الفهرس 
LITERATURE REVIEWOnly 14 pages are availabe for public view
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Abstract
The cotton leaf worm, S. littoralis is an important insect pest; represents a relatively important group of plant-feeding insects causing a considerable loss in the national economic yield. Also, this pest became more resistant against biological control agents, especially Bt formulations. Many studies have demonstrated that commensal gut mi-crobiota modulate the expression of several host genes that actively influence the host gut physiology and immunity.
The present study has been conducted to isolate and identify the characterization of midgut bacteria isolated from the 5th larval instars of S. littoralis reared on three diets. To investigate their immune role by oral administration of two probiotic immune bacteria (E. casseliflavus and L. acidophilus) for larvae before challenging with Bt.
1- Effect of different diets on the development of S. littoralis:
Three different diets (castor bean, clover leaves, and artificial diet) were used to examine the effects of different diets on growth and development of S. littoralis. The clover leaves showed the highest larval period and adult longevity than artificial diet and castor bean. However, the pupal weight was significantly high for larvae fed on artificial diet followed by castor bean and clover leaves.
2- Isolation and identification of midgut bacteria isolated from the 5th larval instar of S. littoralis according to the diets:
13 bacterial isolates were extracted from the 5th instar larvae of S. littoralis reared on different three diets and were identified as bacillus, cocco-bacillus, and coccus. Five bacterial isolates were GN, and the others were GP. According to different diets, the high significant number of gut bacteria was extracted from the 5th instar larvae fed on castor bean leaves followed by clover reared larvae and artificial diet. The isolated bacteria were categorized into a total of 2 phyla (Firmicutes and Proteobacteria), 11 genera, and 13 species. a higher abundance percentage of the Proteobacteria was found when S. littoralis larvae were reared on either the castor or clover leaves. In contract, when larvae were reared on artificial diet, they harbored higher bacterial communities of Firmicutes phyla than Proteobacteria. Insects reared on the castor bean leaves harbored four isolates, S. gallinarum, B. agrestis, R. pickettii, and E. casseliflavus. For the insects reared on the artificial diet, a total of five isolates were detected, L. acidophillus, P. polymyxa, L. fusiformis, S. plymuthica, and M. sciuri. While the isolated bacteria from larvae reared on clover leaves were K. oxytoca, E. gallinarum, E. casseliflavus, and M. sciuri.
3- Antibiotic susceptibility test of the midgut bacterial isolates:
The antibiotic susceptibility test of midgut bacteria isolated from the 5th larval instar of S. littoralis according to the three diet was studied .S. gallinarum, R. picketii, L. fusiformsi, L. acidophilus, and E. gallinarum were susceptible to VA (30 µg). While S. gallinarum, E. casseliflavus, K. oxytoca, M. scuri, L. fusiformsi, P. polymyxa, S. plymuthica, and L. acidophilus were susceptible to FOX (30 µg). However, B. agrestis, E. casseliflavus, M. scuri, and L. acidophilus were susceptible to CN (10 µg).
4- The effects of antibiotic mixture on the biological parameters of S. littoralis and midgut bacterial isolates:
With increasing of antibiotic concentration, the larval, pupal, and adult duration increased. While the quantity of midgut bacteria isolated from the 5th larval instar decreased in all diets. The strains of midgut bacteria were decreased after rearing with antibiotic mixture. Among the three diets, the 500 µg/ml concentration of antibiotic mixture removed all midgut bacteria isolated from of 5th larval instar of S. littoralis.
5- Susceptibility of the 5th larval instar of S. littoralis reared on different diets to Bt after 48 h of oral infection:
The susceptibility test of the 5th larval instar of S. littoralis reared on the three diets amended with or without antibiotics to Bt after 48 h of oral infection was estimated. The LC50 values for larvae reared without antibiotics were 1.811, 1.558, and 1.988 mg/ml. While larvae reared with antibiotics were13.470, 28.501, and 62.092 mg/ml for castor bean, clover leaves, and artificial diet, respectively. The estimated LC20 for larvae fed without antibiotics were 0.632, 0.738, and 0.965 mg/ml but, the larvae fed with antibiotics were 2.454, 5.058, and 7.659 mg/ml for castor bean, clover leaves, and artificial diet, respectively. It’s clear that from the present results that the estimated LC20 and LC50 values in bacterial free larvae were significantly higher than the control larvae in all the three diets.
6- Antagonistic interaction between the culturable midgut bacteria and Bt:
The result showed that the two probiotic immune E. casseliflavus (the midgut bacteria isolated from the 5th larval instar of S. littoralis reared on either the castor bean leaves or the clover leaves) and L. acidophilus (the midgut bacteria isolated from the 5th larval instar of S. littoralis reared on the artificial diet) were significantly antagonize the growth of Bt.
7- Role of midgut probiotic immune isolated from the 5th larval instar of S. littoralis on insect immunity:
Both probiotic immune bacteria isolates (E. casseliflavus and L. acidophilus) increased the cellular (THC and PO activity) response of larvae reared on the three diets. While humoral immunity (Cec and Def gene expression) was studied only on the 5th larval instar reared only on castor bean leaves after Bt infection.
In the control insects reared on clover leaves and artificial diet registered the highest number of THC (10825 and 10585 cells/mm3, respectively). While larvae fed on castor bean had the lowest number (8287.5 cells/mm3). After 24h of treatment with of Bt, the THC values of bacterial free+ Bt and midgut probiotic immune bacteria treated larvae increased in all diets, castor bean leaves, artificial diet, and clover leaves, (18987.5, 22715.5, and 22221.75 cells/mm3, respectively) compared with the control. However, PO activity of the 5th larval instar reared on clover leaves and artificial diet registered the highest PO activity (13.8 and 12.6 O.D. units/min /ml, respectively). But those fed on castor bean had the lowest PO activity (5.9 O.D. units/min /ml). After 24h of treatment with of Bt, the PO activity of bacterial free+ Bt and midgut probiotic immune bacteria treated larvae showed a significant increase in all experimental diets, castor bean, artificial diet, and clover, (25.3, 27.37, and 34.8 O.D. units/min /ml, respectively) compared with the control. While the expression of both Cec and Def genes (1.3 and 1.43-fold, respectively) up-regulated in bacterial free+ Bt and midgut probiotic immune bacteria treated larvae reared on castor bean leaves than control (1 and 1-fold, respectively). It is clear from the present results that the cellular and humoral immunity of the bacterial free, Bt and midgut probiotic immune bacteria treated larvae increased than control.
8- Larvicidal and bactericidal effects of EOs on the 5th larval instar of S. littoralis reared on the three diets:
The larvicidal effect of two EOs, R. communis and C. camphora on the 5th instar larvae of S. littoralis was recorded after 48 h of oral treatment. The LC50 of castor EO was 0.605, 0.386, and 0.319 mg/ml. however, LC50 of camphor EO was 1.327, 0.579, and 0.530 mg/ml for larvae reared on castor, clover leaves, and artificial diet, respectively. Both EOs had bactericidal effect. The result showed a reduction in all midgut bacterial isolates expect the two midgut probiotic immune bacteria (E. casseliflavus and L. acidophilus) were increased after treatment with of LC50 of castor and camphor EOs for 48 h.
9- The effects of EOs and Bt on midgut probiotic immune bacteria
The antimicrobial activity of EOs, Bt, mixture A (mixture of Bt with castor EO), and B (mixture of Bt and camphor EO) was determined by disc diffusion method. The data showed that the inhibition zone was varied around the midgut probiotic immune bacteria. The inhibition zone of mixture B increased around E. casseliflavus. However, the mixture A inhibited L. acidophilus.
10- GC–MS analysis
The chemical constituents of the two EOs, R. communis and C. camphora were identified by GC–MS analysis. Data indicating that C. camphora and R. communis contains 23 and 43 chemical compounds, respectively. The percentage of terpenes in castor EO was 53.99%. However, camphor EO consisted mainly of terpenes (27.94%).
In conclusion, the results showed that castor bean was the most suitable diet for rearing S. littoralis larvae. Also, two midgut probiotic immune bacteria (E. casseliflavus and L. acidophilus) that isolated from the 5th larval instar can activate both host immune system (cellular and humoral) against Bt. To control this pest, we recommended that the use of a combination of Bt and Eos could be get rid of these probiotic immune bacteria.