety mixture, made from the endosperm only) was used as food in the experiments.The concrete surface was treated with the formulation of chlorantraniliprole, Coragen ® SC (suspension concentrate) with 200 g/L active ingredient (a.i.), which was provided by Dupont (Halandri, Greece).

Bioassays
The concrete surface of individual dishes was sprayed with 1 ml of an aqueous solution, as a fine mist, that contained the appropriate volume for each dose of chlorantraniliprole (i.e.0.01, 0.05, 0.1 and 0.5 mg a.i./cm 2 ), using an AG-4 airbrush (Mecafer S.A., Valence, France).Then, 10 T. confusum adults or larvae were placed on each sprayed dish, which contained 0.5 g white soft wheat flour.Before the beginning of the experiments, the moisture content of the flour was adjusted to 13.5 ± 0.5% as determined by a calibrated moisture meter (mini GAC plus, Dickey-John Europe S.A.S., Colombes, France) either dried inside oven at 50 •C or hydrated with distilled water according to its initial moisture content [26,27].Then, it was sprinkled over the surface area of the concrete one day after spraying.An additional series of dishes were prepared and sprayed, as described above, with distilled water, and served as controls.Afterwards, all dishes were placed in incubators set at 25 o C and 65% relative humidity and continuous darkness.Mortality was determined by prodding the insects gently with a brush to detect movement under an Olympus stereomicroscope (Olympus SZX9, Bacacos S.A., Athens, Greece) after 1, 2, 3, 4 and 5 d of exposure on the treated substrate.The brush was carefully washed after the examination of each dish.After the 5-days interval, the surviving adults or larvae were transferred to new dishes with untreated concrete surfaces, containing 0.5 g white soft wheat flour for 7 days more at the same conditions.After this period, the new dishes were opened and the number of dead adults or larvae was counted as described above.

Data Analysis
Immediate or delayed mortality of control was low (<3%), so no correction was considered necessary.Data were analyzed separately for immediate and delayed mortality by following the repeated measures model [28].All analyses were conducted using the JMP 14 software [29].

Immediate and delayed mortality of T. confusum adults
Mortality was very low after 3 days of exposure in all tested doses, not exceeding 27.8% (0.5 mg a.i./cm 2 ) (Table 1).After 4 days, mortality increased further, reaching 46.7%, at 0.5 a.i./cm 2 , while at the rest doses it ranged between 27.8 and 33.3%.One day later, chlorantraniliprole killed 70.0% of exposed adults at the highest dose and caused moderate mortality to the lower doses (i.e.0.01, 0.05 and 0.1 mg a.i./cm 2 ),.Delayed mortality was high, even in the low doses, killing more than 86.5% of the survived adults (Table 2).

Immediate and delayed mortality of T. confusum larvae
After 2 days of exposure mortality was in very low levels, reaching 18.9% of the exposed larvae (Table 1).One day later, mortality increased further for all tested doses, ranging between 17.8 and 37.8%.After 4 days of exposure, moderate (53.3%) mortality was noted at 0.5 mg a.i./cm 2 , while at doses between 0.01 and 0.1 mg a.i./cm 2 , mortality did not exceed 38.9%.At the 5-days post exposure, the proportion of dead larvae increased significantly, reaching 78.9% at 0.5 mg a.i./cm 2 .Concerning delayed mortality, chlorantraniliprole killed 91.7% of the survived larvae at 0.01 mg a.i./cm 2 , while with the increase of the dose mortality reached 100.0%(0.5 mg a.i./cm 2 ) (Table 2).

Discussion
Chlorantraniliprole is a promising formulation for the control of adults and larvae of T. confusum, when they are exposed on concrete surfaces.It has a unique mode of action, activating the ryanodine receptor in the muscles of insects that causes the release of cellular calcium.The insect stops feeding, its muscles are paralyzed and eventually dies [30][31][32].In the present study, chlorantraniliprole killed 70.0 and 78.9% of the exposed adults and larvae on treated concrete with the highest dose, after 5 days, respectively.On the other hand, 97.2 and 100.0% of surviving adults and larvae, respectively, died when they were transferred on untreated surfaces, indicating the high delayed effects of this insecticide to the non-toxic areas.This is an important finding considering that the movement of individuals from treated to untreated areas may lead to their colonization by insects [33].
In a previous study, two Chlorantraniliprole formulations (WG and SC) have been examined as grain protectant against a wide range of insect pest, including T. confusum adults and larvae [22].Chlorantraniliprole SC was able to kill a sufficient number of larvae (94.4% on maize, 83.3% on barley and 86.1% on oat) and adults (80.6% on maize) at the dose of 10 mg chlorantraniliprole SC kg −1 grain, and reduced the progeny production.Moreover, in the same study, both chlorantraniliprole formulations that tested were very effective for the control of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), the booklouse, Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae), R. dominica and S. oryzae, in six commodities (e.g.barley, maize, oats, whole rice, peeled rice and hard wheat.Similar results were recorded by Boukouvala and Kavallieratos [23], when they examined chlorantraniliprole as maize protectant against the larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrychidae), at different temperatures.
To conclude, the results of the current study indicate that chlorantraniliprole is an effective management tool against adults and larvae of T. confusum on concrete surfaces, with a remarkable delayed effect.

Citation:
Kavallieratos, N.G.; Boukouvala, M.C.; Nika, E.P. Immediate and delayed mortality of Tribolium confusum adults and larvae on concrete surfaces treated with chlorantraniliprole, in Proceedings of the 1st International Electronic Conference on Entomology, 1-15 July 2021, MDPI: Basel, Switzerland, doi:10.3390/IECE-10362Published: 30 June 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.Copyright: © 2021 by the authors.Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses /by/4.0/).