الفهرس 
Bacteria attacking insectsOnly 14 pages are availabe for public view
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Abstract
The present investigation has been conducted to evaluate the cellular and humoral defense mechanisms in the hybrid Craniolian race of the honey bee, Apis mellifera (L.) following injection with the bacterium, Paenibacillus larvae larvae White (P. l. larvae) and different intensities (low, medium and high) of naturally infected colonies. Also, to determine the changes in some physical and biochemical properties of the haemolymph following infection due to its important role in the immune response.
1-Susceptibility of the honey bees to P. l. larvae injection:
The estimated LD50 values were 1.350026, 327.7239 and 3.750162 CFU/larva for the third, fourth and fifth honey bees’ larval instars, respectively. This indicates different levels of resistance in all instars toward the bacteria. The third larval instar has proven to be more susceptible than the fourth and fifth larval instars; but the fourth larval instar is the most susceptible one between tested instars. The LD20 for the third, fourth and fifth honey bees larval instars were 1.67X10-6, 2.91X10-2 and 4.49X10-3 CFU/larva, respectively. These doses were found to induce the immune response of larvae and at the same time did not cause high mortality rate in all tested instars so they used in the subsequent tests during the course of study.
2-Effect of P. l. larvae injection on the total body weight and body water content:
After bacterial-injection, the fresh (total) body weight of third larval instar decreased significantly at 6 and 12 hr post-injection, the dry body weight decreased only at 12 hr post-injection, while the body water content didn’t induce any significant change.
The bacterial treatment in fourth larval instar, the fresh (total) body weight and body water content decreased significantly at 12 hr post-injection but, the dry body did not induce any significant change.
After fifth larval instar injection, the fresh (total) body weight and the body water content increased significantly at 3 hr post-injection also, the body water content decreased significantly at 24 hr post-injection while, the dry body weight did not induce any significant change.
3-Effect of P. l. larvae injection on some physical parameters of the haemolymph:
3-1-Haemolymph volume:
The bacterial treatment caused no significant change in the haemolymph volume of both fourth and fifth larval instar but, it induced a significant increase in the haemolymph volume of the third larval instar at 3 hr post-injection. This increase may be attributed to the increase in blood THC.
3-2-Haemolymph density:
In injected third larval instar, the haemolymph density increased significantly at 6 and 24 hr post-injection. This increase may be due to the increase of bacterial metabolites. While, in naturally infected larvae, there were a significant decrease at all infection intensities (low, medium and high), this may be due to the decrease in THC and increase in haemolymph volume.
The injected fourth larval instar haemolymph density decreased significantly at 12 and 24 hr and in naturally infected larvae at high intensity of infection, this may be due to the decrease in THC and increase in haemolymph volume.
The injected and naturally infected fifth larval instar caused no significant change in the haemolymph density.
4-Effect of P. l. larvae injection on the total haemolymph proteins:
The bacterial-injection caused a significant decrease in the haemolymph protein content at 3, 12 and 24 hr for the third larval instar, at 3 and 6 hr post-injection for fourth larval instar and at 6, 12 and 24 hr post-injection for fifth larval instar. This may be due to the elimination of some proteins and/or their involvement in immune reactions.
In naturally infected larvae, third larval instar at low and high intensities caused a significant increase in the haemolymph protein content. But the fourth larval instar and fifth larval instar cause a significant decrease at low, medium and low intensities of infection, respectively. This may be due o the difference in larval age.
5-Effect of B. t. injection on the haemocytes:
5-1-Description of the haemocytes:
Thirteen type of haemocytes were described in the third, fourth and fifth larval instars of A. mellifera: Prohaemocytes (PRs), granulocytes (GRs), eosinophil cells (EOs), oenocytes (OEs), spindle shaped cells (SPLs), plasmatocytes (PLs), micronucleocytes (MIs), macronucleocytes (MAs), spherulocytes (SPs), pycnonucleocytes (PYs), Basophil cells (BAs), Adipohaemocyte cells (ADs) and Neutrophil cells (NEs).
5-2-Pathological changes:
Due to injection and natural infection with P. l. larvae of honey bees, certain pathological consequences were observed in the defined haemocytes. These changes include variation in the cell volume; vacuolization in the cytoplasm; distortion of the cell membrane, pycnosis in the nuclei and cells dwarfing. These changes may be due to the activity or toxins of P. l. larvae.
5-3-Differential haemocyte counts (DHCs):
After bacterial-injection into the third larval instar, percentages of GRs and MIs at 6 hr and Eos at 3 and 24 hr post-injection increased significantly, In naturally infected larvae, percentages of GRs increased significantly at high intensity of infection, In contrast, SPLs decreased significantly at high intensity of infection.
The injection of bacteria into the fourth larval instar, induced a singificant increase in percentages of OEs at 6 hr following injection. Percentages of GRs showed a significant decrease at all periods post-injection. In naturally infected larvae, SPLs decreased significantly at high intensity of infection.
Injection of fifth larval instar with bacteria induced a singificant increase in percentage of EOs at 12 and 24 hr following injection. But a significant decrease in percentage of SPLs at 12 hr post-injection. In naturally infected larvae, PLs percentages increased significantly at all intensities of infection. In contrast, percentages of PRs decreased significantly at all intensities of infection.
5-4-Total haemocyte counts (THCs):
There was a significant increase in THCs of the third larval instar, at 3 hr post-injection period. In contrast, in naturally infected larvae a significant decrease in THCs at all intensities of disease (low, medium and high).
The infection caused a significant decrease in THCs of the fourth and fifth larval instars at 24 hr and 6 hr post-injection, respectively. Also, a significant decrease at high intensity of infection for both instars. This decrease may be due to the involvement of the haemocytes in cellular defense mechanisms or may be due to the action of the released toxins and its lytic action in the haemocoel and degradation of haemocytes.
6-Effect of P. l. larvae injection on the defense reactions:
6-1-Cellular defense reactions:
6-1-1-Estimation of phagocytosis:
The results indicated that PLs were the essential phagocytic cell type followed by the GRs. Phagocytosis in A. mellifera was shown to be composed of three phases: attachment and recognition; activation of pseudopodia and engulfing and then digestion.
Phagocytosis response was observed in all tested instars at all post-injection periods, the maximum phagocytic rate recorded in different instars was at 24 hr post-injection. Also, in naturally infected larvae, the phagocytosis response was observed at all intensities of infection, the maximum phagocytic rate recorded in different instars was at the high intensity of infection.
6-1-2-Estimation of nodule formation:
Injection of A. mellifera with P. l. larvae caused a significant increase in nodule formation in third and fourth larval instars at 12, 24 hr and 3, 24 hr post-injection, respectively. In contrast, a significant decrease in nodule formation in fifth larval instar at 6 hr.
6-2-Humoral defense reactions:
6-2-1-Antibacterial activity:
This investigation resulted in the appearance of antibacterial substances in the haemolymph of the bacterial-injected, naturally infected larvae as well as the un-injected larvae.
This antibacterial activity increased significantly at 3 hr post-bacterial injection for the third larval instar, but decreased significantly at low and medium intensities of infection. The fourth and fifth larval instars did not induce any significant change at all periods post-injection and at all intensities of infection (low, medium and high).
7- Haemolymph electrophoresis:
The haemolymph proteins of A. mellifera third, fourth and fifth larval instars were electrophoretically separated into 10, 12 and 7 bands, respectively, according to their rate of flow from 0.142 - 0.538, 0415 - 0.745 and 0.058 - 0.74, respectively, and to their molecular weight from 122.49 - 61.79, 74 – 50.15 and 162.5 – 54.286 kDa.
In the third larval instar, injury by the needle caused a release of 12 new protein bands (2, 4, 7, 9, 11, 14, 15, 17, 18, 20, 21 and 22) and a disappearance of only one band (1). In fourth larval instar,11 bands (6, 7, 8, 9, 11, 12, 14, 15, 16, 18 and 22) were appeared after injury by the needle, where, 1 protein (4) were disappeared. While, in fifth larval instar, 16 bands (1, 4, 6, 7, 9, 10, 11, 13, 15, 16, 18, 19, 20, 21, 22 and 23) were appeared after injury by the needle, where, 1 protein (17) were disappeared. water-injection induced the appearance of twelve proteins (2, 4, 7, 9, 11, 14, 15, 17, 18, 20, 21 and 22) in the third larval instar with high percentage at 12 hr post-injection. While, it induced the synthesis of 5 new proteins (6, 11, 14, 18 and 22) in the fourth larval instar with high percentage at 6 hr post-injection. In the fifth larval instar it induced the synthesis of 14 new proteins (1, 4, 6, 7, 9, 11, 13, 15, 16, 18, 19, 20, 22 and 23) with high percentage at 24 hr post-injection. Also, bacterial injection didn’t induce the synthesis of any new protein in the third larval instar; however, in the fourth larval instar synthesis of 6 new proteins were recoded (7, 8, 9, 12, 15 and 16) with high percentage at 6 hr post-injection. But in the fifth larval instar, only one new band (21) was detected after baterial injection. Some of these new proteins may have a role in the immune response against P. l. larvae injection.
In naturally infected larvae, the third larval instar infection cause a complete clearance to 4 protein bands (1, 5, 8 and 13), while, induce synthesis of 6 bands (2, 4, 7, 14, 15 and 20) at all intensities of disease. The fourth larval instar infection cleared 3 protein bands (3, 10 and 20), and induce synthesis of 8 bands (1, 6, 7, 9, 11, 12, 14 and 16). But the fifth larval instar cause a complete clearance for 3 bands (5, 8 and 17), and induce the synthesis of 9 bands (1, 4, 7, 10, 11, 15, 18, 19 and 21).
from the overall results, it was concluded that, the honey bees, A. mellifera has efficient immune mechanisms (cellular and humoral) against the bacterium, P. l. larvae.