The development of resistance in many species, the demand for residue free food, the increased concerns about worker safety and the gradual withdrawal of several commonly used insecticides, lead researchers to evaluate the potential use of other reduced-risk, control methods. Recently, alternative methods are being emphasized to reduce use of insecticides to lessen the potential for human exposure and to slow the development of insecticide resistance in pests (Aldryhim, 1993). Diatomaceous Earths (DEs) are among the most promising alternatives to traditional grain protectants, because they are easy to use and combine low mammalian toxicity with natural origin, doesnt break down rapidly and dose not affect grain end-use quality (Athanassiou et al., 2004). DEs are almost pure silicon dioxide made up of fossilized diatoms which absorb wax from insects cuticle resulting in water loss and death through desiccation (Korunic, 1998; Nikpay, 2006). DEs can be applied directly to the grain, without specialized equipment using much the same technology as far residual insecticides (Athanassiou et al., 2005). Vayias and Athanassiou (2004) exposed adults, young larvae (1-3 instars) and old larvae (4-7 instars) of Tribolium confusum Du Val. to SilicoSec® at the rates of 0.25, 0.5, 1 and 1.5 g kg-1. High level of mortality was observed for T. confusum larvae after 24 h of exposure even at the lowest dose rate. Athanassiou et al. (2005) in the other research investigated the impact of SilicoSec® against adults of Sitophilus oryzae L. and T. confusum. The authors stated that adults of T. confusum were more tolerant to DE SilicoSec® than S. oryzae.
The red flour beetle, Tribolium castaneum (Herbst) is probably one of the most common and the least susceptible stored-product pests to DE, so a DE formulation able to control flour beetles should be able to control most insects occurring in stored food (Korunic, 1998; Fields and Korunic, 2000; Arnaud et al., 2005).
The objective of present study is to evaluate the insecticidal efficacy of SilicoSec®, a commercial DE product, against adults, young and old larvae of T. castaneum under laboratory conditions.
MATERIALS AND METHODS
Beetles: Beetles were reared at 28°C and 65±5% r.h (relative
humidity) in continues darkness. Adults of T. castaneum were reared on
wheat flour plus 5% brewers yeast (by weight). All adults were used in the experiment
were 7-14 days old of mixed sex. Young larvae of T. castaneum were obtained
by placing 100 unsexed adults of mixed ages on 100 g wheat flour plus 5% brewers
yeast (by weight) diet in glass jars. After 7 days, young larvae were separated
from the diet by using appropriate sieves with the mean±SE (n = 30) weight
of 0.6±0.1 mg and old larvae separated after 20 days from the diet with
the mean±SE (n = 30) weight of 3.4±0.1 mg. Beetles were obtained
from cultures maintained in the Entomology laboratory of Urmia University for
at least 3 years, with no history of exposure to insecticides.
DE formulation: SilicoSec® is a freshwater formulation of diatomaceous earth (Biofa GmbH, Munsingen, Germany) and is composed of 92% SiO2, 3% Al2O3, 1% Fe2O3 and 1% Na2O. The median particle size is between 8-12 μm. DE was stored in the laboratory at ambient conditions, until the beginning of the experiment (approximately for a month).
Bioassays: Experiments were conducted in the Entomology laboratory of Urmia University in 2006.
Susceptibility of adults (Test 1): Fifteen glass vials with the volume of 0.5 L were provided and poured with sixty grams of clean wheat (variety Zarin) (95% whole wheat plus 5% cracked wheat), three vials for each dose rate. The moisture content (m.c) of the wheat was measured using Dickey-John moisture meter ranged about 11.4% m.c which is equilibrium to 55% r.h (Pixton and Warburton, 1971). Wheat treated with four dose rates of SilicoSec®: 0.7, 1, 1.3 ans 1.7 g kg-1. Doses were determined with a preliminary test and untreated wheat with a similar ratio of whole to cracked wheat served as a control treatment. The vials were shaken for one minute to distribute the DE in the entire product. Subsequently, 30 adults were introduced into each sample and vials were covered with muslin cloth for sufficient ventilation. The vials were then placed in incubator set at 27°C and 55±5% r.h. Adults mortality was measured after 2, 7 and 14 days of exposure. After 14 days mortality count, all adults were removed and samples retained under the same conditions for a further 60 day to assess progeny production.
Susceptibility of larvae (Test 2): The method of this experiment was similar to that employed for adults, but in order to that the susceptibility of stages is different, therefore four doses of SilicoSec® were determined separately for each of the stages with a preliminary test. The 0.35, 0.6, 0.9, 1.2 g kg-1 of SilicoSec® were used for young larvae and in the case of old larvae 0.35, 0.6, 1, 1.5 g kg-1 of SilicoSec® was treated with 95% whole wheat + 5% cracked wheat and placed in the appropriate conditions of previous experiment after introducing individuals. Mortality of young and old larvae was counted after 1, 2 and 7 days interval.
Adherence of SilicoSec® to wheat: The method of determining DE adherence to wheat kernels was similar to that employed by Korunic (1997). First, 500 g of wheat was cleaned by sieving for a minute using a No. 10 sieve (2 mm openings, Retsch GmbH and Co KG, Germany). Subsequently, the cleaned wheat was mixed with 1 g kg-1 of SilicoSec® (0.5 g per 500 g) in a tightly closed glass jar and shaken for a minute. The treated grain was then sieved thoroughly using laboratory sieve No. 10 with a lid and bottom for one minute. The dust collected and weighed. The weight was subtracted from 500 mg and the value was expressed as a percentage of adherence of DE on the wheat kernels.
Data analysis: The mortality counts were corrected by using Abbotts (1925) formula. The data were analyzed using Analysis of Variance and means were separated by using the Tukey-Kramer adjustment at p = 0.05 (SAS, 2000). To equalize variances, mortality percentage of adults, young and old larvae were transformed using the square root of the arcsine and the data of adults progeny production was transformed to log (x +1) scale. The dose required to kill 50% of the insects (LC50) was estimated using probit analysis (SPSS, 1999). Linear lines were drawn to define all dose-mortality relations for each exposure time. Percentage of reduction in progeny production was determined by Aldryhim (1990) is given as:
RESULTS AND DISCUSSION
The main effects for adults: Dose (F = 127.09, df = 4); exposure interval
(F = 701.8, df = 2), young larvae: Dose (F = 128.1, df = 4); exposure interval
(F = 276, df = 2) and old larvae: Dose (F = 201.1, df = 4); exposure interval
(F = 242.05, df = 2) were all significant. In addition, all associated interactions;
dose x exposure interval for adults (F = 35.8, df = 8), young larvae (F = 7.1,
df = 8) and old larvae (F = 4.1, df = 8) were also significant. The mortality
percentage for adults of T. castaneum after 2, 7 and 14 days of exposure
and in the case of young and old larvae after 1, 2 and 7 days exposed to different
doses of SilicoSec® has been shown on Table 1.
||Mean mortality (%) ±SE of adults, young and old larvae
of T. castaneum exposed to SilicoSec® after 2, 7 and
14 days of exposure for adults and 1, 2 and 7 days in the case of larvae
|Means followed by the same letter on each table are not significantly
different; Tukey-Kramer at p = 0.05
||The LC50 values (g kg-1) for adults,
young and old larvae of T.castaneum exposed to wheat treated with
SilicoSec® (Significant Chi-square for interaction = 5.99)
|NC: Confidence limits could not be calculated; NS: Non Significant
difference; *: Indicate significant difference at p<0.05
||Relationship between dose rates of SilicoSec®
and mortality of T. castaneum adults for 2 (a), 7 (b) and 14 (c)
days exposure time
Dose-mortality lines for each exposure time were presented on Fig.
1 for adults and Fig. 2 in the case of young and old larvae
of T. castaneum. The determined coefficient (R2) indicated
that what percentage of variation in the mortality can be accounted by the present
linear model (Fig. 1 and 2).
||Relationship between dose rates of SilicoSec®
and mortality of T. castaneum young larvae on the left and old larvae
in the right for 1 (a), 2 (b) and 7 (c) days exposure time
||The mean (number of individuals per vial±SE) and percentage
of reduction in progeny production (f1) of T. castaneum exposed
to wheat treated with SilicoSec®
|Means followed by the same letter are not significantly different;
Tukey-Kramer at p = 0.05
The LC50 values decreased with increases in time of exposure. The
7 days LC50 for T. castaneum adults was 0.913 g kg-1,
in the case of young larvae the LC50 value after 7 days was 0.32
g kg-1, however; 0.446 g kg-1 of SilicoSec® was
needed to achieve 50% mortality for old larvae of T. castaneum after
7 days interval. Chi-square analyses of expected and observed mortalities of
beetles indicated that observed mortality was the same as expected from the
experiments at all testes (α = 0.05) with the exception of T. castaneum
adults after 7 days of exposure to SilicoSec® (Table
2). The mean number±SE of progeny in the control was 7.66±0.10
individuals per sample and significant suppression in progeny production was
reported even at the lowest dose rate of SilicoSec® (Table
3). The retention of SilicoSec® to wheat kernels with the
11.4% m.c was recorded 78.62%.
Present study indicates that mortality of T. castaneum on wheat treated with SilicoSec® increases with exposure time, this stands in accordance with previous reports by Aldryhim (1990 and 1993), Vayias and Athanassiou (2004), Athanassiou et al. (2005) and Ziaee et al. (2006).
In the present research longer exposure interval is needed to achieve 100% mortality for adults of T. castaneum, because the longer the insects walk over the treated substrate the more dust particles are trapped by their bodies; resulting in water loss and death through desiccation (Arthur, 2000).
Results indicated that larvae of T. castaneum are more sensitive to
SilicoSec® than adults; however this effect is determined by
the larval stages. Young larvae are significantly susceptible than older ones
and this difference is apparent after 7 days of exposure. This agrees with that
experiment of Vayias and Athanassiou (2004). After 24 h of exposure to DE SilicoSec®;
approximately 61% of young larvae were dead, while the respective mortality
for old larvae was only 26%. In young larvae the cuticle may be softer than
in older ones and thus, DE may cause more rapid cuticle damage which may result
in more desiccation. Also, young larvae are particularly agile; a fact which
increases the contact with the dust particles, as compared to older larvae stages
prior to pupation which is less active (Vayias and Athanassiou, 2004).
DE dose rate is crucial not only for efficacy but also for the physical properties of the grain. High dose rates provide a satisfactory level of protection but dramatically affect the bulk density less, but may not be sufficient for long term protection (Korunic, 1998).
In present study more than 85% of the variation in the adult and young larvae mortality can be accounted by SilicoSec® dose rate with linear lines which indicated that there is a direct relation between the mortality and DE dose rate; so the mortality increases with dose rate (Fig. 1 and 2).
The comparison of LC50 values after 7 days interval indicated that high amount of SilicoSec® (0.913 g kg-1) was required to achieve 50% adult mortality; while, the less dose rates of 0.446 and 0.32 g kg-1 is sufficient for controlling 50% of old and young larvae, respectively. Therefore, adults of T. castaneum were more tolerant to SilicoSec® than larvae and can survive at application rates and exposure intervals that are lethal to all larval stages; so the application rate recommended for controlling adults can control different larval stages of T. castaneum. Results confirmed that 1.3 and 1.7 g kg-1 of SilicoSec® were sufficient enough to control adults of T. castaneum because with 1.7 g kg-1 of SilicoSec®, 100% mortality and completely progeny suppression and in the case of 1.3 g kg-1, 96.3% mortality after 14 days of exposure and 95.65% reduction in progeny production was recorded and these doses ranged in the same group; therefore we recommend 1.3 g kg-1 of SilicoSec® to control infestations of T. castaneum. Athanassiou et al. (2005) reported that 1 g kg-1 of SilicoSec® was equally effective with 1.5 g kg-1 against S. oryzae and T. confusum and this is in agreement with present results.
Stored product insect pests show a wide range of susceptibility to DE (Aldryhim, 1990, 1993). Fields and Korunic (2000) found that T. castaneum had noticeably less DE attached to its cuticle than other storage beetles, so T. castaneum appeared more tolerant stored grain species to DE and the application rate for this species can be used for protecting grain in the storage facilities.
However, not only the species of the beetles but also the strain of it may be important to its susceptibility to DEs. According to Vayias et al. (2006) different susceptibility to Des was observed among strains of T. confusum. In the same study strains of T. confusum from Denmark, United Kingdom and Germany were the most susceptible to Insecto, Protect-It, Protectot, PyriSec and SilicoSec whereas the strain from Portugal was the least susceptible.
Exposure to SilicoSec® suppressed reproductive potential of adults significantly. The adults probably were killed before they were able to lay eggs and therefore the SilicoSec® could provide stored wheat grain with complete protection from infestation.
One of the characteristics that determine the insecticidal efficacy of DE is the degree of adherence to the kernels.
Aldryhim (1993) reported that two factors contributed to the effectiveness of DE; (1) the degree of adhesion of DE particles to different commodities and (2) the rate that DE particles are picked up by beetles. Korunic (1997) speculated that among other characteristics of DEs, adherence to commodities correlates well with the insecticidal activity of a given DE. Kavallieratos et al. (2005) reported different degrees of adherence among wheat, whole barley, peeled barley, oats, rye, triticale, rice and maize for the DEs SilicoSec® and Insecto®. The retention of SilicoSec® was significantly higher than the corresponding figure for Insecto®. In the present study, the degree of SilicoSec® adherence to wheat kernels was noted 78.62% (>70%) which is marked plus (+) due to the criteria for the prediction of potential insecticidal value of diatomaceous earth recorded by Korunic (1997). The high retention level of SilicoSec® on wheat reflects the typical high adhesion rates achieved by all fine diatomaceous earth based inert-dust insecticides.
Research carried out by Desmarchelier et al. (1996) on Dryacide®, which has very similar chemical and physical properties and particle size distribution as SilicoSec®, clearly demonstrated that conventional cleaning in a flour mill completely removes the dust even at high dosage levels. At the end of the wheat cleaning process 98.8% of Dryacide® was removed from the highest dosage rate (1000 g t-1), while, 100% was removed from the lower doses (175 and 10 g t-1). End products of the highest treatment dose (1000 g t-1), produced no significant dough handling or bread quality problems and there was no difference in flavor or aroma between the control and the highest treatment dose.
However, filed tests using the similar design should be done to confirm the laboratory findings.
Present gratitude is extended to Dr. Bernosy for his assistance in analyzing data.