MATERIALS AND METHODS

Survey of parasitoids and estimation of parasitism rate in the field on certain aphid species: To survey the aphid parasitoids, random samples of the following aphid species B. brassicae, A. craccivora, A. nerii and Hyalopterus pruni were collected from different host plants cultivated at the experimental farm located at Kafr Sakr District, Sharkia Governorate, Egypt during the two successive seasons, 2011-2013. These host plants were cabbage, faba bean, reed plants (Hagna) and Dafla, which were kept free from any pesticide application. Weekly samples were chosen from heavily infested plant parts with the previous aphid species collected. Infested plant parts were placed in tight closed paper bags and transferred to the laboratory, the size of samples of each crop were as follows: from cabbage seven leaves, each of about 20 inch², from faba bean 20 leaves and from reed plant (Hagna) and Dafla fifteen leaves. All individuals from each aphid species found on the host plant samples were counted. Aphids were fed on their host plant and kept in Petri dishes (50 aphid individuals/petri dish) until formation of mummies. The mummies were isolated and kept in small glass tubes until emergence of adult parasitoids at the same time, hyperparasitoid adults emerged from mummies were classified, counted and their percentages were also calculated. Emerged parasitoids were mounted and identified at the Biological Control Department, ARC, Giza, Egypt. Percentage of parasitism was calculated as monthly means according to Farrell and Stufkens (1990) also sex ratio was calculated.

Biology
Life cycle: A laboratory culture of the aphid species (B. brassicae, A. craccivora, A. nerii, H. pruni and Hyperomyzus lactucae L.) were maintained under laboratory conditions. Fifty nymphs, almost 3rd nymphal instars from the last aphid species were placed on the last host plants under small cages (10 replicates/treatment). In each cage or jar, aphids were exposed to five mated females for 2 h. Afterwards, parasitoid females were removed and then the cages or jars were placed in the laboratory at 21±1°C and 68±5% R.H. to determine the durations of different parasitoid stages (egg-mummy, mummy-adult and egg-adult). Parasitized aphids were dissected daily by a very fine needle, in a drop of ringer's solution.

Host preference: The experiment was carried out under laboratory conditions of 21.0±1°C and 68.0±5% R.H. the aphid species tested were B. brassicae, A. craccivora, A. nerii, H. pruni and H. lactucae. Five mated females of D. rapae were exposed to 150 aphids, almost 3rd nymphal instars. The experiment was replicated five times in plastic jars. The experiments of host suitability were examined and counts of aphid mummies, No. of emerged adults and No. of non emerged mummies were recorded. The percentages of parasitism were calculated.

Biochemical assessment: Sample preparation included 150 apterous adult use of each aphid species was assembled and placed in clean jars. Apterous adults were homogenized for 3 min. In distilled water (150 adults 5 mL^-1) using a teflon homogenizer surrounded with a jacket of crushed ice. The homogenates were centrifuged at 3500 r.p.m for 10 min at 5°C to remove the haemocytes. The samples were divided into three small portions and kept in deep freezer at (-20°C) until required. The supernatant were immediately assayed to determine total soluble protein, total carbohydrate and free amino acid. The haemolymph was assayed to determine total lipids:

Statistical analysis: The significance of the main effects was determined by analysis of variance (ANOVA). The significance of various treatments was evaluated by Duncan’s multiple range test (p<0.05) (Snedecor and Cochran, 1980). Data were subjected to statistical analysis using a software package CoStat Statistical Software (2005). A product of Cohort Software, Monterey, California.

RESULTS

Survey of aphid parasitoids on certain plants
Cabbage aphid: (B. brassicae):
Primary parasitoids: Diaeretilla rapae (Mcintosh)
Secondary parasitoids: Pachyneuron sp. and Alloxysta sp.

Faba bean aphid (A. craccivora):
Primary parasitoids: D. rapae, Ephedrus persicae Frogatt and Trioxys sp.
Secondary parasitoids: Aphidencyrtus sp.

Oleander aphid (A. nerii):
Primary parasitoids: D. rapae, Aphidius matricariae Hal. and Aphelinus sp.
Secondary parasitoids: Pachyneuron sp., Alloxysta and Aphidencyrtus sp.

Hagna aphid (H. pruni):
Primary parasitoids: D. rapae, Aphidius colemani Viereck and Aphelinus sp.

Role of D. rapae for controlling B. brassicae, A. craccivora, A. nerii, H. pruni on B. brassicae: The primary parasitoid D. rapae was the demonstrate species with high relative densities during the two successive seasons. The highest total percentage of parasitism was 83.4% in the fourth week of February in the first season and 91.21% in the second week of March on cabbage in the second season. Seasonal means of the aphid's count were 447±32.39 and 444±48.26 while the mean percentages of parasitism were 39.32±12.46 and 43.45±12.93 on cabbage during the two seasons, respectively (Table 1).

Aphis craccivora: The primary parasitoid D. rapae was the highest relative densities (60.87 and 72.73%) during the third week of January and the second week of December during the both studied seasons, respectively. The mean relative densities was (55.25 and 63.77%) during the both seasons, respectively. The highest percentages of parasitism were 13.94% in the fourth week of January during the first season 2011-12 and 15.26% in the first week of March. Meanwhile, the mean percentages of parasitism were 9.14±4.78 and 8.2±4.42 during the two seasons, respectively (Table 2).

Aphis nerii: The parasitoid D. rapae was the highest relative densities (61.91 and 62.50%) during the second week of November in the first season, while the first week of December in the second season, respectively. The mean relative densities were (51.20 and 52.61%) during both seasons, respectively.

Hyalopterus pruni: The parasitoid D. rapae was the highest relative densities (66.67 and 83.3%) in the second week of October in the first season and the third week of October. The mean relative densities were (54.95 and 32.97%) during both seasons, respectively. The highest percentage of parasitism was 8.39% in the third week of February and 7.71 in the second week of March. The mean percentage of parasitism was 4.66±1.15 and 5.37±2.15 during the both seasons, respectively (Table 4).

Data in Table 5 indicated that the mean seasonal percentages of parasitism were ranged between 4.72-83.84, 2.58-13.94, 1.01-35.14, 2.22-6.36% and 4.01-91.21, 1.37-15.01, 1.85-40.78, 0.76-7.25% on B. brassicae (cabbage), A. craccivora (faba bean), A. nerii (Dafla) and H. pruni (Reed plant) during the two seasons, respectively. Ratio between mummies: aphids were 1:2.78, 1:12.23, 1:9.51, 1:23.18 and 1:2.61, 1:13.41, 1:10.84, 1:19.22 on the same last aphid species during 2011-2013 seasons, respectively.

Biology: Presented data included the life cycle, longevity and sex ratio of the parasitoid female on the aphid species B. brassicae, A. craccivora, A. nerii, H. pruni and H. lactucae under laboratory conditions. Females of D. rapae parasitized and developed to adults in all last aphid species except H. lactucae, they could not develop to adulthood in it.

Life cycle: Data presented in (Table 6) indicate life cycle of the parasitoid D. rapae on the tested aphid species B. brassicae, A. craccivora, A. nerii, H. pruni. Developmental time from (Sting-Mummy) were 11.6±0.62, 9.05±0.63, 12.37±0.84, 11.67±0.82 days, meanwhile, the period from (Mummy-Adult) lasted 4.42±0.67, 3.12±0.67, 5.15±0.33 and 3.70±0.43 days. On the other hand, total developmental times (Sting-Adult) were 15.58±0.69, 12.17±0.75, 17.52±0.70 and 15.37±0.71 days on last aphid species, respectively.

Table 1:	Monthly percentage of parasitism of Brevicoryre brassicae by Diaeretiella rapae on cabbage plants at Sharqia Governorate during successive seasons 2011-2012 and 2012-2013
A: No. of mummies counted at the date of inspection, B: No. of mummified host appeared during laboratory rearing, RD: Relative density, SE: Standard error

Table 2:	Monthly percentage of parasitism of Aphis craccivora by Diaeretiella rapae on faba bean plants at Sharqia Governorate during successive seasons 2011-2012 and 2012-2013
B: No. of mummified host appeared during laboratory rearingm RD: Relative density, SE: Standard error, A: No. of mummies counted at the date of inspection

Table 3:	Monthly percentage of parasitism of Aphis nerii by Diaeretiella rapae on oleander plants (Dafla) at Sharqia Governorate during the two successive seasons 2011-2012 and 2012-2013
A: No. of mummies counted at the date of inspection, B: No. of mummified host appeared during laboratory rearing, RD: Relative density

Table 4:	Monthly percentage of parasitism of Hyalopterus purni by Diaeretiella rapae on (Hagna) plants at Sharqia Governorate during two successive seasons 2011-2012 and 2012-2013
A: No. of mummies counted at the date of inspection, B: No. of mummified host appeared during laboratory rearing, RD: Relative density

Table 5:	Mean seasonal percentage of parasitism of aphid parasitoids on certain aphid species on some plants at Sharqia Governorate during the two successive seasons 2011-2012 and 2012-2013

Longevity: From data presented in Table 6, it could be generally observed that adult females lived for a longer period than male ones.

Sex ratio: Data presented show the sex ratio of adult parasitoids of D. rapae in the two generations on the last aphid species its were (Female:Male) 1.2:1, 1.07:1, 1.1014 and 1.09:1 on B. brassicae, A. craccivora, A. nerii and H. pruni, respectively.

Table 6:	Development time in days of the parasitoid Diaeretiella rapae on certain aphid species at 20± 1°C and 50 RH (%)
Data expressed as Mean±SD, *p≤0.05 **,***p≤0.01. Mean under each variety having different letters in the same raw denote a significant different (p≤0.05)

Table 7:	Performance of the parasitoid Diaeretiella rapae towards certain aphid species under laboratory conditions 20±1 C and 68±5 % RH

Host preference: Data presented in (Table 7) indicated performance the parasitoid D. rapae towards certain aphid species under laboratory conditions. Total numbers of mummies were 66.78±2.3, 42.44±3.59, 28.42±3.35 and 17.52±1.39 on B. brassicae, A. nerii, A. craccivora and H. pruni, respectively. Statistical analysis of data showed significant differences among the parasitoid and aphid species at 20±1°C. highest percentage of parasitoid reached 44.52% at B. brassicae while the minimum was 11.79% at H. pruni. Aslo, the highest percentage of adult emergence was 81.42 on B. brassicae while the minimum was 57.05% on H. pruni.

Nutrient compounds of different aphid species: Data presented in (Table 8) show the level of total protein in the supernatant of the homogenated aphid species Hypermoyzus lactucae and H. pruni recorded the highest significant level (34.733±1.22 and 34.533±1.23 mg g^-1 t. wt., respectively). While B. brassicae gave the lowest significant one (12.89±0.560 mg g^-1 t. wt.), p = 0.0000.

Results also indicate significant increase in the total carbohydrate was regarded in the case of H. pruni (23.10±0.95 mg g^-1. t. wt). Reversely, A. nerii, B. brassicae and A. craccivora recorded the lowest significant reduction (10.73±0.45, 10.49±0.33 and 10.42±0.40 mg g^-1. t. wt., respectively p = 0.0000 (Table 8).

As for total lipids, B. brassicae manifested the highest significant level of total lipids (4.616±0.27 mg g^-1 t. wt.) followed by H. pruni 1.573±0.09, A. craccivora 1.21±0.006, Hypermoyzus lactucae 1.143±0.04 and A. nerii 0.88±0.04 mg g^-1. t. wt. ) p = 0.0000 (Table 8).

A. craccivora and B. brassicae produced the highest significant increase in free amino acids (4.266±0.15 and 1.65±0.09 mg g^-1. b.wt.), respectively (Table 8). On the other hand, Hypermoyzus lactucae recorded the least significant decrease (0.556±0.04 mg g^-1 t. wt., p = 0.0000).

Table 8:	Nutrient compounds of different aphid species
Data expressed as Mean±SD, *p≤0.05 **-***p≤0.01, mean under each variety having different letters in the same raw denote a significant different (p≤0.05)

DISCUSSION

In the current study, we found that the primary parasitoids associated with four aphid species in the field namely, B. brassicae, A. craccivora, A. nerii, H. pruni. These species infested cabbage, faba bean, Dafla and reed plants (Hagna). The results indicated those three aphids parasitoids were emerged from the mummified aphids of B. brassicae namely; D. rapae, Pachyneuron sp. and Alloxysta sp. Four aphids parasitoids were emerged from the mummified aphids of A. craccivora namely; D. rapae, Ephedrus persicae, Trioxys sp. and Aphidencyrtus sp. Aphis nerii was the dominant aphid species infesting Dafla plants. Six aphids parasitoids were emerged from its mummies namely; D. rapae, Aphidius matricariae, Aphelinus sp., Pachyneuron sp., Alloxysta and Aphidencyrtus sp. Meanwhile, three aphid parasitoids were emerged from the mummified aphids of H. pruni namely; D. rapae, Aphidius colemani and Aphelinus sp. Obtained results showed that D. rapae was the dominant aphid species in this study.

The results agree with (El-Maghraby, 1993; Abdel-Megid, 1999; Zhang and Hassan, 2003) and Saleh (2012) found that B. brassicae was the aphid species infesting cabbage and cauliflower crops and the main parasitoid emerged from the mummified aphid was D. rapae. On the other hand, Abdel-Samad (1996), Ragab et al. (2002a), Rakhshani et al. (2006) and Saleh et al. (2009b) mentioned the parasitoid, D. rapae, L. fabarum, Ephedrus sp. and a hyperparasitoid, Aphidencyrtus sp., emerged from mummified aphid A. craccivora. Meanwhile on oleander plants, Kavallieratos et al. (2001) and Maghraby (2012) showed that the most common parasitoid species attacking A. nerii were A. colemani, Binodoxys angelicae, D. rapae and P. volucre. Also, (Vaz et al., 2004) found that parasitoids on A. nerii were L. testaceipes and A. colemani and the hyperparasitoids were Pachyneuron sp. and S. aphidivorus. These results agree with those of (Ibrahim and Afifi, 1994; Megahed, 2000) and Saleh et al. (2006) who mentioned that the aphid H. pruni is one of the important aphid species infesting common reed and plum trees in Egypt. On reed plants (Stary, 1970; Ibrahim and Afifi 1994; Megahed, 2000) and (Maghraby, 2012) who showed that A. colemani and D. rapae are considered among important parasitoid species on the aphid H. pruni.

The results in this investigation indicated that the mean seasonal percentages of parasitism were ranged between 4.72-83.84, 2.58-13.94, 1.01-35.14, 2.22-6.36% and 4.01-91.21, 1.37-15.01, 1.85-40.78, 0.76-7.25% on B. brassicae (cabbage), A. craccivora (faba bean), A. nerii (Dafla) and H. pruni (Reed plant) during both seasons, respectively (Table 5).

However (Vaz et al., 2004; Saleh, 2012) and (Maghraby, 2012) showed that the aphid B. brassicae is a major pest on crucifer plants in several parts of the world especially cabbage in Egypt, also, reported that D. rapae played the major role towards suppressing B. brassicae population. (Saleh, 2012) mentioned that the total means of parasitism rates of D. rapae were 23.58 and 28.06 on cauliflower plants during two seasons. In addition (Maghraby, 2012) in Egypt showed that the mean parasitism rates were 34.6, 36.13 and 28.73 and 32.24 on cabbage and cauliflower plants during 2010-2012 seasons, respectively.

However, Abdel-Samad (1996) in Egypt, reported that the rate of parasitism on A. craccivora ranged between 15.4 and 22.0% during March on this aphid species. Also, Ragab et al. (2002b) stated that the highest total percentage of parasitism was 15.14% in February in the first season and 17.40% in January in the second season. (Saleh et al., 2009a) showed that the Ephedrus sp., on A. craccivora were 8.17 and 6.45% during the two seasons of their study.

The highest percentages of parasitism on A. nerii were 40.57% in the first of March 2011-12 and 34.49% during the third week of March in the second one 2012-13, respectively. The mean percentage of parasitism was 16.08±5.45 and 13.22±5.48 during both seasons, respectively (Table 3). These results agree with those of (Mackauer and Volki, 1993) who mentioned that the parasitism rates on A. nerii tend to range between 1.0 and 10.0%. Although parasitism rates ranged between 30.0 and 45.69% on some host plants Asclepias and Dafla plants (Sandra et al., 2004) and (Saleh et al., 2009b).

Results agree with findings of Megahed (2000) and (Saleh et al., 2006), who mentioned that the percentage of parasitism mentioned that the percentage of parasitism on H. pruni ranged between 6.64-7.5%.

In the respect of biological parameters (Saleh et al., 2009a) reported that sex ratio of the parasitoid D. rapae (females: Males) was 1.7:1 by rearing the parasitoid for five successive generations, sex ratio was almost 1:1 in the first three generations, but males dominated in the 4th and 5th generations.

However, El-Batran et al. (1996) mentioned that the parasatoid D. rapae completed its life cycle in B. brassicae. Also, Ragab et al. (2002b) reported that D. rapae completed its life cycle in a period of 12-18 days at 19.5°C on B. brassicae and 11-15 days on A. craccivora. On the other hand, Saleh (2008) reported that the total development period of the parasitoid D. rapae lasted 16-24 days, with an average of 19.87, 24.39, 16.34 and 18.55 days in B. brassicae, A. nerii, A. craccivora and H. pruni, respectively at 16°C and 77% R.H.

The results in this research indicated that performance of the parasitoid D. rapae towards certain aphid species under laboratory conditions. Total numbers of mummies were 66.78±2.3, 42.44±3.59, 28.42±3.35 and 17.52±1.39 on B. brassicae, A. nerii, A. craccivora and H. pruni, respectively.

Similar finding was recorded by Elliot et al. (1994) in USA and Ragab et al. (2002a) in Egypt who mentioned that D. rapae parasitized on many aphid species (B. brassicae, A. craccivora, A. nerii, M. persicae, S. avane, H. pruni and A. gossypii). On the other hand El-Naggar et al. (2008) and (Ralec et al., 2011) showed that the parasitoid density in relation to host density had influenced percentage of parasitism and emergence. The percentage of parasitism increased with the increase in numbers of parasitoid.

Due to the nutrient compounds of different aphid species, available informations indicated that, there are few researches about the relation between parasitism percentages and nutrition components of different species of aphids. A result of the ongoing needs of parasitoids to get their food to continue to grow and develop, they are looking for a suitable host that ensures it is available source of nutrition. Parasitoids larvae grow at different rates in different aphid hosts of similar sizes, which makes us suggest that this preference is linked to the quality of the nutrition they provided these hosts (Sequeira and Mackauer, 1993). The host species may influence the rate of development and the survival of a parasitoid. A host may be unsuitable due to the lack of some necessary nutritional or hormonal resource (Carver and Sullivan, 1988; Kant et al., 2008). Our data on D. rapae showed that host aphid species, B. brassicae, A. nerii and A. craccivora were nutritionally and physiologically suitable for parasitoid development but B. brassicae was considered the best host among the species tested, adult emergence and higher parasitization.

That is probably attributed to its higher nutrient composition of total lipids and free amino acids. These amino acids rapidly incorporate to produce large amounts of proteins which are necessary for the developing parasitoid. Lipids can convert to proteins to substitute the reduction in protein content or produce supplementary energy used for growth and development. In addition, they include important hormones and pheromones (Downer, 1978).

However (Abdul Rehman and Powell, 2010) mentioned that aphid parasitoids have considerable potential as biological control agents but their efficiency is dependent upon their presence in the right place at the right time and right host: parasitoid ratio. Understanding parasitoid behavior, together with identification of physical and chemical cues regulating the behavior, is providing exciting opportunities for manipulation of parasitoids in the field, as populations introduced through inundative releases.

CONCLUSION

Diaeretialla rapae is an important primary parasitoid of a wide range of aphid species and is considered a promising biological control agent against aphid species especially cabbage aphid in cabbage and cauliflower fields and it recommended to be an item of Integrated Pest Management Programs in Egyptians fields designed to control B. brassicae.

ACKNOWLEDGMENTS

Many thanks to Prof. Dr. A. El-Heneidy, Biological Control Department. Agricultural Research Center. Giza, Egypt for the identification of the parasitoid species in this investigation.