MATERIALS AND METHODS

Tested flies: The house flies, Musca domestica reared in Medical Entomology Insectary, Animal House, Department of Zoology, Faculty of Science, Al-Azhar University under controlled conditions of temperature (25-27°C), relative humidity (55- 60%) and (12-12) light-dark regime. Larvae were reared on an artificial diet (wheat bran, milk, powder yeast; 200:100:5 g) per 200 mL distilled water, while emerged flies were fed on dry diet (milk powder) and sucrose solution (cotton pads soaked in 10% sucrose solution)⁹.

Plants and extraction: Trigonella stellata and Eucalyptus citriodora belonging to Fabaceae and Verbenaceae collected during June, 2017 from El- Fayoum Governorate, Egypt. Leaves of two plants washed and dried under shade at room temperature and then ground into a powder. Samples of 100 g of powder were extracted using ethanol 70%, chloroform and hexane at a rate of 3 mL g^–1 for 24 h extraction period at room temperature, then the supernatant were decanted and filtered through Whatman filter paper (No. 5) and the solvents were evaporated to dryness under vacuum using a rotary evaporator with a water bath adjusted¹⁰ to 40°C. The dry extracts were kept in a deep freezer (-18) until used.

Larvicidal bioassay: Different concentrations of each extract were mixed with larval artificial diet instead of water to detect mortality percepts. Ethanol 70% tested materials were dissolved in 0.1 mL of ethanol 70%, while the tested materials of chloroform and hexane extracts were dissolved in 2 drops of Tween80 as emulsifier to facilitate the dissolving oils of tested material in water. About 25 of third instar larvae were put immediately into plastic cups contained media with different concentrations of extracts. Three replicates were used for each tested concentration. Control larvae received 0.1 mL of ethanol 70% or 2 drop of Tween80 in 100 mL water¹¹. Mortality in larvae and pupae was recorded daily until adult emergence. Larval and pupal mortality was calculated using the equation of Briggs¹². Growth index was estimated after Hassan et al.¹¹.

Reproductive potential of resulted females: Females that succeeded to emerge from third larval instar treated with each concentration were collected and transferred with untreated males obtained from the colony and were fed for 5 days. After oviposition, the eggs were counted using a binocular and then the mean values were taken. Sterility index was estimated according to the formula of Tappozada et al.¹³.

Statistical analysis: Data were subjected to ANOVA to find out the differences among the activity of plant extracts using Tucky’s HSD test at 5% probability level. The LC₅₀ were calculated using probit analysis¹⁴. All the statistical analyses were carried out using Statistical Package Social Science (SPSS) software¹⁵ version 23. Results represented as Mean±SD.

Ethical approval: The data of this study was approved by National Research Centre Ethics Committee (approved at date of 1/7/2015).

RESULTS

Ethanol extract of Trigonella stellata (leaves) recorded complete larval mortality (100.0%) at 700 ppm and the pupal mortality recorded 100.0% at 600 ppm. Also, the adult emergence recorded 66.3 and 27.8% at the highest concentrations (400 and 500 ppm), respectively. Meanwhile, at 50, 100, 150, 200, 250, 300 and 350 ppm the chloroform extract recorded 6.7, 20.0, 33.3, 46.7, 65.3, 90.7 and 100.0% larval mortality, the highest pupal mortality (58.3%) induced by chloroform extract at the highest concentration (300 ppm). In addition, the highest and lowest larval mortality (100 and 6.7%) recorded by hexane extract at 150 and 30 ppm and pupal mortality recorded 44.4% at the highest concentration (130 ppm). Also, T. stellata tested extracts insignificantly (p>0.05) affected the larval period except for hexane extract at the higher concentrations (90, 110 and 130 ppm), where the larval period prolonged significantly (p<0.05) to record 2.38±0.15, 2.55±0.06 and 2.71±0.04 days vs. 1.95±0.05 days for the control group. Also, chloroform and hexane extracts significantly affected (p<0.001) the pupal period at all concentrations used. A very retarded effect on growth of larvae, pupae and adult M. domestica was observed by T. stellata ethanol, chloroform and hexane extracts, where the growth index recorded 3.57, 4.87 and 6.08 at 500, 300 and 130 ppm, respectively, compared with 15.46, 16.50 and 16.34 for the untreated groups (Table 1).

The complete larval mortality (100%) attained by ethanol, chloroform and hexane extracts of E. citriodora leaves at 900, 500 and 350 ppm. Chloroform extract recorded the highest pupal mortalities (100 and 70%) at 450 and 400 ppm, respectively. In addition, E. citriodora tested extracts significantly (p<0.05) prolonged the developmental period of larvae and pupae as compared with the untreated groups (Table 2).

Based on LC₅₀ and LC₉₀ values recorded that, hexane extract of T. stellata and E. citriodora (leaves) were the most effective extract against M. domestica larvae compared with chloroform and ethanol extracts (Table 3).

Data arranged in Table 4 indicated that, there was a significant (p<0.05) decrease in fecundity of females resulted from larvae treated with T. stellata tested extracts. Also, the lowest hatchability percentages of eggs laid (77.23 and 78.46) recorded for females resulted from larvae treated with 130 and 110 ppm of hexane extract. The sterility index recorded 50.78, 62.22 and 64.21 at 500, 250 and 130 ppm by T. stellata ethanol, chloroform and hexane extracts, respectively (Table 4). On the other hand, ethanol extract of E. citriodora (leaves) significantly (p<0.001) decreased the fecundity of females resulted from treated pupae at 600 and 700 ppm as the number of eggs laid/female recorded 41.63±1.30 and 39.20±3.03 vs. 59.0±3.95 egg/female for the control group. Also, the lowest hatching percent recorded by hexane extract was 80.22% at 250 ppm, respectively, compared with 98.22% for the untreated group.

Table 1:	Effect of T. stellata (leaves) tested extracts on different stages of M. domestica
Means followed by the same letter are not significantly different (p>0.05)

Table 2:	Effect of E. citriodora (leaves) tested extracts on different stages of M. domestica
Means followed by the same letter are not significantly different (p>0.05)

Table 3:	Relative efficiency of T. stellata and E. citriodora (leaves) tested extracts against M. domestica larvae
χ2: Chi square value, n.s: Non-significant (p>0.05)

Table 4:	Effect of T. stellata (leaves) tested extracts on reproductive potential of M. domestica females
Means followed by the same letter are not significantly different (p>0.05)

Fig. 1(a-f):	(a, d) Normal larvae and pupae, (b, c) Malformations in larval stage and (e, f) Malformations in pupal stage

Table 5:	Effect of E. citriodora (leaves) tested extracts on reproductive potential of M. domestica females
Means followed by the same letter are not significantly different (p>0.05)

In addition, a remarkable increase in the percentage of sterility index was observed, where it was 39.33, 45.22 and 59.28% by E. citriodora (leaves) ethanol, chloroform and hexane extracts at 700, 400 and 250 ppm, respectively (Table 5).

Clear malformations in M. domestica larvae and pupae induced after treatment of the third larval instar with tested extracts were represented in Fig. 1. Morphological larval abnormalities (Fig. 1b , c) include brown pigmentation and a weakness in cuticle. Morphological pupal abnormalities (Fig. 1e, f) include larvi form pupation, irregular shaped and shrinkage pupae and larval-pupal intermediates.

DISCUSSION

Results of the present study showed that, the toxicity ethanol 70%, chloroform and hexane extracts of Trigonella stellata and Eucalyptus citriodora (leaves) against M. domestica larvae and resulted pupae varied according to solvent used in extraction and the concentration of the extract, hexane extract from tested plant species was the most effective extract as compared with ethanol 70% and chloroform extracts. These results confirmed earlier reports for Calotropis procera and Anonna squamosa leaves crude ethanolic extracts where, LC₅₀ values recorded 282.5 and 550 ppm against 3rd larval instar of M. domestica¹⁶, C. procera (seeds) ethanolic extracts where, complete mortality (100.0%) in M. domestica third larval instar achieved¹⁷ by 500 ppm, C. procera, Piper longum and Polygonum hydropiper whole-plant boiled extracts where, LC₅₀ were 557.89, 981.02 and 773.27 μL against M. domestica larvae¹⁸, Ocimum basilicum crude extract where, LC₅₀ recorded 110 ppm against M. domestica larvae¹⁹, Lantana camara leaves ethanol 70%, acetone and petroleum ether extracts which recorded 1462.6, 959.3 and 607.3 ppm (LC₅₀) against M. domestica third larval instar²⁰ and petroleum ether extract of Lagenaria siceraria which was found to be more effective against third larval instar of M. domestica with LC₅₀ 101.4 ppm than chloroform, acetone and methanol extracts with LC₅₀ 433.8, 432.1 and 468.5 ppm, respectively¹¹. In addition, prolongation in M. domestica larval and pupal periods by all tested extracts agree the previous results of Lupinustermis, C. procera and Atriplex inflate^21,22, using Artemisia monosperma, Conyza dioscoridis, Clerodedron inerme and Clocasia antiqorum²³, L. camara and Cupressus macrocarpa (leaves) powders²⁴ and L. camara (leaves and stems)²⁰.

Also, a reduction in M. domestica adult emergence percent as a result of tested extracts was observed. Similar observations noted by chloroform extract of Ricinus communis which recorded 12.75 and 25.55% reduction in M. domestica adult emergence after treating third larval instar²⁵, Petiveria alliacea and Flueggae virosa which induced 10.0, 9.0, 11.9, 11.9 and 13.8% reduction in M. domestica adult emergence at the concentration²⁶ of 15.0% and L. siceraria (leaves) methanol and chloroform extracts which recorded the lowest M. domestica adult emergence percent (16.5%) at 1000 and 800 ppm, respectively¹¹. In addition, the growth index of M. domestica was decreased as the concentration of the extract increased; such results are in agreement with the results obtained by Fouda et al.²⁰ using L. camara (leaves and stems) extracts against M. domestica.

On the other hand, the effect of tested extracts extended to females resulted from treated larvae, fecundity of resulted females decreased and the sterility index increased as compared with the control groups. Earlier authors revealing the possible reasons for the reduction of insect fecundity, as a result of treatment with plant extracts to the weakened physical stage of the treated insects²⁷, mild suppressing effect exerted by the plant extract on the insect's mating-decisive factor²⁸, reduction in the number of normal sperms produced by males²⁹, a blockage in ovarian activity, as the tested botanical products may interfere with oogenesis which, in turn, results in a complete and irreversible sterility of insect female flies³⁰and a delay or reduction of ova giving some opportunities not for retention but for possible egg re-sorption within ovaries. Also, delay could be due, in part, to a lower metabolic rate^31,32. Reduction in fecundity and increasing sterility index of M. domestica were similar to observations reported by A. inflate²², aqueous leaf extract of Nicotiana tabacum³³and methanol, acetone, chloroform and petroleum ether extract of L. siceraria leaves¹¹.

Malformations in M. domestica larvae and pupae as a result of tested extracts including brown pigmentation and a weakness in larval cuticle, larviform pupation and irregular shaped, shrinkage pupae are similar to those obtained by using essential oils against Lucilia sericata and M. domestica different stages^34,35.

CONCLUSION

The findings of the present study revealed that, Trigonella stellata and Eucalyptus citriodora tested extracts considered as important alternative agents for the control of the house fly, Musca domestica. Also, more studies are needed to reach the bioactive compounds in two plant species.

SIGNIFICANCE STATEMENT

The findings of the present study suggested that Trigonella stellata and Eucalyptus citriodora tested extracts considered to be promising alternative agents for the control of Musca domestica. The hexane extracts from tested plants recorded the highest activity against M. domestica as compared with ethanol and chloroform extracts.