A Novel Use for Potassium Alum as Controlling Agent Against Periplaneta americana (Dictyoptera: Blattidae

Journal of Economic Entomology Advance Access published August 12, 2015 ECOTOXICOLOGY

A Novel Use for Potassium Alum as Controlling Agent Against Periplaneta americana (Dictyoptera: Blattidae) ELHAM M. SALAMA1 Department of Entomology, Faculty of Science, Benha University, P.O. Box 13518, Egypt.

J. Econ. Entomol. 1–10 (2015); DOI: 10.1093/jee/tov239

ABSTRACT In this work, we present our results on the use of potassium alum as an environmentally friendly insecticide. This compound has the potential to rid our homes, schools, hotels, restaurants, and ships of cockroach infestations. This compound is environmentally friendly and has no hazardous effects on plant, animal, or human ecosystems. Alum was approved for medical use a long time ago. In our laboratory, we developed a novel method using potassium alum as an environmentally friendly insecticide to kill the most common cockroach in the subtropical region, Periplaneta americana (L.). Adult and nymph-staged cockroaches were left to feed on potassium alum per individual insect after a period of food deprivation. The mortality was recorded as LT50. The younger nymphs the third and early fourth instars died within 4 d of feeding after consuming an average of 0.3 mg per individual insect. Gravid females were highly susceptible to alum toxicity and experienced a higher mortality rate, with an average of 3 mg per individual female. The oothecae of the normal untreated females were 8.1 mm long and 4.13 mm wide and weighed 94 mg. The eggs laid by the treated gravid females were underweight and exhibited a dwarfism shape, and these eggs did not hatched if the females consumed the potassium alum before laying eggs. The results revealed that the adult male and female cockroaches have to consume 1 mg and 2.7 mg, respectively, of potassium alum to kill 100% of them after 1 month of ingestion. The potassium alum had to be ingested by the cockroaches to affect mortality. The effect of potassium alum was attributed to chronic toxicity and not acute toxicity. The potential applications of this novel technique will be discussed. KEY WORDS can cockroach

environmentally friendly insecticide, potassium alum, Periplaneta americana, Ameri-

Cockroaches have successfully adapted to various habitats, including rodent burrows, sand dunes, caves, and human dwellings (Roth and Willis 1960). These insects act as an active carrier of many pathogenic organisms (Britter and Williams 1949, Roth and Willis 1957, Rueger and Olson 1969) and may cause allergic reactions in some people (Rosa`rio Filho et al. 1999, Wu et al. 2000, Arruda et al. 2001). The American cockroach is an omnivorous and opportunistic feeder. This insect mostly consumes decaying organic matter, but because the cockroach is a scavenger, it will eat almost anything. The American cockroach prefers sweet food (carbohydrates) and has been observed eating paper, boots, hair, bread, fruit, book bindings, fish, peanuts, old rice, putrid sake, the soft part on the inside of animal hides, cloth, and dead insects (Bell and Adiyodi 1981). Potassium alum is a natural white crystalline mineral salt (double sulfate) of potassium. Potassium alum is a specific chemical compound in a class of alum chemical compounds. The term “alum” refers to various double sulfates plus an additional element, such as potassium,

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Corresponding author, e-mail: [email protected].

which results in aluminum potassium sulfate dodecahydrate: Al K (SO4)2.12H2O. Potassium alum, like other alums, crystallizes into a regular octahedral shape. It is soluble in water and liquefies when heated. If the heating is continued, an unstructured powder will remain. Potassium alum crystals are colorless and glassy (vitreous), and in its purest form, alum is completely translucent with small fractures. Potassium alum has been a popular natural mineral salt for thousands of years due to its uses in water purification, pickling, tanning, medical procedures, and cosmetics, such as aftershave or deodorant. Additionally, owing to its attractive crystal appearance, potassium alum has become a favorite among mineral collectors. Potassium alum has antimicrobial properties and can, therefore, be used as a natural deodorant by inhibiting the growth of the bacteria and fungi responsible for body odor. It is also used in medical procedures as a local astringent and styptic. Potassium alum has been commonly used in folk medicine in ancient Egypt, Saudi Arabia, India, China, and Iran. The use of potash alum as a flocculent and disinfectant agent in households has been reviewed in a number of studies, such as Oo et al. (1993), Wrigley (2002), and Crump et al. (2004). Additionally, potash alum has been tested for its larvicidal efficacy against Anopheles stephensi

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(Preet and Seema 2010) and Aedes aegypti (L.) (Preet and Sneha 2011). The aim of the present work is to investigate the efficacy of potassium alum (also known as potash alum, alumbre, or Shabah, the Arabic name) as an environmentally friendly insecticide against Periplaneta americana (L.) adults and nymphs and its impact on the amylase, protease, and lipase activities, under laboratory conditions. Materials and Methods Test Insect. P. americana were collected from the Wadi Eldawaser region in the southern Riyadh area of Saudi Arabia in 2007. The rearing conditions for P. americana were as previously described (Edwards et al. 1987). This colony was successfully maintained and provided us with nymphs and adults of both sexes. The mean temperature in the rearing room during the experiment was 25 C 6 5 with 70% 6 5 relative humidity (RH) under a photoperiod of 12:12 (L:D) h. Food preferences of cockroaches (adults of both sexes and nymphs) were examined independently by offering 7-d starved individuals for normal food (white bread) and potassium alum. Sony video camera Model No CCD- TRV27E-0Lux, made in Japan, was used to count the number of stages during feeding. Adults males and females (virgin or at the time of extrusion) after emergence were transferred to transparent plastic cups (10.0 cm diameter, 4.5 cm high) to avoid the cannibalism among the cockroaches. The cups contained a bottle filled with water to avoid desiccation, and the cockroaches were kept individually for 0, 1, 2, and 4 wk with either 1) no food/starved for one control, 2) potassium alum, or 3) normal food for a second control. To avoid being pinched, the cockroaches were cooled on ice before dissection for subsequent experiments. Newly molted nymphs (third and fourth instars) and adult cockroaches were collected for experimental tests from stock cultures and were kept in small containers (five individuals per 2 kg Kilner jar) at 25 C 6 5 and 70% 6 5 RH during the experiment with potassium alum. Two types of control were used: one group with normal food and another group without food (starved). The LT50 and LT95 values of cumulative mortality were plotted against the time of the all stages of roaches fed with potassium alum and were estimated using probit analyses according to Finney (1971). Mated P. americana females were collected when they were extorting on ootheca for additional experiments. The oothecae produced by these treated females were measured and compared to those of the control group (mated females group with normal food). The container containing cockroaches was covered with a muslin cloth and tied with a rubber ring. All the tested cockroaches were provided with water from a cotton pad socked with water. Adult females were staged according to the time after ootheca formation, with the time of oothecae formation designated as day 1. Staged females were selected every 24 h after ootheca formation. The length

and width of the ootheca were measured for both the normal and potassium alum-treated female groups, and the amount of food consumption was estimated by weighing the potassium alum and before and after the 4 d of treatment. The biochemical activity of the amylase, lipase, and protease enzymes was measured according to the methods described by Mikani et al. (2012). Newly emerged adults (7 d old) of both sexes were used for the experiments for the biochemical studies after treatment (the potassium alum-fed group and two controls: one control group with normal food and the other control without food, i.e., starved). All the cockroaches either normal, treated, or under treatment were provided with water from a cotton pad socked with water. Extraction. Male and female cockroaches from each of the three groups (that fed upon the potassium alum for 4 h, starved for 1, 2, and 4 wk, and with normal food as a control) had their midgut dissected in 50 mm Tris hydrochloride (Tris-HCL; pH 7.4) or phosphate buffered saline (PBS), and its contents were removed. After dissection, the male and female cockroach midgut tissues were homogenized and ultracentrifuged at 16,000 rpm for 10 min at 4 C. The supernatant was placed in a centrifuge tube and kept at 4 C for enzymatic quantification. Each experiment was repeated three times. The tissue extraction was incubated in 50 mm TrisHCL (pH 7.4) at room temperature for 30 min to measure the a-amylase activity in the midgut (the different concentrations of the tested enzymes were made from the prepared stock using PBS buffer for dilution; 1 ml was added to each sample). The activity of the released a-amylase was quantified. The samples (0.1 ml) were incubated at 37 C for 10 min, with 0.5 ml of substrate buffer containing 2-chloro-4-nitrophenyl 65 -azido-beta-maltopentaoside (N3-G5-CNP) and 0.5 ml of coworking enzyme solution containing glucoamylase and b-glucosidase. Then, the reaction was stopped by adding 2.0 ml of stop solution containing sodium carbonate. In all of the assays, a-amylase activity was calibrated proportionally to protein concentration. One unit (u) of a-amylase activity was defined as the amount of enzyme that produces 1 mmol 2-chloro4-nitrophenol (CNP) from N3-G5-CNP in 1 min at room temperature. The absorbance was measured at 400 nm using a PerkinElmer’s LAMBDA UV/Vis spectrophotometer. Lipase activity of the cockroach midgut was measured using a lipase measuring kit (Quantichrom Lipase Assay Kit, Bio Assay System, Hayward, CA). The tissue extracted was incubated individually in cold PBS for 30 min. Lipase activity released was quantified. To prepare the working reagent, 5 mg of color reagent was mixed in 140 ml of assay buffer and 8 ml of dimercaptopropanol tributyrate (BALB). The working reagent was freshly prepared and used within 1 h. Then, 140 ml of working reagent was added to 10 ml of each sample. The assay was based on an improved BALB method, in which SH groups formed from lipase cleavage of BALB react with 5,5’-dithiobis (2-nitrobenzoic acid). The optical density at 412 nm was proportionate to the

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SALAMA: POTASSIUM ALUM AS CONTROLLING AGENT AGAINST P. americana

enzyme activity of the sample, which was read using a microplate reader (SH-9000, Corona Electric, Ibaraki, Japan). One unit of enzyme catalyzed the cleavage of 1 ml of substrate per minute under the assay conditions. Protease activity of the midgut was measured by the digestion of azocasein, according to Sakai et al. 2006. The midgut tissues were incubated in 50 mm Tris-HCL (pH 7.4) at room temperature for 20 min. Protease activity was quantified. Samples of 300 ml with 300 ml of 0.5% (w/v) azocasein solution in Tris-HCL (pH 7.4) were incubated at 37 C for 30 min. Then, the reaction was stopped by adding 800 ml of 20% trichloroacetic acid and cooled on ice for 10 min. After removal of precipitated azocasein in the incubation mixture by centrifugation (4,000 g at 4 C, 15 min), the optical density of the supernatant at 335 nm was read. One unit (U) of hydrolytic activity of the protease was defined as the amount of enzyme required to cause an increase of 0.01 A335 units per minute in 1 ml of reaction mixture. The protein content in the resulted fraction was determined using the Lowry et al. (1951) method as the following: Reagents Used. 1. Alkaline sodium carbonate solution (20 g/liter Na2CO3 in 0.1 mol/1NaOH) 100/31. 2. Copper sulfate sodium potassium tartrate solution (5 g/liter CuSO4.5H2O in 10 g/liter Na, K tartrate) was prepared fresh by mixing stock solutions. 3. Alkaline solution was prepared on the day of use by mixing 50 ml of 1) and 1 ml of 2). 4. Folin–Ciocalteu reagent (stock solution). A solution of sodium tungstate (100 g), sodium molybdate (25 g), distilled water (700 ml), phosphoric acid (85%, 50 ml), and concentrated HCl (100 ml) was gently refluxed for 10 h. Then, lithium sulfate (150 g), distilled water (50 ml), and a few drops of bromine were added to the solution. The mixture was boiled for 15 min without a condenser to remove excess bromine. The solution was then cooled, brought to 1 liter with distilled water, and filtered. Finally, the resulting solution was titrated against 1NaOH to a phenolphthalein end point. Before use, the solution was diluted with distilled water (approximately twofold). 5. Bovine serum albumin (0.2 mg/ml) was used as a protein standard to determine the amount of protein in each sample. Chemicals Used. All of the chemicals were of analytical grade and were obtained from Sigma, Aldrich, and Fluka (England) unless otherwise stated. Potassium alum was supplied from Elnaser Pharmaceutical Chemicals Company at Abu Zaabal (Egypt). The enzymatic activities were measured as follows: amylase was measured using an a-amylase measuring kit (Kikkoman Corp) Chiba, Japan), lipase was measured by the Quantichrom Lipase Assay Kit (Bio Assay System, Corporate place, Hayward, CA), and protease activity of the midgut tissue extract was measured by digestion of azocasein. All of the reagents were freshly prepared to properly quantify enzymatic activity. Different

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concentrations were prepared using Tris-HCL (pH 7.4) for a-amylase and PBS for lipase. The dissected male and female cockroaches (from each of the starved, control, and potassium alum-fed groups) were placed in cold PBS to remove the contents from the midgut. After incubation for 30 min, the enzymatic activity was quantified. All the above solutions were filtered before use. Statistical Analysis. Data obtained were subjected to one-way analysis of variance (ANOVA; P < 0.05) after checking for normality and number of visits to normal food and potassium alum by male, gravid female, and third-instar nymphs. Means were compared by Duncan’s multiple range test, admitting significant differences (P < 0.05, df ¼ 4, F ¼ 12.01). The percentage of cockroach mortality was plotted against the recorded time, and the LT50 and LT95 values were determined graphically using SPSS 20, for Windows. Mortality percentage was corrected using Abbott’s formula (1925) if the mortality in control exceeds 5%. Data obtained were subjected to one-way ANOVA (P < 0.01, df ¼ 2, F ¼ 8.12) after checking for normality. Activities of digestive enzymes were subjected to ANOVA, and mean values were shown as bar charts. For each sex, standard error bars (6SE) were used to compare activities of each enzyme tested. Enzyme activities were also compared between male and female cockroaches. Statistical analysis was performed using SPSS for all analyses (SPSS version 20). Results The results of preliminary tests showed that contact or topical application of the saturated aqueous solution of potassium alum had no significant effect in inducing mortality, as the integument of the cockroaches was the perfect water proof to be applied in such way. Potassium Alum as a Stomach Poison. During this study, we had tested the preference of the American cockroaches to normal food and potassium alum, as presented in Table 1. Mean cumulative number of visits were recorded and indicated no significance in all tested stages (adult males, gravid females, and thirdinstar nymphs). The susceptibility levels of the different instars of P. americana after ingestion of potassium alum are presented in Tables 2, 3, and 4 and graphically illustrated in Figs. 1, 2, and 3. The results showed that the third nymphal instars and gravid females were the most susceptible stages with high statistical significance Table 1. Mean cumulative number of visits to normal food and potassium alum by males, gravid females, and third-instar nymphs (20 individuals each). Stages

No.

Normal food (white bread)

Potassium alum

Adult / Adult ? Nymph

20 20 20

11.6a (2.80) 14.8b (1.82) 10.2c (1.20)

10.4a (1.18) 13.2b (2.45) 8.8c (1.24)

Means within the same row followed by the same letter are not significantly different (P > 0.05; Duncan’s multiple range test [SPSS version 20]).

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Table 2. Mortality of different P. americana stages due to the effect of potassium alum based on the time LT50 and LT95 of the average for six treatments (40 individuals each) P. americana

LT50 (d)

Confidence interval 95% Lower limits

Slope 6 SE

v2

r

LT95 (d)

Upper limits

Third-instar 4.0 6 0.275 3.780 4.220 nymphs Gravid females 4.1 6 0.141 3.986 4.213 Females 19.0 6 0.547 18.562 19.438 Males 22.0 6 1.414 20.869 23.131

Confidence interval

Slope 6 SE

v2

r

Lower Upper limits limits 2.589 6 0.866

3. 678 0.897 10.0 6 0.209

9.833 10.167 0.981 6 0.16 3.897 0.9884

2. 379 6 0.4 63 3. 421 0.986 10.1 6 0.167 9.967 10.233 0.987 6 0.32 3.457 0.9683 2.895 6 0.567 3.967 0.945 28.0 6 1.549 26.761 29.239 0.89 6 0.15 0.976 0.9984 2.421 6 0.667 3.987 0.974 29.9 6 0.817 29.247 30.553 0.869 6 0.24 0.889 0.9876

Most of the adult cockroaches in control can live on water only for 60 d, but nymphs died within 20 d. Cumulative mortality at the end of the experiment was analyzed using Tukey’s test.

Table 3. The oothecae produced by normal females of P. americana and those fed with potassium alum at extrusion (the average of 25 oothecae 6 SE) P. americana oothecae

Normal strain mm or mg 6 SE

Length in mm Width in mm Weight in mg Percentage of hatching (%)

Fiducial limits

8.2 6 0.12 5.1 6 0.13 94.2 6 0.12 99.0 6 1.00

Lower limits

Upper limits

8.08 4.97 94.08 98.0

8.32 5.23 94.32 100

Potassium alum-fed females mm or mg 6 SE 5.3 6 0.31 3.0 6 0.45 70.0 6 0.56 15 6 0.51

Fiducial limits Lower limits

Upper limits

4.99 2.55 69.44 14.49

5.61 3.45 70.56 15.51

Table 4. Percentage of oothecae hatching according to the time of potassium alum feeding on the reproductive cycle of P. americana females at extrusion time P. americana oothecae percentage of hatching (%) Normal strain oothecae

99% 6 1.0

Lower limits

Fiducial limits Upper limits

Females at early stage of the cycle (48 h)

Females at the middle of the cycle (96 h)

98%

100%

0.0% 6 0.0

30.0% 6 3.56

(P < 0.05), followed by the females and males, according to the calculated LT50, which was found to be 4, 4.1, 19, and 22 d, respectively. Fig. 2A showed that newly molted P. americana nymphs died immediately after hatching with a pale color, but older nymphs with darker brown integument responded weakly to mechanical stimuli after feeding with potassium alum. Fig. 1 and Table 2 show that the mortality rate of the gravid females was significantly higher (P < 0.05) than that of normally fed controls, and the produced oothecae were severely underweight and exhibited a dwarfism shape after the gravid females fed on potassium alum. Normal females exhibited cyclical feeding patterns. They fed intensively prior to mating and the production of an ootheca, during ovulation and only minimally after oviposition. After depositing an egg case, females resumed feeding until the production of the next ootheca. However, in potassium alum-fed group, this feeding pattern ceased. In some cases, the female would deposit an undeveloped ootheca to save her life, but these females died soon after. This premature dropping of oothecae by gravid females, which lowered the survival rate of the oothecae, was caused by the sublethal effects of potassium alum due to

Fiducial limits Lower limits

Upper limits

26.44%

33.56%

Females at late stage of the cycle (144 h) 70.0% 6 2.56

Fiducial limits Lower limits

Upper limits

67.44%

72.56%

feeding during pregnancy (i.e., time of extrusion). In some cases, the undeveloped ootheca did not hatch, indicating that the potassium alum significantly (P < 0.05) affected not only the female but also the oothecae formed during feeding on potassium alum. The potassium alum consumption after 4 d of feeding by different instars of P. americana indicated that the ingested amount greatly affected the mortality rate: gravid females consumed 3 mg/weight of potassium alum per individual insect; third nymphal instars consumed 0.3 mg/weight of potassium alum per individual insect; and adult males and females consumed 0.1 and 0.3 mg/weight of potassium alum per individual insect, respectively. After treatment (i.e., ingestion or feed upon) with potassium alum, the cockroaches were stopped feeding. Those roaches never ate again even if they were offered normal food. The treated cockroaches also exhibited irreversible effects, such as severe paralysis (the insects respond weakly to mechanical stimuli), which can potentially impact cockroach populations through a combination of adult and nymphal mortality as well as ovicidal activity. This extended effect is considered phenomenal in P. americana control programs.

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Fig. 1. Mated females were selected from stock colonies at extrusion (i.e., beginning of ootheca formation). The produced oothecae were underweight and exhibited a dwarfism shape after feeding with potassium alum (Fig. 1B). Figure 1A shows a normal ootheca.

Fig. 2. P. americana nymphs with pale brown integument died shortly after molting, whereas the others with darker brown integument did not respond well to mechanical stimuli, especially after feeding with potassium alum (Fig. 2A). Figure 2B shows that the post nymphal instars failed to expand their wing pads to become fully adult.

The results showed that older nymphs, after feeding with potassium alum, died during molting or did not achieve expanded wing pads (Fig. 2B) and died prematurely. The effect of potassium alum was attributed to chronic toxicity not acute toxicity. We examined the effect of 4 wk of starvation and refeeding on a-amylase, protease, and lipase activities. Our results showed that the activities of all three enzymes decreased in the starved cockroaches in a

time-dependent manner. The activity gradually decreased during 4 wk of starvation. Starvation for 4 wk caused loss of 62%, 63%, and 44% of a-amylase, protease, and lipase activities, respectively, in the midgut of cockroaches (Fig. 4). Interestingly, refeeding increased a-amylase activity levels almost 60% in 3 h, illustrating that this effect is reversible (Fig. 4). Lipase activity was also affected by refeeding, showing an increase of 44% in 3 h of refeeding (Fig. 4). Protease activity also

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showed a similar change, and the females always exhibited higher enzymatic activity than the males did at 2 df and Significant F 12.01 at P < 0.05 levels of probability. However, the cockroaches’ digestive enzymes were greatly affected by treatment with potassium alum; therefore, these roaches did not recover enzymatic activity as the starved controls did. The effect of potassium alum was not reversed (Fig. 5), and the activities of a-amylase, protease, and lipase were greatly inhibited by 70%, 51%, and 31%, respectively, after 4 h of feeding with potassium alum. This is a significant loss of activity

7 Treated Normal

6

mg

5 4 3 2 1 0 3rd nymphal instars

Gravid females

Female

Male

Fig. 3. The potassium alum consumption of different P. americana instars after 4 d of feeding (25 individual each, and the bars represent SE) in comparison with normal one which never stops feeding only at time of molting or according to the reproductive cycle.

in comparison with the normally fed roaches. The enzymatic activity of the females was greatly reduced compared to that of the males at 2 df and Significant F 8.12 at P < 0.01 levels of probability. Discussion The food preference test showed that the potassium alum has a promising effect as a potential food poison, which found to be in accordance with Rust and Reierson (1977), Ballard and Gold (1982), and Brenner and Patterson (1988) concerning bait preference. Also, the results of this study showed that treatment with potassium alum affects not only the nymphs and adults of P. americana but also the oothecae produced by gravid females that had ingested potassium alum at the time of extrusion. This is the first study that shows an extended effect on the waterproof ootheca, also known as an ovicidal effect. This is the first study to show the extended ovicidal effects of potassium alum on P. americana, and the result is nearly conclusive. After treatment with potassium alum, P. americana offspring cannot build colonies in our homes. However, the results of this study also showed that potassium alum has no contact toxicity on the eggs of P. americana. One possible explanation for this result could be that this type of pianistic ovariole acts as a transferable link between the active ingredient and the ootheca when the ootheca is still attached to the female body, but this link no longer exists when the ootheca is separated from the female. However, in addition to effects in nymphs and adults, the results showed that the oothecae produced by gravid females that had eaten the potassium alum at the time of extrusion (the female

Fig. 4. Change in amylase, protease, and lipase activity in P. americana during 0, 1, 2, and 4 wk of starvation and after refeeding with normal food, compared with normal fed cockroach as a control. Each point represents three measurements 6 SE. The females consistently showed higher enzymatic activity than male roaches (P < 0.05, F ¼ 12,01, df ¼ 2).

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Fig. 5. The effect of potassium alum-fed males and females with references to normal food-fed and starved roaches on digestive enzyme activity—a-amylase, protease, and lipase activities. The activity of all three enzymes decreased in the starved group in a time-dependent manner. The enzymatic activity gradually decreased during 4 wk of starvation, and the potassium alum greatly affected the activity of digestive enzymes by 70, 51, and 31% for a-amylase, protease, and lipase, respectively, after 4 h. Each point represents three measurements 6 SE. The female’s enzymatic activity was significantly less than that of the male roaches (P < 0.01, F ¼ 8.12, df ¼ 2).

carries an ootheca and oocyte development is inhibited during pregnancy); the produced ootheca were underweight and exhibited a dwarfism shape. Additionally, few to no nymphs emerged from these eggs. This result may be due to the reduced water content of the ootheca caused by the potassium alum. The deposited ootheca contains enough water for the eggs to develop without receiving additional water from the substrate (Bell and Adiyodi 1981). Normal females exhibited cyclical feeding patterns. They fed intensively prior to mating and the production of an ootheca, during ovulation, and only minimally on oviposition. After depositing an egg case, females resume feeding until the production of the next ootheca. However, in the potassium alumtreated group, this feeding pattern ceased. In some cases, the female had to drop an underdeveloped ootheca to save her life, although these females died soon after. This finding was in accordance with results found by Hamilton and Schal (1990), Harmon and Ross (1987, 1988), Haynes (1988), and Wooster and Ross (1989), who reported the premature dropping of oothecae by gravid females by German cockroaches, leading to a lower survival rate of the oothecae due to the sublethal effects of insecticide treatments. Similar results were observed by Bressan-Nascimento et al. (2008). They reported that extreme temperatures affected embryonic development, resulting in enviable ootheca. Our results were also in accordance with those reported by the previously mentioned authors concerning the normal oothecae dimension, which is usually 8 mm long

and 4 mm wide, weighing 95 mg. Our results were also in accordance with King (2005), who evaluated the ovicidal activity of the benzoylphenylurea noviflumuron in the laboratory on three adult groups (virgin females, virgin males, and fertilized, nongravid females) of the German cockroaches, Blattella germanica (L.), through ingestion of treated bait. He reported that noviflumuron caused a significant ovicidal effect. Noviflumuron is an insect growth regulator that prevents successful molting. Potassium alum presents more advantages as an insecticide, as it induces death in adults at the nonmolting stage and in the molting nymphs. The high susceptibility of the nymphs and gravid females is due to their increased feeding habits, which leads to them ingesting more potassium alum. This observation is in accordance with the results of Woodruff (1938) and Eidmann (1924), who concluded that insects (cockroaches) approximately double in weight between instars. Additionally, the gravid females in gonotrophic cycle require more food during their reproductive cycle. A key finding when comparing the effect of potassium alum on P. americana with the effects of other insecticides that are usually used to control cockroach infestation is that the usual insecticides lower the population number but do not affect the hardened egg cases of the cockroaches, which are impervious to most insecticides and are frequently placed in protected locations. Most conventional insecticides only reduce the population without affecting the ootheca, which

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allows P. americana to build a new colony after treatment. The results of this study suggest that potassium alum can safely control nymphs, adults, and the oothecae. This affect could be a revolutionary change in control programs against cockroach infestation. As feeding upon potassium alum make the insect dies peacefully, giving no sign of pain to other individuals. In our attempt to provide a logical explanation for the phenomenon of death of the cockroaches treated with potassium alum, we might say that this compound works as an oxidizing agent, making the body of the insect work as a reducing agent. As an electron donator, this distribution of the osmotic balance inside the insect leads to a loss of its own symbiotic flora, which help the insect to digest its food, resulting in reduced enzymatic release. In normal cockroaches, this leads to mortality of the insect. This theory is in agreement with Peleg et al. (1998), who reported that the chelation of the aluminum ion in alum by acids reduces its availability for interacting with salivary proteins or epithelial proteins, indicating that the alums have an astringent effect. Lee et al. (2012) also found that the alum astringency might be due to the precipitation of salivary proteins. The extended effect from the female at time of extrusion to the oothecae may be due to the reduced water content in the oothecae. This reduced water content also explains the reduced size of the ootheca and the reduced chance of nymph emergence. Female cockroaches fed potassium alum before mating and after the ootheca had protruded from the abdomen were highly affected. Comparison of these data revealed that high mortality due to potassium alum is most likely caused by the interaction of several factors. Digestive enzymes were detected in the midgut of P. americana, and the activity of a-amylase was generally found to be higher than that of males in the midgut. A similar trend was observed for proteinase activity in the midgut. However, for lipases, the activity was higher in the midgut of females than in that of males, a result also found by Oyebanji et al. (2014). This result could explain the great susceptibility of the females with lower LT50 than the male cockroaches, which shows the highest LT50 among all the instars tested. Our results were in agreement with those reported by Hivrale et al. (2005), indicated by the presence of 11 proteinases in the gut of P. americana. Vinokurov et al. (2007) also recorded high proteolytic and amylolytic activities in the midgut of P. americana with a moderate activity in the crop. Cockroaches have been the most popular group of insects for studies of lipid digestion, and a number of early studies indicated lipolytic activity in the fore- and midgut. It has also been demonstrated that lipase originates in the epithelial cells of the midgut. Thus, the presence of lipolytic activity in the foregut results from regurgitation of midgut contents into the foregut (Downer, 1978). The digestive enzymes in insects are adapted to the diet on which they feed. Omnivorous insects such as the cockroach secrete protease, lipase, amylase, invertase, and maltase, to digest hydrolysing respectivly

natural proteins, fats, starch, cane sugar, and maltose, respectively, as reported by Swingle (1925) and Wigglesworth (1927). In our study, the cockroaches were inclined to eat potassium alum because of its sweet taste. Our results showed that potassium alum lead to inhibition of the digestive enzymes a-amylase, protease, and lipase, resulting in their death. Starvation exerts fatal stress on animals and affects various aspects of life such as behavior, development, reproduction, and metabolism, as reported by Park and Takeda (2008) and Park et al. (2009). Starvation for 10 d reduced a-amylase and protease activities by 60 and 67%, respectively, in the midgut of Gryllus rubens. Starvation reduced lipase activity in a manner similar to its effect upon a-amylase and protease in the midgut, as indicated in our study, which is in accordance with the results reported by Thomas and Nation (1984). However, the activities of these enzymes drastically changed after refeeding. The mechanism of enzymatic release comes from the brain of cockroaches, but feeding on potassium alum greatly inhibited the activity of these enzymes, which lead to cockroach mortality, i.e., irreversible damages. It is possible that the sweet taste of potassium alum made the cockroaches think that it is a carbohydrate, a substance that is easily digested by cockroaches. Potassium alum could actively harm the symbiotic microorganisms found in the midgut, which produce a number of carbohydrates that had been reported by Scrivener et al. (1998) and Genta et al. (2003). Unlike c-amylases, b-amylase is not produced by animals, although it may be present in microorganisms found the digestive tract (Adachi et al. 1998, Mikami et al. 1999). The detection of a-amylase in the present study may indicate that some symbiotic microorganisms reside in the midgut of P. americana. This in accordance with Zurek and Keddie (1996), who reported that the gut bacteria in P. americana played a functional role in the development and survival of the insect species. Additionally, their omnivorous, detritophagous feeding habit and association with symbiotic bacteria (Cruden and Markovetz, 1987) has contributed to their survival for at least 350 million years (Thorne and Carpenter 1992). The higher lipase activity in the midgut of females with greater inhibition after potassium alum feeding explains the early mortality observed in the gravid female during our study and the accompanied dwarfism of the oothecae produced by these females, which agrees with the results of Kawooya and Law (1988) and Briegel (1990). They reported that it is possible that female wasps maximize the use of lipase substrates in the midgut to meet specific reproductive requirements. Lipids, mostly triacylglycerol (TAG), and smaller amounts of phospholipids (PL) and cholesterol make up 30–40% of the dry weight of the insect oocyte. Our view meets with that of Weaver et al. (1995) who interpret that the rapid increase in juvenile hormone (JH) biosynthesis and JH titer was required to support reproductive processes such as vitellogenin production by body fat, vitellogenin uptake by the oocytes, and synthesis of oothecal proteins by the left colleterial gland. Additionally, lipids are the main sources of

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SALAMA: POTASSIUM ALUM AS CONTROLLING AGENT AGAINST P. americana

energy for the developing embryo (Beenakkers et al. 1981), and PL is needed for the formation of membranes. Insect oocytes synthesize TAG using fatty acids (FA) (Lubzens et al. 1981, Ferenz 1985), but because the ability of oocytes to synthesize FA de novo is limited, Ziegler and Van Antwerpen (2006) concluded that nearly all the lipids must be imported from ingested food. This requirement might necessitate the maximal digestion of lipase substrates in the female midgut. We will publish a detailed account of the histopathological effects in a separate paper. Additionally, the effect of potassium alum against Bellatella germanic is currently under investigation in our laboratory, and the results will be in press soon. In conclusion, the results of this study confirmed the insecticidal efficiency of potassium alum on P. americana adults and nymphal instars. The American cockroach has the longest life span, often lasting up to two and half years from egg to adult. Because adult female cockroaches produce egg cases repeatedly over relatively short lengths of time throughout their lives (over 10–12 oothecae), potassium alum would be the best option for eliminating infestations caused by the fast reproductive potential of female cockroaches. Our results show that the potassium alum can control both the nymphs and adults, with an extended effect on offspring (oothecae; i.e., ovicidal effect); thus, it may be recommended to use potassium alum in control management strategies, which will generally remain effective under field conditions for extended periods (i.e., until they are consumed by the cockroaches), and as a protective measure against cockroach infestation without environmental effects against humans or other nontargeted organisms.

Acknowledgments We thank the Ministry of Higher Education at Saudi Arabia for the chance to conduct a research in this area. Special thanks to Dr. Zinab Salama, Dr. Ahmed Salama, Dr. Mohamed Elsesi, and Dr. Amira Alaa for their technical help and kind support. Deep appreciation goes to the staff members and the faculty of Science, Benha University, for encouragements and sincere help.

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