Effect of neem oil extract on pupal development

To obtain the pre-pupae and pupae, larvae of D. saccharalis were placed on artificial diet (^{HENSLEY; HAMMOND JUNIOR, 1968}) and maintained in controlled conditions of 25 ± 1ºC, relative humidity of 70 ± 10% and photoperiod of 12 hours, at the Department of Cellular Biology and Genetics of the State University of Maringa, Paraná, Brazil. Pre-pupae and pupae of different ages were removed from the diet and separated into the following groups: pre-pupae (intermediate stage between caterpillars and pupae, beginning the melanization and chitinization); young pupae (one to six days of development); and old pupae (seven to fourteen days of development).

The bioinseticide used was NATUNEEM^® (neem virgin oil with azadirachtin content above 1,500 ppm), an organic product certified by BCS OKO Garantie, Doc number Natur 9009/09.05/7331-BR. The neem oil extract was diluted in distilled water into the following concentrations: 0.0; 0.3; 0.5; 1.0 and 2.0%. Twelve individuals were used for each treatment, with three replicates for each concentration (n = 36) and a total of 180 individuals per group. Approximately 100 µL of the product was topically sprayed onto each organism.

After the treatments, all individuals were placed in polyethylene containers containing moistened cotton lined with filter paper and kept under the experimental conditions previously mentioned. Observations were made daily, up to 15 days after treatment, under a stereoscopic microscope (Zeiss). Abnormalities, mortality and longevity rates of the groups were recorded for later analysis.

Residual effect of neem oil extract in stages subsequent to treatments

The emergency rate, abnormalities and adult mortality for each treatment were evaluated in the three experimental groups during 15 days after treatment. Females that emerged from treated young pupae were separated into pairs and placed with two normal males in polyethylene boxes, lined with filter paper (posture chamber) and containing cotton soaked in sucrose solution, remaining incubated under experimental conditions for 48 hours. The experiment was repeated three times and the number of eggs deposited on the filter paper was quantified and compared to the number of the controls. The number of embryos that did not develop among the deposited eggs was also verified in relation to controls.

Data analysis

Experimental data from groups PP (pre-pupae), YP (young pupae) and OP (old pupae) were compared by analysis of variance (ANOVA). Results indicating differences between the values were analyzed by Tukey’s multiple comparison test. In all cases, values of p < 0.05 were considered significant. The most frequent morphologic alterations found were documented photographically with a digital camera (Canon 7.0 MG) coupled to the stereoscopic microscope (Zeiss).

Effect of neem oil extract on pupal development

The treatment of pre-pupae and pupae of D. saccharalis of at different ages with neem oil extract caused differentiated and concentration-dependent mortality (Table 1). Similar results were found in pre-pupae of R. ferrugineus (Coleoptera: Curculionidae) and pupae of Corcyra cephalonica (Staint) (Lepidoptera: Pyralidae), in which mortality was higher, with increasing doses of topically applied extract (^{EL-BOKL et al. 2010}; ^{PATHAK; TIWARI, 2010}).

According to Table 1, at the concentrations of 0.3 and 0.5%, the PP group was more susceptible to the product and was statistically different from other groups (F = 19.44, df = 8, p < 0.01 and F = 39.92, df = 8, p < 0.01, respectively). All groups differed statistically at the concentration of 1.0% (F = 61.11, df = 8, p < 0.0001), and the PP group did not differ statistically from the YP group at the 2.0% concentration, but both groups differed from the OP group (F = 20.34, df = 8, p < 0.01).

^{TORRES et al. (2001}) analyzed the effect of various aqueous plant extracts on the development of Plutella xylostella ( Plutellidae), in which A. indica caused 100% mortality in caterpillars, not reaching the pupae stage. On the other hand, the topical application of neem oil extract and azadirachtin did not affect the survival of pupae of the predator Cycloneda sanginea (L.) (Coleoptera: Coccinellidae) (^{SILVA; MARTINEZ, 2004}; ^{COSME et al., 2007}).

In this study, we observed that younger stages are more susceptible to neem treatment, as all treatments caused 100% mortality in the pre-pupae stage (Table 1). ^{LADD et al. (1984}) obtained similar results when azadirachtin was topically applied on pre-pupae and pupae of Popillia japonica (Coleoptera: Scarabaeidae) at different ages, in which the extract was effective in pre-pupae and one-day-old pupae, but had no effect on three-day-old ones.

Inhibition of ecdysis and consequent death of the pre-pupae can be attributed to the reduction of ecdysteroids peak or an interference in the release of the ecdysis hormone (^{SIEBER; REMBOLD, 1983}). This may occur because azadirachtin has a structural formula similar to that of ecdysone, which can compete with this hormone and block its effect (^{NAKANISHI, 1975}). Several studies described the anti-ecdysteroid action of azadirachtin, especially in the immature stages (^{SIEBER; REMBOLD, 1983}; ^{COUDRIET et al., 1985}; ^{ANDRADE-COELHO et al., 2006}; ^{NATHAN et al., 2007}).

In the present study, mortality was related to deformities of pre-pupae and pupae treated with neem (Table 1), in which morphologic alterations may be the cause of molt inhibition and death. All experimental groups differed at the concentration of 0.3% (F = 496.0, df = 8, p < 0.0001), and the PP group was the most affected by the neem oil extract at 0.5%, being different from the YP and OP groups (F = 58.49, df = 8, p < 0.0001). In higher concentrations, the OP group was less affected by the treatment, differing from the other groups at the concentrations of 1.0% (F = 16.0, df = 8, p < 0.01) and 2.0% (F = 430.6, df = 8, p < 0.0001).

The most frequent abnormalities in pre-pupae, young pupae and old pupae are shown in Figure 1. After a few hours of treatment, it can be observed in the pre-pupae that the wings were free from the body and with changes in coloration, becoming darker. The abdomen remained lighter in color, typical of recent pre-pupae (Fig. 1A), indicating they did not continue the pupation process. There are several studies in the literature reporting the reduction of pupation after treatment with neem extracts in caterpillars, preventing the emergence of adults (^{KHAN et al., 2007}; ^{PATHAK; TIWARI, 2010}; ^{WAKIL et al., 2012}).

Figure 1: Morphological alterations caused by the neem oil extract (1.0 e 2.0%) in pre-pupae after 24 hours of treatment (A); young pupae after 10 days of treatment (B-E); old pupae after 15 days of treatment (F); adults emerged from young pupae (G-H) and old pupae (I-J). 

In the young pupae, the most frequent abnormalities were abdominal deformations with the appearance of regions in which the cuticle remained thinner and with small black spots near the wing. In old pupae, increased melanization was observed throughout the cuticle (Figs. 1B, 1C, 1D, 1E and 1F). Some reports in the literature also demonstrate abnormalities in pre-pupae and pupae, resulting from treatment with azadirachtin or neem oil, as in Spodoptera littoralis (Lepidoptera: Noctuidae) (^{MARTINEZ; EMDEN, 2001}) and R. ferrugineus (^{BREAM et al., 2001}).

These changes have also been reported in experiments performed on Bombyx mori larvae, suggesting some interference with ecdysteroid and juvenile hormone levels (^{KOUL et al., 1987}; ^{HORI et al., 1984}). Another possible cause of the alterations in vital physiological events may be due to the water loss of the pupae. ^{BREAM et al. (2001}) found that desiccation increased proportionally to the neem extract dosage and to pupal mortality.

Because there was 100% mortality in the PP group, it was not possible to calculate the longevity at this stage, unlike the YP and OP groups, in which the means of longevity did not differ in relation to the control (F = 0.47, df = 14 and p > 0.05 in group YP; F = 1.95, df = 14 and p > 0.05 in group OP; Table 2). These results correspond to some studies that did not find any change in the duration of the pupae development period after larvae were treated with neem oil extracts (^{HERNANDEZ; VENDRAMIN, 1996}; ^{VENDRAMIN; SCAMPINI, 1997}; ^{TORRES et al., 2001}).

Residual effect of neem oil extract in stages subsequent to treatments

The adult emergence rate is shown in Table 2. In the YP group, the rate decreased with increasing concentrations, with high statistical differences when comparing concentrations (F = 35.53, df = 14, p < 0.0001). In the OP group, the statistical difference of the adult emergency rate was lower (F = 9.63, df = 14, p < 0.01; Table 2). Topical treatment of neem extract in R. ferrugineus pre-pupae resulted in reduced adult emergence rate, which exhibited various morphological abnormalities such as reduced body length (^{EL-BOKL et al., 2010}). Topical treatment in Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) and Ephestia cautella (Lepidoptera: Pyralidae) caterpillars also caused reduced adult emergence rate (^{KHAN et al., 2007}; ^{SHEHU et al., 2010}). Artificial diets containing different concentrations of the aqueous extract of A. indica also decreased the survival of D. saccharalis caterpillars (^{MAZZONETTO et al., 2013}).

The percentage of abnormalities in adults is also described in Table 2. In the YP group, the abnormality rate was zero for both the 0.3% concentration and the controls, and at concentrations of 0.5 to 2.0%, the rate was less than or equal to 50%, in relation to the emergency rate (F = 14.83, df = 14, p < 0.01). In the OP group, the abnormality rate in adults was rather variable, not being concentration-dependent (F = 133.30, df = 14, p < 0.0001), which may be a result of morphogenetic alterations due to interferences in hormonal events (Figs. 1G and 1H) (^{ALINIAZEE et al., 1997}; ^{GHONEIM et al., 2000}).

^{SALLES; RECH (1999}) observed that adults of Anastrepha fraterculus (Diptera: Tephritidae) resulting from treatment with neem had abnormal wings, which did not fully expand, characteristics also being described by ^{LADD et al. (1984}) and ^{MARTINEZ; EMDEN (2001}). Some adults of E. cautella also emerged deformed after treatment of the larvae with Calneem® oil (^{SHEHU et al., 2010}).

In the treatment of young pupae with higher concentrations of neem, some adults were unable to completely get rid of the exuvia, with some regions of the body remaining with parts of the old cuticle (pupa), such as wings, legs or abdomen (Figs. 1I and 1J). The occurrence of this fact, originating an intermediate pupa-adult, is a rare event. ^{BREAM et al. (2001}) reported no pupae-adult intermediates after neem treatment in R. ferrugineus pre-pupae, although they found this abnormality after the treatment of Culex pipiens larvae with Echinochloa stagninum (Poaceae) petroleum ether extract (^{BREAM et al., 2010}). Intermediate larva-pupae were observed when the treatment with azadirachtin was applied in the last instar of the larva of Spodoptera litura, Spodoptera mauritia, Ephestia kuehniella, Manduca sexta and Spodoptera littoralis (^{GUJAR; MEHROTRA, 1983}; ^{JAGANNADH; NAIR, 1992}; ^{MARTINEZ; EMDEN, 2001}).

The mortality rate in adults was increased with higher concentrations of neem (F = 10.24, df = 14, p < 0.01, Table 2). In the experimental groups YP and OP, adult mortality was related to the appearance of abnormalities, except at the concentration of 1.0% (Table 2). This fact can be explained due to the changes caused, being harmful to vital activities of the insect, such as locomotion, flight and, consequently, feeding (^{MARTINEZ; EMDEN, 2001}).

The fecundity and viability of the eggs were analyzed by the oviposition of emerged females of the YP group that did not present abnormalities. The standard mean of the number of deposited eggs after the oviposition period, the number of undeveloped eggs and the percentage of undeveloped eggs were different in all treatments (F = 2429, df = 14, p < 0.0001; F = 152.9 df = 14, p < 0.0001; F = 1247, df = 14, p < 0.0001; Table 3). These results were similar to those of ^{DAD et al. (2011}), who observed reduced fecundity, limitation in the number of deposited eggs and totally inhibited eclosion after the treatment with neem in Musca domestica larvae.

Studies with azadirachtin or neem oil on Lepidoptera also demonstrated changes in fertility and fecundity of S. littoralis (^{PINEDA et al., 2009}), anti-oviposition and ovicidal effects against E. cautella (^{SHEHU et al., 2010}), ovicide with changes in the embryonic development against D. saccharalis (^{SILVA et al., 2013}) and extended embryonic period in D. saccharalis (^{OLIVEIRA et al., 2013}).

In this work, the fecundity of females from the YP group and the viability of deposited eggs decreased with higher doses of the neem extract, probably because azadirachtin inhibits oogenesis and the production of ecdysteroids (^{REMBOLD; SIEBER, 1981}) synthesized by follicular cells during ovarian development and later incorporated into the eggs (^{GLASS et al., 1978}). The inhibitory action of neem in vitellogenesis (^{DENARDI et al., 2011}) and in the follicular cells by degeneration, hyperplasia and necrosis (^{EL-BOKL et al., 2010}) also justifies the reduction of fecundity and egg viability. ^{KHAN et al. (2007}) also observed inhibition in the ovarian development in adults of B. cucurbitae due to reduction of ovarian size after treatment with neem formulations.