ISJ 3: 89-96, 2006 ISJ 3: 97-102, 2006 ISSN 1824-307X SHORT COMMUNICATION Morphological abnormalities in Tribolium castaneum (Herbst) and Tribolium confusum Duval due to cyromazine and pirimiphos-methyl treatments alone or in combination AHM Kamaruzzaman1, AMS Reza2, KAMSH Mondal3, S Parween2 1Institute of Biological Sciences, Rajshahi University, Rajshahi 6205, Bangladesh 2 Department of Zoology, Rajshahi University, Rajshahi 6205, Bangladesh 3Member, Bangladesh Public Service Commission, Agargaon, Dhaka, Bangladesh Accepted November 03, 2006 Abstract Newly hatched (24 h old) larvae of Tribolium castaneum and T. confusum were allowed to feed on different doses of cyromazine or pirimiphos-methyl, or on a combined dose of both compounds up to pupation. All the treatments produced deformities at all the life stages. Cyromazine produced a number of abnormalities in the larval stage (P < 0.001) of the two species. Both the compounds produced similar type of deformities in the adults, but the effect was slightly more in the female T. confusum. The combined action (10 ppm cyromazine + 0.1 ppm pirimiphos-methyl) of the compounds also produced deformities at each stage; and the effects were more pronounced than the effect caused by a single dose of either 10 ppm cyromazine or 0.1 ppm pirimiphos-methyl, and this will produce less stress on the environment and human health. Key words: abnormalities; Tribolium; cyromazine; pirimiphos-methyl Introduction Insects surviving insecticidal treatments very often become variously deformed, and the formation of chimeric individuals and elytral deformities in adults are very common. The organophosphorous insecticide pirimiphos-methyl has been reported to produce morphogenetic abnormalities in treated insects including Tribolium species (Khan, 1981; Mondal, 1984; Rahman, 1992). The insect growth regulators (IGRs) are also able to produce various morphological abnormalities in treated insects (Stall, 1975). Among the IGRs, chitin synthesis inhibitors (CSIs) interfere with the formation of new cuticle (Hajjar, 1985), and disturb the process of ecdysis. A number of these compounds affects moulting in insects (Hajjar, 1985), among which the triazine compounds are effectively used to control dipteran insects in poultry (Bloomcamp et al., 1987), animal house (El-Oshar et al., 1985) and public health (Awad and Mulla, 1984; Nelson et al., 1986). Cyromazine, derived from azido-triazine herbicides (Shen and Flapp, 1990), is effective against dipteran larvae (Fox, 1990); but was ___________________________________________________________________________ Corresponding author: Dr. A.M. Saleh Reza Department of Zoology, Rajshahi University Rajshahi 6205, Bangladesh E-mail: salehbgd@yahoo.com found to be active against the larvae of Colorado potato beetle, Leptinotarsa decemlineata (Say) (Bishop et al., 1990; Sirota et al., 1993; Sirota and Grafius, 1994). As a CSI compound, cyromazine affects growth and development in different insect species, including T. castaneum (Herbst) (Mondal and Port, 1995). The present study is aimed to assess the abnormalities produced in flour beetles Tribolium castaneum (Herbst) and T. confusum Duval due to the activities of cyromazine and pirimiphos-methyl alone or in combination, using low doses of both the compounds for protection of the stored products destined for human consumption. Materials and Methods Insects used Laboratory strains of Tribolium castaneum and Tribolium confusum were used in the present experiment. The insects were collected from the IPM Laboratory, Institute of Biological Sciences, Rajshahi University. The stock cultures of these species were maintained in the laboratory for the last 15 years. Compounds used The tested compounds were a chitin synthesis 97 mailto:salehbgd@yahoo.com Table 1 Larval deformities produced by cyromazine or pirimiphos-methyl alone and by their combination in T. castaneum (N = 250) Treatment (ppm) Pupal recovery (%) No. of abnormal larvae No. of larval- pupal intermediates Total no. of abnormal larvae (%) Control (0) 241 (96.40) - - - Cyromazine 10 20 30 228 (91.20) 215 (86.00) 197 (78.80) 09±0.23a 14±0.04b 17±0.31b 04±0.64a 07±0.5a 09±0.33ab 13±0.42 (5.20)a 21 ± 0.67 (8.40)b 26±0.34 (10.40)bc Pirimiphos-methyl 0.1 0.2 0.4 230 (92.00) 196 (78.40) 180 (72.00) 07±0.41a 15±0.36b 19±0.1b 00 04±0.06a 05±0.72a 07± 0.48 (2.80)a 19 ± 0.39 (7.60)b 24±0.05 (9.60)b Cyromazine + Pirimiphos-methyl 10 + 0.1 190 (76.00) 16±0.7 08±0.48 24±0.55 (9.60)b Note: data with the same letters do not differ significantly from each other (P > 0.05 DMRT) inhibitor, cyromazine and an organophosphorous insecticide pirimiphos-methyl. Cyromazine was kindly supplied by the Ciba Geigy as Larvedex ca 98.4% wp formulation. Pirimiphos-methyl was purchased from the local agrochemical shop. Doses used Doses were prepared by mixing required amount of each of the compound and mixed with standard food medium (19:1, whole wheat flour: Brewers’ yeast) to obtain the doses in ppm unit. The doses prepared for cyromazine were 10, 20 and 30 ppm and those for pirimiphos-methyl were 0.1, 0.2 and 0.4 ppm. The single combined dose used was prepared as 10 ppm cyromazine + 0.1 ppm pirimiphos-methyl (lowest doses of both the compounds). The doses of cyromazine and pirimiphos-methyl chosen were based on the mortality tests of the compounds on the larvae of T. castaneum and T. confusum (Kamaruzzaman et al., 1999; Kamaruzzaman, 2000). Experimentation Newly hatched larvae (12 h old) of both species were collected separately from sub-cultures of the beetles. The larvae were released in food medium treated with different doses of either cyromazine or pirimiphos-methyl or cyromazine + pirimiphos- methyl. The larvae were reared up to pupation. After every three days the food material was replaced by a fresh one treated with same dose and compound. The pupal recovery (%) was recorded. The pupae were sexed according to Halstead (1963) and kept separately for emergence of adults. Adult recovery (%) was recorded. The morphologically abnormal individuals were separated. The deformed characters were studied, and the number of abnormal individuals was counted for each life stage. A set of control larvae was reared similarly on untreated food. Fifty larvae of each species were used for each treatment, each dose and control. The experiments were carried at 30±1 0C in an incubator, without controlling light and humidity, and replicated five times. Statistical analyses The abnormalities produced by different treatments alone or in combination and in different species of Tribolium were tested for significance using analysis of variance (ANOVA). The effect of the different doses of each treatment with respect to control was tested with Duncan’s Multiple Range Test (DMRT). Results Abnormal characters produced Cyromazine treatment produced various types of abnormalities in the larvae and adults of both species. Abnormalities produced by cyromazine were recorded as follows: a) Larval abnormalities i) reduced body size, ii) swelling in the integument/cuticular lesions, iii) stiffness of the cuticle, iv) incomplete metamorphosis: larviform pupae, pupal head with larval body, and pupa with larval skin. 98 Table 2 Larval deformities produced by cyromazine pirimiphos-methyl alone and by their combination in T. confusum (N = 250) Treatment (ppm) Pupal recovery (%) No. of abnormal larvae No. of larval- pupal intermediates Total no. of abnormal larvae (%) Control (0) 240 (96.00) - - - Cyromazine 10 20 30 230 (92.00) 221 (84.40) 209 (83.60) 10±0.88a 15±0.53ab 21±0.55b 05±0.72a 06±0.6a 08±0.46a 15±0.49 (6.00)a 21±0.55 (8.40)b 29±0.32 (11.60)c Pirimiphos-methyl 0.1 0.2 0.4 227 (90.80) 198 (79.20) 195 (78.00) 08±0.52a 12±0.62ab 14±0.55b 00 05±0.46a 06±0.31a 08±0.51 (3.20)a 17±0.33 (6.80)b 20±0.05 (8.00)bc Cyromazine + Pirimiphos-methyl 10 + 0.1 200 (80.00) 15±0.43b 07±0.06a 22±0.71 (8.80)bc Note: data with the same letters do not differ significantly from each other (P > 0.05 DMRT) b) Adult abnormalities i) bent abdomen, ii) incomplete elytra. Pirimiphos-methyl alone or in combination with cyromazine produced a similar type of deformities in both species, as follows: i) reduced larval body size, ii) larval-pupal intermediates, iii) adultoids and incomplete elytra in adults. The abnormal individuals with incomplete metamorphosis at larval-pupal transformation were categorized as larval abnormality and the adultoids as pupal abnormality. Percentages of abnormalities produced Cyromazine produced a higher percentage of abnormal larvae in T. confusum than T. castaneum (Tables 1, 2). Effect of cyromazine on the normal growth of larvae was found to be dose related (P < 0.001, F = 87.219), and was similar in both species (P > 0.05, F = 0.981). The number of abnormal larvae was higher than the number of larval-pupal intermediates, in both species of Tribolium. Pirimiphos-methyl produced comparatively less number of abnormal larvae in both species (P > 0.05, F = 1.31), though the effect varied with the doses (P < 0.001, F = 84.283) (Tables 1, 2). The combined effect of the IGR and the insecticide produced a greater effect on the morphogenesis of the larvae of both species (P > 0.05, F = 0.4). The effects of the combined treatment was more than the effects recorded with the lower doses of either cyromazine or pirimiphos- methyl (P < 0.001, F = 211.6) (Tables 1, 2). Cyromazine produced similar abnormal adults at same extent in both sexes of T. castaneum (Table 3), whereas the effect was slightly greater in the females of T. confusum (Table 4). The percentages of abnormal adults produced were not significant between the species (P > 0.05, F = 0.067); but significant differences of the effect was observed between the doses in T. confusum (P < 0.05, F = 9.25). Pirimiphos-methyl treatments also produced similar percentage of adult abnormalities in both the species (P > 0.05, F = 0.72), which did not vary between the sexes of T. castaneum (Table 3), but the females were more affected than the males of T. confusum (Table 4). The adult morphogenesis of the beetles slightly varied among the doses (P < 0.01, F = 10.47). The combined treatment of cyromazine (10 ppm) and pirimiphos-methyl (0.1 ppm) produced a greater effect on the adult morphogenesis than the single treatment with either cyromazine or pirimiphos 99 Table 3 Adult abnormalities produced by cyromazine or pirimiphos-methyl alone and by their combination in T. castaneum (N = 250) No. of abnormal individuals (%) Treatment (ppm) Total Adult recovery (%) Male (no.) Female (no.) Male Female Control (0) 237 (94.80) 115 122 - - Cyromazine 10 20 30 218 (87.20) 204 (81.60) 188 (75.20) 117 109 100 101 95 88 09±0.06 (7.69)a 09±0.2 (8.26)a 09±0.11 (9.00)a 06±0.21 (5.94)a 07±0.13 (7.37)a 12±0.31 (13.64)a Pirimiphos-methyl 0.1 0.2 0.4 209 (83.60) 189 (75.60) 168 (67.20) 115 95 96 94 94 72 08±0.21 (6.96)a 09±0.05 (9.47)a 09±0.21 (9.37)a 07±0.07 (7.45)a 09±0.23 (9.57)a 13±0.15 (18.05)b Cyromazine + Pirimiphos-methyl 10 + 0.1 185 (74.00) 107 78 19±0.25 (17.76)b 15±0.22 (19.23)b Note: data with the same letters do not differ significantly from each other (P > 0.05 DMRT) methyl at the same dosage (Tables 3, 4). The combined treatment produced similar effects in both species of Tribolium (P > 0.05, F = 0.034). Discussion The mode of action of cyromazine is different from that of pirimiphos-methyl. Being a chitin synthesis inhibitor, cyromazine affects the mechanical properties of the insect cuticle and produces abnormalities in the skin, and resists moulting (Fox, 1990). Inhibition of moulting results in increase of the internal body pressure in the larvae (Fox, 1990), producing swellings on the cuticle (cuticular lesions) (Kotze and Reynolds, 1993; Sirota et al., 1993; Sirota and Grafius, 1994). So, larval deformities are common in CSI treated insects. The abnormal characteristics as noted in the present experiment, have been also reported in cyromazine treated Colorado Potato beele (Sirota and Grafius, 1994) and triflumuron (a CSI compound) treated T. castaneum (Parween, 1998). Due to CSI activity insect cuticle often becomes stiff (Fox, 1990). Consequently feeding is often hampered, as reported by Neuman and Guyer (1988), Soltani (1984) and Parween (1996) in different insects. Even is some could survive, the starved larvae were reduced in body size. Pirimiphos-methyl had been reported to avoid feeding on the treated medium Tribolium species, and as a result adults loose weight and length (Mondal, 1984b; Rahman, 1992). Pirimiphos-methyl affected the larval-pupal transformation and produced intermediate forms along with adult abnormalities. Both cyromazine and pirimiphos-methyl affected growth of the emerged adults, which were superficially normal but with deformed elytrae. The combined action of cyromazine and pirimiphos-methyl to some extent was greater on the morphogenesis of Tribolium species, than the action of single treatment of either compound. All the abnormal larvae and larval-pupal intermediates failed to survive long, and the abnormal adults were found to be uncapable to mate or oviposite. Cyromazine is easily miscible with most of the standard insecticides and fungicides (Fox, 1990), and in the present study was found to reduce the doses of insecticide used, producing the same effect. So, it can be used combined along with traditional insecticides at minimal doses for the protection of the stored products against beetle infestation, which may produce less stress on the environment and human health. Both T. castaneum and T. confusum have become resistant against most of the insecticides used. For insect management in the grain and cereal stores, high doses of these insecticides are needed, which affect the human health, the environment and its biota. To overcome this problem, cyromazine could be used with pirimiphos-methyl against Tribolium species, since this association would be able to produce abnormal individuals. The abnormal larvae or adults either would die or fail to 100 Table 4. Adult abnormalities produced by cyromazine or pirimiphos-methyl alone and by their combination in T. confusum (N = 250) No. of abnormal individuals (%) Treatment (ppm) Total Adult recovery (%) Male (no.) Female (no.) Male Female Control (0) 237 (94.80) 116 121 - - Cyromazine 10 20 30 219 (87.60) 207 (82.80) 197 (78.80) 114 110 105 105 97 92 08±0.21 (7.02)a 08±0.03 (7.27)a 11±0.21 (10.48)b 06±0.04 (5.71)a 10±0.22 (10.31)b 14±0.5 (15.22)bc Pirimiphos-methyl 0.1 0.2 0.4 221 (88.40) 194 (77.60) 188 (75.20) 116 101 102 105 93 86 06±0.06 (5.17)a 10±0.22 (9.90)b 14±0.22 (13.72)bc 10±0.15 (9.52)b 11±0.21 (11.83)b 10±0.03 (11.63)b Cyromazine + Pirimiphos-methyl 10 + 0.1 195 (72.78) 103 92 21±0.15 (20.39)c 11±0.3 (11.96)b Note: data with the same letters do not differ significantly from each other (P > 0.05 DMRT) develop further or to reproduce, and ultimately the population would be controlled. Moreover, as very low doses of these compounds could be used, the residues may not create hazard to the stored commodities as well as to the environment. References Awad TI, Mulla MS. Morphogenic and histopathogenic effects induced by the insect growth regulator cyromazine in Musca domestica (Diptera: Muscidae). J. 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