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19 archives of phytopathology and plant protection.pdfArchives of Phytopathology and Plant ProtectionVol. 45, No. 14, August 2012, 1741–1763 Sublethal effects of pyriproxyfen, a juvenile hormone analogue, onPlutella xylostella (Lepidoptera: Plutellidae): life table study M. Alizadeha*, J. Karimzadehb, G.R. Rassouliana, H. Farazmandc,V. Hoseini-Naveha and H.R. Pouriana aDepartment of Plant Protection, Campus of Agriculture and Natural Resources, University ofTehran, PO Box 4111, Karaj 31587-11167, Iran; bDepartment of Plant Protection, Isfahan Research Centre for Agriculture and Natural Resources, PO Box 199, Isfahan 81785, Iran; cDepartment of Agricultural Entomology, Iranian Research Institute of Plant Protection, (Received 10 May 2012; final version received 15 May 2012) The sublethal effects of pyriproxyfen (PYR, a juvenile hormone analogue) wereinvestigated for two consecutive generations on life-history parameters, such as developmental time, pupal weight, fecundity, fertility and longevity of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Aftertopical application bioassay of third instar larvae, the LC25 and LC50 values (assublethal doses) were determined to be 1.49 and 2.01 mg/ml, respectively. The results showed that the egg incubation period, larval and pupal duration times and oviposition period were increased in treated groups compared with thecontrol (treated with distilled water). Pupal weight, adult longevity, fecundity and fertility were significantly reduced in the treatment groups compared with thecontrol. Using the age-stage, two-sex life table analysis, we found that the intrinsic rate of increase (r), finite rate of increase (l), gross reproduction rate(GRR) and net reproductive rate (R0) significantly decreased while the meangeneration time (T) and doubling time (DT) increased in two treatment groups compared with the control. In addition, administration of PYR induced morphogenetic abnormalities including untanned pupae, larval–pupal intermedi- ates and various defective adults. The LC50 value was more effective on all of theabove biological characteristics than the LC25 for both generations, although parents were more affected than the offspring. In general, our results revealed that PYR was highly effective against P. xylostella in the laboratory both directly (causing mortality) and indirectly (disruption of normal growth and develop- ment). We conclude that PYR is an excellent candidate for suppressing populations of P. xylostella through its sublethal effects.
Keywords: Plutella xylostella; pyriproxyfen; sublethal concentrations; age-stage; two-sex life tables; sublethal effects; life table The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), isone of the most destructive cosmopolitan insect pests of cruciferous plants in manyparts of the world (Talekar and Shelton 1993). In many countries, P. xylostella has *Corresponding author. Email: firstname.lastname@example.org ISSN 0323-5408 print/ISSN 1477-2906 onlineÓ 2012 Taylor & Francishttp://dx.doi.org/10.1080/03235408.2012.706426http://www.tandfonline.com M. Alizadeh et al.
developed multiple- and cross-resistance to a wide range of conventional organicinsecticides as well as Bacillus thuringiensis (Bt) products (Tabashnik et al. 1990;Tabashnik 1994; Zhao et al. 2002, 2006; Sarfraz and Keddie 2005; Raymond et al.
2007; Qian et al. 2008; Sayyed et al. 2008; Gassmann et al. 2009; Nehare et al. 2010;Santos et al. 2011). The development of insecticide resistance in field populations ofDBM due to the high frequency of insecticides application has necessitated the use ofalternative strategies and new management tactics. The use of very low doses ofinsecticides having strong sublethal effects represents an environmentally friendlyoption to improve existing integrated pest management strategies (Sial and Brunner2010). The sublethal effects of pesticides influence the physiological or behaviouralresponses of individuals that survived prior exposure to pesticides (Desneux et al.
2004). The sublethal concentrations of insecticides may affect a number ofphysiological parameters including weight of larvae, pupae and adults (Yin et al.
2008; Nasr et al. 2010), fecundity, fertility (Perveen and Miyata 2000; Liu and Trumble 2005), egg size (Yin et al. 2008), adult longevity (Gerig 1975) anddevelopment speed (Cripe et al. 2003). They may also alter sex ratios (Shaalan et al.
2005) and affect behavioural parameters influencing feeding (Nasr et al. 2010), searching and oviposition (Dabrowski 1969; Fujiwara et al. 2002). The sublethal doses also affect reproduction parameters such as the intrinsic rate of increase (r),finite rate of increase (l), gross reproduction rate (GRR), net reproductive rate (R0), mean generation time (T) and doubling time (DT) (Zanuncio et al. 2005; Yin et al.
2008). A useful component of an integrated pest management is the use of insect growth regulators (IGRs) such as juvenile hormone analogues (JHAs) (Kostyu- kovsky et al. 2000; Sial and Brunner 2010). Pyriproxyfen (PYR), a JHA, mimics theaction of juvenile hormones in a number of physiological processes and is a potent inhibitor of embryogenesis, metamorphosis and adult formation (Ishaaya and Horowitz 1992). In addition, it has been shown to have sterilising and toxic activitieson many insects in a variety of orders, including Dictyoptera (Koehler and Patterson1991), Hymenoptera (Reimer et al. 1991), Diptera (Langley et al. 1993), Orthoptera (Vennard et al. 1998), Homoptera (Liu and Chen 2001; Boina et al. 2009), Siphonoptera (Rajapakse et al. 2002), Coleoptera (Abo-Elghar et al. 2004) and Lepidoptera (Hatakoshi et al. 1991; Yokoyama and Miller 1991; Oouchi 2005; Kwon and Kim 2007; Sial and Brunner 2010). The objective of the present work was to evaluate the insecticidal activity and sublethal effects of PYR on life-history parameters of P. xylostella. In particular, the effects of PYR were observed on the mortality, developmental time of immature stages, pupal weight, adult fecundity, fertility, longevity and the incidences of various abnormalities in P. xylostella for two Materials and methods Insects culturing procedure Plutella xylostella larvae and adults with no history of exposure to insecticides wereoriginally collected from Brassica fields at the Horticultural Investigation Center ofTehran University in Karaj (Alborz province, Iran) during the 2009–2010 growthseasons. Chinese cabbage (B. pekinensis) cv. Spring Smile (Kumochon-Dong Co.,Seoul, South Korea) was grown under greenhouse conditions (25 + 58C, 65 + 10%RH and a photoperiod of 16L:8D). The stock culture of DBM was maintained on Archives of Phytopathology and Plant Protection 8-week-old Chinese cabbage in screened hyaline cages (40 6 40 6 40 cm) understandard constant environment (25 + 18C, 65 + 5% RH and a photoperiod of16L:8D h).
Bioassay and determination of sublethal doses The concentrations of 1.5, 1.75, 2, 2.4, 2.8 and 3 mg/ml of PYR (ADMIRAL1 10%EC; Sumitomo Chemical Co, Japan) were made using distilled water. To perform abioassay, 0.5 ml of the chemical was topically applied onto the dorsum of the thoraxof early third instar larva using a Hand micro-applicator (Burkard ManufacturingCo., Rickmansworth, UK). Batches of 15 treated larvae were transferred to freshChinese cabbage leaf discs (6 cm diameter) within individual Petri dishes (9 cmdiameter, with a 20-mesh polyester net embedded in the lid for ventilation) and maintained in a growth chamber set at 25 + 18C, 65 + 5% RH and a photoperiod of 16L:8D h. All treatments were replicated four times, and mortality was recordedafter 96 h. Finally, the LC25 and LC50 values were selected as sublethal con-centrations for the experiments.
Effects of sublethal doses on biological characteristics of parent generation The sublethal doses of PYR were applied on 100 early third instar larva as explainedin the previous section. Thereafter, larvae were individually placed on fresh Chinese cabbage leaf discs (3 cm diameter) in Petri dishes (5 cm diameter) and allowed to develop to pupation. To prevent starvation of larvae, the leaf discs were replacedevery 12 h. The early pupae were preserved at 48C until all the larvae had pupated and then the pupae were kept at room temperature until adult emergence. The experiments were conducted under controlled environment conditions (25 + 18C,65 + 5% RH and 16L:8D h). Life stage, mortality and deformities were recordedevery 4 h until all the insects had either died or emerged as adults. The pupal weight was measured using an OHAUS Analytical Plus AP250D digital scale (OHAUS1 To measure fecundity and biological parameters of the DBM, a pair of moths (male and female) that emerged on the same day was placed into a Petri dish (5 cm diameter) containing a fresh Chinese cabbage leaf disc (3 cm diameter) to mate and lay eggs. The eggs laid on the Petri dishes wall and leaves were counted every 12 h and the Petri dish replaced with a fresh one until the females died. Twenty-five pairs of P. xylostella adults were used for each treatment. At the end of the experiment, adult sizes were measured using a stereoscopic microscope (Stemi V6, ZIESSGermany) fitted with a drawing tube.
Effects of sublethal doses on biological characteristics of offspring generation One hundred eggs were obtained from parent adults of each treatment. Each egg wasplaced in a Petri dish similar to that described in the previous section. The eggincubation, larval and pupal periods, pupal weight, mortality and abnormalitieswere recorded as in the previous generation. Following the emergence of adults, wepaired one male with one female as above. Thereafter, we supplied fresh Chinesecabbage leaf discs for oviposition and recorded the fecundity every 12 h until thedeath of all individuals.
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Age-stage, two-sex life table analysis All data obtained on the survival rates of adults and those individuals that diedbefore maturity, and the female daily fecundity as described above, were analysedaccording to the age-stage, two-stage life table theory (Chi and Liu 1985; Chi 1988).
The age-stage specific survival rate (sxj) (where x ¼ age in days and j ¼ stage; the first,second, third, fourth and fifth stages are the egg, larval, pupal, adult including femaleand male, respectively), the age-stage specific fecundity (fxj), the age-specific survivalrate (lx), the age specific fecundity (mx) and the population parameters (r, theintrinsic rate of increase; l, the finite rate of increase, l ¼ er; R0, the net reproductiverate; T, the mean generation time) of the offspring are calculated accordingly. Theintrinsic rate of increase is calculated iteratively from the Euler–Lotka equation withage indexed from 0 (Goodman 1982): eÿrðxþ1Þlx mx ¼ 1 In the age-stage, two-sex life table, according to Chi and Liu (1985), the lx and mx areestimated as: where k is the number of stages (Chi and Liu 1985). The GRR is defined as follows, where d is the last age of the cohort.
The net reproduction rate (R0) is calculated as R0 ¼ F(Nf/N), where F is the meanfemale fecundity, N is the total number of individuals used for the life table studyand Nf the number of female adults (Chi 1988). The mean generation time (T) is theperiod of time that a population needs to increase to R0-fold of its size atthe stable age-stage distribution and is estimated by using the following formula:T ¼ [(ln R0)/r]. To simplify analysis of the raw data, the computer program,TWOSEX-MSChart (Chi 2008), was used for data analysis and the jackknifemethod (Sokal and Rohlf 1995) in Visual BASIC for the Windows operating system.
[The computer program is available at http://nhsbig.inhs.uiuc.edu/wes/chi.html(Illinois Natural History Survey)].
Archives of Phytopathology and Plant Protection Statistical analysis The data on larval cumulative percentage mortality were corrected for controlmortality by using the Schneider Orelli's formula (Pu¨ntener 1981). The LC25 andLC50 values were estimated using probit analysis, carried out by the statisticalprogram POLO-PC (Leora Software 1987). The sublethal effects of PYR on themoth biological characteristics were analysed using the PROC General LinearModel [The same applies for following tests, as well]. Pairwise comparisons wereperformed using Fisher's Least Significant Difference (F-LSD) test. All statisticalanalyses (GLM and pairwise comparisons) were completed in SYSTAT 12.02(SYSTAT Software 2007).
Insecticidal activity of PYR and determination of sublethal concentration The responses of third instar larvae of P. xylostella to PYR were dose-dependent(Figure 1). The LC25 and LC50 values measured 96 h after treatments were 1.49 and2.01 mg/ml, respectively, and these concentrations were selected as sublethal doses for the experiments (Table 1).
Effect of sublethal doses on developmental period of P. xylostella Developmental times of subimago stages including egg, larva and pupa were significantly increased by PYR treatment during both generations (Table 2). Due to direct application of sublethal doses on parents, PYR affected the developmentaltimes of parents more than the offspring generation (see Table 2 for more details).
Percentage mortality, pupal weight, adult emergence, size and sex ratio In both generations and especially in the parent, mortality of larva and pupa was remarkably increased by sublethal concentrations of PYR (see Tables 3 and 4 for Corrected mortality percentage rate equivalent to probit unit of the third-instar larvae of Plutella xylostella caused by preparation of pyriproxyfen at different concentrations.
M. Alizadeh et al.
Comparison of results of probit, Polo-PC analysis of dose response data for pyriproxyfen applied to early third instar larvae of P. xylostella.
2.01 (1.868–2.15) 1Number of P. xylostella larvae assayed.
more details). In the parent generation, the pupal mortality of groups treated withthe LC25 and LC50 doses was 15.73- and 20.60-fold more than those of the controltreatment. The pupal weight was diminished by 1.11- and 1.22-fold, respectively.
Adult emergence of those given the LC25 and LC50 treatments decreased to 0.75 and 0.64 of that in the control (Table 3). In the parent generation, adult abnormalities such as twisted wings increased from zero in the control to 13.75%and 25.12% in the LC25 and LC50 treatments, respectively (Table 3). The adult sizeof LC25 and LC50 groups were 1.13- and 1.19-fold (male wingspan), 1.16- and 1.20- fold (female wingspan), 1.08- and 1.16-fold (male length) and 1.09- and 1.14-fold (female length) smaller than the untreated group (Table 3). The indirect effects ofthe two sublethal doses of PYR on the next generation (offspring) obtained from treated parents included an increase in egg mortality (unhatched eggs). This was5.5- and 9.01-times greater in LC25 and LC50 treatment groups than in the control (Table 4). The LC25 and LC50 values led to 4.13- and 6.81-fold enhancement in larval mortality compared with the control (Table 4). The offspring pupalmortality caused by LC25 and LC50 values was 7.39- and 9.38- times higher than in the untreated group. Moreover, in the offspring, normal adult emergence from pupae was significantly reduced in PYR-treated groups. Indirect effects of PYRled to other anomalous conditions in adults in the LC25- and LC50-treated groupsabout 3.44- and 5.96-times more than in the controls (Table 4). Furthermore, treatment with the LC25 and LC50 values of PYR significantly reduced the pupal weight to 1.07- and 1.16-times lower than those in the control group (Table 4).
There was also an obvious decrease in the adult sizes of P. xylostella treated with the LC25 and LC50 dose of PYR. Wingspan decreased to between 0.89 and 0.93 times (males) and 0.88 and 0.92 times (females) that of untreated moths. Overall body length was 0.89–0.96 times (males), and 0.91 and 0.97 times (females) that of untreated moths (Table 4). The number of emerged females and males showed that the sex ratio [number of female/(female þ male)] was close to 1:1 in all treatments, and there were no differences between treatments (see Table 4 formore details).
Effect of sublethal doses on adult performance Treatment with the LC25 and LC50 values of PYR significantly influenced theduration of adult pre-oviposition, oviposition, post-oviposition periods, longevityand fecundity of P. xylostella in both generations, though this was especially evidentin the parent generation (see Tables 5 and 6 for more details). Compared with thecontrol, pre- and oviposition periods of PYR-treated females were prolonged, butthere was no significant difference between the LC25 and LC50 treatments. The post-oviposition period was shortened in both generations and it was significantlty shorter Downloaded by [University of Tehran], [M. Alizadeh] at 20:04 12 August 2012 The effects of pyriproxyfen on developmental period of immature stages of P. xylostella in two successive generations.
Developmental time (days; mean + SE; n1 ¼ 100) Parent generation Offspring (F1 generation) 2.07 + 0.02a2 2.56 + 0.01a 4.63 + 0.02a 4.30 + 0.02a 2.39 + 0.02a 2.18 + 0.02a 2.05 + 0.03a 2.14 + 0.02a 2.54 + 0.02a 11.30 + 0.05a 4.25 + 0.03a 3.11 + 0.05b 6.75 + 0.09b 5.26 + 0.06b 3.11 + 0.02b 2.36 + 0.04b 2.25 + 0.04b 2.36 + 0.03b 2.82 + 0.03b 12.82 + 0.09b 5.12 + 0.04b 3.79 + 0.05c 7.86 + 0.07c 5.81 + 0.07c 3.83 + 0.04c 2.93 + 0.04c 2.86 + 0.04c 2.99 + 0.04c 3.87 + 0.05c 16.45 + 0.12c 5.25 + 0.05b 1Total number of third instar larvae that assayed by estimated LC25 and LC50 of PYR., Control, distilled water. L 1, L2, L3 and L4 denote the first, second, third and fourth instar moth larvae, respectively. 2Means followed by the same letter within columns are not significantly different (p 5 0. 05; Fisher-LSD Test). 3The numbers show the degrees of freedom of treatment and error, respectively.
Downloaded by [University of Tehran], [M. Alizadeh] at 20:04 12 August 2012 The effect of pyriproxyfen on larval mortality, pupation, adult emergence and size (mean + SE) of P. xylostella (Parent).
Larvae (n1 ¼ 100) 1Total number of third instar larvae that assayed by estimated LC25 and LC50 of PYR. 2The numbers given show the degrees of freedom of treatment and error, respectively.
PW, pupal weight (mean + SE), weight that was recorded within 24 h after pupation; MW, male wingspan; FW, female wingspan; ML, male length; FL, female length.
3Means followed by the same letter within columns are not significantly different (p 5 0. 05; Fisher-LSD Test).
Downloaded by [University of Tehran], [M. Alizadeh] at 20:04 12 August 2012 The effect of pyriproxyfen on larval mortality, pupation, adult emergence, sex ratios and size (mean + SE) of P. xylostella (offspring).
Treatment (mean + SE) (mean + SE) (mean + SE) 608.7+12.45a 97.05+1.57a 50.50+3.41 13.72+0.11a 14.95+0.10a 5.93+0.08a 5.81+0.08a 28.73+2.02b 22.58+2.37b 558.33+12.91b 84.83+5.32b 9.5+3.08bb 48.42+4.15 12.07+0.06b 13.68+0.07b 5.68+0.06b 5.62+0.05ab 75.92+6.14c 14.81+0.11b 45.38+3.18 12.19+0.08b 13.17+0.04c 5.26+0.07c 5.28+0.05bc PW, pupal weight (mean + SE), weight that was recorded within 24 h after pupation; MW, male wingspan; FW, female wingspan; ML, male length; FL, female length.
1Means followed by the same letter within columns are not significantly different (p 5 0. 05; Fisher-LSD Test). 2The numbers given show the degrees of freedom of treatment and error, respectively.
Downloaded by [University of Tehran], [M. Alizadeh] at 20:04 12 August 2012 The effect of pyriproxyfen on adult fitness and performance of P. xylostella in parent.
Parent (n ¼ 1001, sex ratio:1:1) Reproduction potential Total oviposition period (mean + SE; days) Longevity (mean + SE; days) (mean + SE; number) 1Total number of third instar larvae that assayed by estimated LC25 and LC50 of PYR. 2Total number of eggs laid by each female. 3Percentage of hatched eggs per adultfemales. 4Means sharing same letters in a column are not significantly different from each other (p 5 0. 05; Fisher-LSD Test). 5The numbers given show the degrees offreedom of treatment and error, respectively.
Downloaded by [University of Tehran], [M. Alizadeh] at 20:04 12 August 2012 The effect of pyriproxyfen on adult fitness and performance of P. xylostella in offspring (F1 generation).
Offspring (n ¼ 1001, sex ratio:1:1) Reproduction potential Total oviposition period (mean + SE; days) Longevity (mean + SE; days) (mean + SE; number) 1Total number of third instar larvae that assayed by estimated LC 25 and LC50 of PYR. 2Total number of eggs laid by each female. 3Percentage of hatched eggs per adult females. 4Means sharing same letters in a column are not significantly different from each other (p 5 0. 05; Fisher-LSD Test). 5The numbers given show the degrees of freedom of treatment and error, respectively.
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with the larger treatment dose (see Tables 5 and 6 for more details). PYR-treatmentcaused a significant reduction in the adult longevity of both sexes in each of the twoconsecutive generations (Tables 5 and 6). Treatment with the LC50 dose was moreeffective than the LC25 dose in reducing adult longevity. There was not muchdifference in adult longevity between the two generation except that the offspringmales of the LC25 treatment had a higher longevity than males of the parentgeneration (Tables 5 and 6). The experimental doses of PYR did, however,significantly reduce female fecundity and the percentage of viable eggs in eachgeneration, especially in the parent generation. In the parent generation, the femalefecundity values in the LC25- and LC50-treated groups were 0.43 and 0.29,respectively, compared to the control number of offspring. In the offspring, thesevalues increased to 0.64 and 0.52 of those found for the control. The percentage ofegg viability affected by the LC25 and LC50 treatments was 0.79 and 0.63 of the control in parent and 0.91 and 0.80 of the control in offspring (Tables 5 and 6).
Age-stage, two-sex life table of P. xylostella In Figure 2, age-stage survival rates (sxj) of P. xylostella in PYR-treated (LC25 and LC50 values) and untreated groups (control) are shown. sxj indicates the probabilitythat a newborn will survive to age x and develop to stage j. According to Chi and Yang (2003), since the variable developmental rates that occur among individuals ofa population are consolidated into the age-stage, two-sex life table, the stage survival rate curves of a cohort show stage overlapping. Equations (2) and (3) were used to calculate the age-specific survival rates (lx) and fecundity rates (mx) (Figure 3). Asingle age-specific survival rate (lx) gives the probability that an egg will survive to age x (Figure 3). The age-stage specific fecundity rate (fxj) of female adult P. xylostella gives the number of eggs produced by adult females (the fourth stage,j ¼ 4) of age x, where the age x is counted from the egg stage. Age specific fecundity(mx) curves show that reproduction began at age 37 and 49 days in LC25 and LC50 treatments, respectively (Figure 3). However, the first reproduction in the untreated group (control) was at age 30 days. The ovipositional period of P. xylostella lasted about 23, 52 and 33 days for the control, LC25 and LC50 treatments respectively, but the number of eggs produced by adult females (fx4) in PYR-treated were much lower than by the control (Figure 3).
Sublethal effects of PYR on biological parameters of the offspring In the two treatment groups (LC25 and LC50), the biological parameters ofP. xylostella including net reproduction rate (R0), GRR, intrinsic rate of increase (r),mean generation time (T), finite rate of increase (l) and DT were significantltyaffected by two the tested sublethal concentrations compared to the control(Table 7). In addition, there were significant differences in all mentioned parametersbetween the LC25 and LC50 treatments except GRR. Treatment with the LC25 andLC50 values significantly decreased the GRR to about 40% and 50%, and R0 toapproximately 70% and 90%, respectively, in comparision to the control. Theintrinsic rate of increase (r) in the LC25 and LC50 groups declined to 0.63 and 0.39 ofthat in the control, respectively (F2,297 ¼ 61.17, p 5 0.001, df ¼ 2, 297). Themean generation time (T) tended to be longer (7.36 and 17.25 days in the LC25and LC50 treated P. xylostella) than that of the control (F2,297 ¼ 144.65, p 5 0.001, Archives of Phytopathology and Plant Protection Age-stage survival rates (sxj) of P. xylostella treated with sublethal concentrations of pyriproxyfen (LC25 and LC50) and control (as untreated group).
df ¼ 2, 297). The DT was affected by the LC25 and LC50 treatments and increased to1.86- and 3.71-fold, respectively, of that in the control (F2,297 ¼ 429.25, p 5 0.01,df ¼ 2, 297).
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Age-specifc survival rate (lx) and age-stage fecundity of female (fx4) (eggs/female), and age-specific fecundity (mx) of P. xylostella treated with sublethal concentrations ofpyriproxyfen (LC25 and LC50) and control (as untreated group) by using the age-stage, two-sex life table.
Downloaded by [University of Tehran], [M. Alizadeh] at 20:04 12 August 2012 Population parameters (mean + SE) of P. xylostella treated with sublethal concentrations of pyriproxyfen and control (as untreated treatment), calculated by using the age-stage, two-sex life table.
1Net reproductive rate, 2Gross reproductive rate, 3Intrinsic rate of increase, 4Mean generation time, 5Finite rate of increase, 6Doubling time, 7Means sharing same letters in a column are not significantly different from each other (p 5 0. 05; Fisher-LSD Test). 8The numbers given show the degrees of freedom of treatment and error, respectively.
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Abnormalities caused by PYR treatment PYR treatment caused a number of abnormalities in DBM larvae. These includefailure of some larvae to completely shed their old cuticles (e.g. inability to withdrawfrom the head capsule – usually caused by entrapment of their mouth parts); larvaewith disordered setae; arrested larval growth due to cessation of feeding; overlappingof the larval anal legs following ecdysis and subsequent death due a combination offactors including the cessation of feeding, failure of the larvae to pupate and theformation of larval–pupal intermediates; untanned pupae or pupae with imperfectsclerotisation; adults that failed to emerge and remain trapped in the cocoon,eventually dying; adults with twisted wings; females with reduced reproductioncapacity due to either being sterile or having reduced fecundity and/or fertility andlaying unviable eggs (Figures 4 and 5).
Other insect species have also been shown to have dose-dependent responses toPYR, including Choristoneura rosaceana (Sial and Brunner 2010) and Plodia interpunctella (Ghasemi et al. 2010). On the other hand, the results obtained by Oochi (2005) on larvae of P. xylostella treated with PYR were the opposite of thoseobtained in the present study, as were those of Hatakoshi et al. (1987) studying Manduca sexta. Oochi (2005) suggested that this inverse relationship may be due toeither a more rapid excretion or more readily breaking down of higher concentrations in the alkaline environment of the larval intestine. Moreover, in our study, the negative effects of the LC25 treatment on biological characteristics ofP. xylostella were lower than the higher sublethal dose (LC50). There are two JH sensitive periods in the last instar larvae of holometabola: in the first, JH must be absent in the hemolymph for the development of pupal characters to begin, and inthe next, JH must be present to stabilise the pupal determined state of imaginal diskstructures and prohibits their commitment to adult development (Nijhout 1994).
Various morphological abnormalities in treated P. xylostella with sublethal concentrations of pyriproxyfen at larval (a–d), prepupal and pupal (e–h) stages: (a) larva withdisordered hairs; (b) inability to shed head capsule, old head capsule (arrow); (c and d)arrested larval growth; (e and f) larval–pupal intermediates; (g and h) untanned pupae, normalpupa (arrow); scale bar: 1 mm.
Archives of Phytopathology and Plant Protection Various morphological abnormalities in treated P. xylostella with sublethal concentrations of pyriproxyfen at eggs and adults: (i and j) twisted wings in adults; (k and l) failure in adult emergence; (m and o) abnormal ovaries; (n) normal ovary; (p) deformedoocytes; (q) eggs without yolk; (r) normal eggs; scale bar: 1 mm.
Therefore, the prolonged duration of larval and pupal periods noticed in DBM, theproduction of larval–pupal intermediates, failure to shed the old larval cuticule, other morphological abnormalities and mortality may be a result of the existence of PYR during the critical time of the DBM life cycle. The production of larval–pupalintermediates as a result of exposure to PYR in C. rosaceana (Sial and Brunner 2010) and Spodoptera littoralis to JHI (Khafagi and Hegazi 1999) have been reported.
Similarly, the increase in time to pupation, without creation of supernumerarylarvae, has been shown in P. interpunctella treated with PYR (Ghasemi et al. 2010)and delay in pupation due to larval–larval intermediates followed by treatment with JH's or JHA's have been observed previously by Khafagi and Hegazi (1999), Oochi (2005), Nomura and Miyata (2000) and Kostyukovsky et al. (2000). In this study, despite of increase in larval period, the weight of pupae declined. Leonardi et al.
(2001) demonstrated that oral and topical applications of fenoxycarb on Bombyx mori decreased larval frass production because of the toxic effect of fenoxycarb on the brush border membrane (BBM) of midgut columnar cells (especially in its anterior–middle regions) and disordered leucine uptake in these areas. It also modified the lipid composition of BBM by changing the balance between saturated and unsaturated fatty acids that both affect amino acid absorption in the larvalmidgut (Leonardi et al. 2001). There is a Kþ/amino acid cotransporter for Lucineuptake in the BBM of midgut columnar cells (Giordana et al. 1994) that it is incharge of the absorption of many other essential amino acids (Parenti et al. 2000).
Moreover, Fenoxycarb severely disturbed lipid synthesis and catabolism in the fatbody of Choristoneura fumiferana (Mulye and Gordon 1993) and reduced intake offood and growth rate in silkworms (Leonardi et al. 1996). Hence, the reduced pupalweight of DBM may be attributed to the disturbed uptake of amino acids, lipidsynthesis and catabolism. In our study, arrested larval growth due to feedinginterruption was similar to the results found by Arthur (2001) in Tribolium castaneum.
At the beginning of the pupal stage of holometabola, there is an additionalJH-sensitive period for pupal versus adult determination that JH must be absent in M. Alizadeh et al.
epidermal cell obligated to adult development (Nijhout 1994). Hence, the presence ofJH (PYR in this study) at this critical time, resulted in the production of deformedpupae and adults (e.g. twisted wings) and failure in adult eclosion in P. xylostella.
These data confirmed prevention of imaginal disk cell growth in P. interpunctellatreated with JHI, JHIII, methoprene, fenoxycarb and farnesol (Oberlander et al.
2000), prohibition of silk gland growth in B. mori treated with JH's and JHA's(Kurata 1981; Garel 1983; Aribi et al. 2006), malformed wings in L. migratoria (DeKort and Koopmanschap 1991), Blattella germanica (Lim and Yap 1996),T. castaneum (Arthur 2003) treated with fenoxycarb, PYR and Hydroprene,respectively, and increased larval and pupal mortality and reduced adult emergenceas a result of JH's or JHA's treatment (Khafagi and Hegazi 1999; Arthur 2001, 2003;Oochi 2005; Sial and Brunner 2010). In the present work, reduction in fecundity, eggviability percentage and adult longevity were observed in both sublethal treated groups at successive generations. Dissection of these females indicated that they had malformed ovaries. Yin et al. (2008) observed the reduction of fecundity inP. xylostella treated with sublethal doses of spinosad not in the offspring but inthe parent. It may be due to the presence of PYR at the early pupal stage preventing the secretion of sufficient ecdyson. Shaaya et al. (1993) suggested that ovaries in P. interpunctella require high level of ecdysteroid in the hemolymph of the earlypupal stage to mature. In other studies, topical application of Chlorfluzuron on Spodoptera litura (Perveen and Miyata 2000), PYR on P. interpunctella (Ghasemiet al. 2010) and Hexaflumuron on Callosobruchus maculatus (Kellouche and Soltani 2006) reduced lipid and protein concentration in ovaries. Kanost et al. (1990) reported that reduction of compounds such as lipids, proteins and carbohydratesmay result in abnormal oogenesis. Pyriproxyfen application to young workers of Aphis mellifera damaged vitellogenin synthesis in the hemolymph (Pinto et al. 2000).
Feeding of A. mellifera workers with Kinoprene contaminated pollen reduced theirlongevity (Gerig 1975). In the beginning of the pupal stage of S. litura, treatmentwith PYR resulted in adults with low ovarian weight and reduction in number of eggs oviposited because of losing an oviposition stimulator factor (a protein with molecular weight more than 14 KDa) in the treated females' hemolymph (Hatakoshi 1992). The reduced reproductive capacity of untreated C. rosaceana (Sial and Brunner 2010) and M. domestica (Chang and Borkovec 1990) via mating with PYR and diflubenzuron (A chitin synthesis inhibitor) treated male, respectively, proved the transfer of IGRs to the eggs via the sperm. This may be justification for continuing to study effects of PYR on subsequent generations in this study.
Pyriproxyfen also influenced the biological parameters of the offspring in the present study. As was previously mentioned in the results, the net reproductive rate (R0) ofDBM was affected by both of the PYR sublethal doses. Reduced female fecundityand longevity led to a reduction in R0 in the current study. This was the reverse of thesublethal effect of bifenthrin on enhancement of the net reproductive rate of cottonaphids (Kerns and Stewart 2000). On the other hand, some insects and mites showincreased vigour (e.g. increase fecundity and the reproductive parameters) whenexposed to sublethal doses of pesticide - a phenomenon known as hormoligosis. Sotaet al. (1998) and Fujiwara et al. (2002) reported the phenomenon of hormoligosis inP. xylostella affected by treatment with different sublethal doses of fanvalerate. Butas previously noted, IGR's reduced the fecundity of insects and Acari duo tophysiological and morphological changes in both sexes (Hatakoshi 1992; Kelloucheand Soltani 2006). In addition to R0, other biological parameters of P. xylostella Archives of Phytopathology and Plant Protection such as GRR, intrinsic rate of increase (r) and finite rate of increase (l) tended to belower in both of the sublethal treatment groups than in the untreated group. Thiswas consistent with the results reported by Yin et al. (2008) on P. xylostella treatedwith spinosad and Lashkari et al. (2007) on Brevicoryne brassicae treated byimidacloprid and pymetrozine. Unlike other population parameters, the meangeneration time of the two sublethal treatments actually increased compared to thecontrol in this investigation. It was due to longer immature stages and adultpreoviposition periods compared with the control. In the present study, the negativeeffects of both tested sublethal concentrations of PYR influenced the biologicalcharacteristics of P. xylostella not only in the parent but also in the next generation,although the mentioned effects were less noticable in the offspring. Yin et al. (2008)reported on the sublethal effects of spinosad on consecutive generations ofP. xylostella. The effects on the second generation, however, were much less apparent than those on the parent generation. In some cases, sublethal concentra- tions do not have an effect on the offspring. For example, in Daphnia carinata, thenegative sublethal effect of chlorpyrifos was absent on the next generation (Zalizniakand Nugegoda 2006). The reduction or loss of effects of insecticides on the next generations of treated parents can be a result of the insect's growth and dilution of residues; in other words, an insect could be restored piecemeal to a normalmetabolism, generation by generation (Yin et al. 2008). The sublethal effects of PYR on vital performance of two successive generation of DBM may generate morebenefits to an integrated pest management program for this major pest than conventional insecticides that have a high acute toxicity (Michaud and Grant 2003).
In conclusion, our findings indicated that PYR was highly effective in controllingP. xylostella in the laboratory. This IGR addition to direct mortality has indirect effects such as disrupting growth and development, reducing reproductive capacity, and is also capable of transferring abnormalities to the next generation. Furtherstudies, however, are needed to investigate the sublethal effects of PYR on latergenerations of DBM and to display the mechanisms by which PYR exerts its sublethal effects in the laboratory and to understand its aggregate effects on population dynamics of P. xylostella and its interaction with natural enemies in the M. Alizadeh was funded by the University of Tehran (UT), Iran. We are grateful to Prof. Dr.
Cecil L. Smith for his help with editing an earlier version of this manuscript. We appreciate Prof. Dr. Hsin Chi, for generously helping with sending a version of his ‘‘Life table, two sex, life table analysis'' software, and we thank Khalil Talebi Jahromi (UT, Iran) for providingpyriproxyfen.
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March 2nd, 2011 MEASURING STRESS REDUCTION USING FAR INFRARED RAY MEDICAL DEVICE BIOMAT FOR 12 SUBJECTS. AUTHOR: DR. GEORGE GRANT, Ph.D, I.M.D., M.Sc., M.Ed., C.CHEM., R.M. Affiliation: Currently in private practice at Champion Integrative Clinic, Toronto, ON Canada. Former Consultant for Health Canada, Ottawa, ON, Professor at Seneca College, North York,