INTRODUCTION

The bean weevil Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae: Bruchinae) is one of the most important pests of many plants from the Fabaceae family, especially the common beans Phaseolus vulgaris (Lineu). Their high reproductive potential and short life cycle allow them to produce several generations within a year, especially under favorable conditions (^{Soares et al., 2015}). The attack of A. obtectus in bean grains begins when they are still in the field and subsequently they are able to infest storage units causing losses ranging from 7 to 40%, which corresponds to 1.59 - 9.12 millions of tons each year in the world caused by bruchids (^{Mbogo et al., 2009}; ^{Jiménez et al., 2017}). The losses caused by A. obtectus is a serious threat to the major protein source of hundreds of millions of people, especially those from Latin America, Asia and Africa (^{Zaugg et al., 2013}; ^{Hnatuszko-Konka et al., 2014}; ^{Pitura and Arntfield, 2019}).

The commonly used strategies to control A. obtectus on small and large stored grain units rely on protectant and fumigant insecticides. However, the indiscriminate use of these products have constantly been questioned, since their use is normally associated with serious problems, such the selection of resistant populations (^{Pimentel et al., 2007}; ^{Boyer et al., 2012}; ^{Haddi et al., 2018}), environmental pollution (^{Stehle and Schulz, 2015}; ^{Tang et al., 2018}) and impacts on the human health (^{Bjørling-Poulsen et al., 2008}; ^{Radwan et al., 2015}; ^{Chen et al., 2016}). Because of these backlashes, the investigation of new products that are less persistent in the environmental and less harmful to other organisms is of extreme importance. Among these alternatives, mechanical control, essential oils, inert dusts or combinations of them have been demonstrated to be effective to control the weevils that attack bean grains (^{Papachristos and Stamopoulos, 2004}; ^{Badii et al., 2014}; ^{Viteri Jumbo et al., 2018}; ^{González Armijos et al., 2019}; ^{Rodríguez-González et al., 2019}), including A. obtectus (^{Bohinc et al., 2013}; ^{Freitas et al., 2016}; ^{Viteri-Jumbo et al., 2018}).

Diatomaceous earths (DEs) are natural inert materials derived from fossilized geological deposits of siliceous; and mainly made of silicon dioxide (^{Korunic, 1997}; ^{Cook et al., 2008}). Additionally, the effectiveness of diatomaceous earth as protectants of different stored beans against their major pests has been already demonstrated (^{Prasantha et al., 2003}; ^{Stathers et al., 2004}; ^{Bohinc et al., 2013}; ^{Badii et al., 2014}). The mode of action of diatomaceous earth is by damaging the insect’s tegument surface (including sensilla and pores) culminating in reduction of body water content and death through desiccation (^{Korunić et al., 2016}; ^{Subramanyam and Roesli, 2000}). Besides being relatively easy to apply, diatomaceous earth toxicity to mammalians is considered low because this chemically unreactive powder is mainly composed by silica, which does not accumulate in mammals as such molecules are excreted in the urine (^{Korunic, 2013}). Additionally, diatomaceous earth does not break down (degrade) rapidly and do not affect end-use quality of grains (^{Subramanyam and Roesli, 2000}; ^{Stathers et al., 2004}; ^{Korunic, 2013}).

The insecticidal efficacy of diatomaceous earth may be affected by abiotic factors e.g., temperature and exposure period or biotic factors e.g., insect species, physiology, and behavior, especially in tropical climate conditions. Thus, this study was conducted aiming to determine whether the exposure period and temperatures levels would affect the efficacy of diatomaceous earth against A. obtectus.

MATERIALS AND METHODS

Insect rearing and diatomaceous earth formulation. Adults of A. obtectus were maintained under controlled conditions (temperature 27 ± 1ºC, relative humidity 65 ± 5% and 12 h of scotophase) on common beans (Phaseolus vulgaris cv Ouro Negro). Newly emerged (<48 h) unsexed adults were used in all the experiments. These insect colonies were reared under controlled conditions and in an insecticide-free environment for at least one year. We used the diatomaceous earth formulation Keepdry™ (Irrigação Dias Cruz Ltda., Santo André, SP, Brazil) which is a diatomaceous earth formulation of freshwater origin containing 86% of silicon dioxide and 14% inert ingredients. The formulation was stored in the dark for three weeks inside sealed plastic bags at 25ºC until the beginning of the experiments.

Bioassays toxicity of diatomaceous earth in different temperatures. To evaluate the susceptibility of A. obtectus to diatomaceous earth (DE) the experimental design was Completely Randomized. Four doses of Keepdry™ which are lower than the recommended field rate (0.25, 0.50, 0.75 and 1.00g kg-1 of beans) were tested under different temperature conditions (25, 28, 30, 32 or 35ºC) and exposure periods (two and five days). Three replicates were used for each dose, temperature, exposure period and a control treatment (grains without DE application) was used for each bioassay. In order to homogenize the DE with the bean grains, the diatomaceous earth amounts were added to a glass jar (2.0L of volume capacity) containing 150g of bean grains. After the application, the jars were rolled manually for 60 seconds. Subsequently, bean grain masses (45g) were placed inside Petri dishes (45cm diameter) and 25 newly emerged unsexed adults of A. obtectus were transferred into the Petri dishes. After the exposure periods, the insects were considered dead when unable of walking or flying when touched with fine brush pods.

Effects of diatomaceous earth on the offspring production of A. obtectus. To evaluate the impacts of DE in the offspring production of A. obtectus, the samples that stayed exposed for five days were maintained after mortality had been assessed. All insects (dead or alive) were removed from the bean grain masses, and the Petri dishes with grains remained in the incubators for an additional period of 60 days. After this period, the number of emerged adults (F1) was recorded and the reduction in progeny or inhibition rate (%IR) was calculated as:

Where Cn is the number of newly emerged insects in the untreated jar and Tn is the number of insects emerged in the treated jar (^{Tapondjou et al., 2002}; ^{Rajashekar et al., 2010}). Three replicates were used for each treatment.

Data analysis. In order to test the effects and the interactions of temperature, dose and exposure interval in the mortality and progeny production of A. obtectus, the data was submitted to three-way Analysis of Variance (ANOVA). Mortality was analyzed using one-way ANOVA to determine significant differences (P < 0.05) among exposure times. Means were separated by Duncan's multiple range test at P < 0.05. The same procedure was followed to analyze for the effects of diatomaceous earth (DE) treatment on the progeny production of A. obtectus.

RESULTS AND DISCUSSION

The Analysis of Variance revealed that temperature, doses, and exposure periods and the associated interactions between them were highly significant (P < 0.0001) for the DE efficacy against A. obtectus (Table 1). Similar results were obtained for the reduction of A. obtectus progeny in bean grain masses that were treated with DE (Table 1).

The A. obtectus mortality caused by DE revealed to be doses, temperature, and exposure period dependent. When the exposure period was of two days, the bean weevils kept at 25 and 28°C exhibited mortality levels of at least 50% in all treatments, excepting the mortality recorded for the treatment of 0.25g kg^-1 at 25°C, where there was no significant difference when compared with the A. obtectus mortality obtained for diatomaceous earth-untreated beans. For temperature treatments above 30°C, the A. obtectus mortality did not differ between treatments, being always superior to 90% (Figure 1A). The mortality results obtained for the exposure period of five days showed that A. obtectus were very susceptible to diatomaceous earth exposure (Figure 1B). For instance, event at lower diatomaceous earth doses and temperatures, the mortality level obtained were never below 40%, resulting in significant differences when compared with the mortality recorded for A. obtectus reared on untreated bean masses (Figure 1B).

Bars grouped under the same horizontal line do not present significant differences (P > 0.05). Histograms of a particular color grouped by similar letters across the different temperatures do not present significant differences (Duncan's multiple range test, P > 0.05).

Figure 1. Mortality levels of A. obtectus when exposed for two (A) and five (B) days to different diatomaceous earth doses and temperature conditions. 

Results obtained for the progeny production showed that the DE caused significant reductions in the progeny of A. obtectus. Even at the lowest diatomaceous earth doses tested (0.25g kg^-1) exposed to 25°C, the suppression of progeny production was above 90% (Table 2). Increase of temperature also affected the number of A. obtectus adults emerged of diatomaceous earth non-treated bean grain masses, where the average number of individuals of 96.0 ± 10.5, 106.0 ± 8.4, 67.7 ± 11.2, 30.3 ± 5.3, 10.0 ± 1.9 when exposed to 25, 28, 30, 32 and 35°C, respectively.

In the present study, it was evaluated the efficacy of diatomaceous earth to control A. obtectus and the contributions of dose, temperature and exposure interval on the efficacy of this inert dust. The results showed that all the doses of DE were toxic to A. obtectus adults and there is a positive correlation between mortality and dose, temperature and exposure period. Such increased mortality rates within exposure time increase are due to the slow-acting mode of action of DE products which are different from traditional grain protectants that may kill insects faster (^{Korunic, 1997}).

Diatomaceous earth is an inert dust mostly composed by small silica particles that are able to abrade the insect cuticle and absorb water, which potentially leads to insect death due to dehydration (^{Korunic, 1997}; ^{Subramanyam and Roesli, 2000}). Since adults of A. obtectus do not feed, it is difficult to recover the water loss caused by DE, which increases the susceptibility of these insects to diatomaceous earth exposures. Moreover, higher temperatures usually increase insect movement inside the grains, causing increased contact with the diatomaceous earth and, consequently, greater cuticular damage. Furthermore, it is important to feature that unlike synthetic insecticides, DE is inert and does not degrade with the increase of temperature, which allow farmers to use DE products combined with elevated temperatures (^{Dowdy, 1999}; ^{Fields and Korunic, 2000}; ^{Athanassiou et al., 2005}; ^{Athanassiou et al., 2011}; ^{Bohinc et al., 2013}; ^{Frederick and Subramanyam 2016}).

Although treatments that caused high A. obtectus mortality resulted in severe progeny reduction, similar reductions were observed in the exposure to lower diatomaceous earth doses, where there were considerably more insects alive. The reasons underlying the aforementioned progeny reduction may potentially be related to the accumulation of diatomaceous earth particles on the ovipositor of A. obtectus females, acting as a physical barrier and disturbing reproduction behaviors (^{Szentesi, 1976}). Furthermore, such progeny reductions may also be attributed to the physiological costs of directing energy to the repairment of damaged cuticle aiming to minimize water losses (^{Subramanyam and Roesli, 2000}; ^{Prasantha et al., 2002}; ^{Prasantha et al., 2015}). Additionally, diatomaceous earth could also have affected the egg hatching or newly emerged larvae. As A. obtectus females lay their eggs freely in the grain masses, the newly emerged larvae generally take few hours selecting and perforating their hosts, which may increase their potential exposure DE particles left on the bean grains (^{Simmonds et al., 1989}; ^{Thiéry et al., 1994}).

Considering that higher doses of diatomaceous earth (> 0.3g kg^-1) are no longer acceptable since they cause negative effects on the grain physical proprieties (causing dusty appearance) and because of the difficulties associated to their sprayed application.(^{Korunic, 1997}; ^{Subramanyam and Roesli, 2000}). Thus, the results showed that lower DE doses as 0.25g kg^-1 of are sufficient to control A. obtectus infestation in bean grain masses in several potential scenarios, without affecting the quality of grains, which reinforce the potential of these inert dust to be used as sustainable alternatives for protecting bean grain masses during on-farm storage phases.

CONCLUSIONS

Diatomaceous earth is a potential alternative to the control of A. obtectus; the mortality of adult weevils was dependent of both temperature and exposure period. Additionally, diatomaceous earth significantly reduced the offspring production (F1) of A. obtectus, regardless of dose and temperature, which makes DE a good candidate to be integrated on management strategies of these bean weevils, especially on small storage units.