CHANGES IN THE HAEMOLYMPH OF THE DESERT LOCUST, SCHISTOCERCA GREGARIA AFTER INJECTION WITH BACILLUS...

CHANGES IN THE HAEMOLYMPH OF THE DESERT LOCUST, SCHISTOCERCA GREGARIA AFTER INJECTION

WITH BACILLUS THURINGIENSIS Emad M. S. Barakat1; Wesam S. Meshrif2 and Magdi. G. Shehata1

1- Department of Entomology, Faculty of Science, Ain Shams Univ., Abbassia, Cairo, Egypt

2- Department of Zoology, Faculty of Science, Tanta Univ., Tanta, Egypt

ABSTRACT

ariable changes in the haemolymph volume, density and pH of the desert locust, Schistocerca gregaria (Forskal) were recorded at

different time intervals post-injection with bacterium, Bacillus thuringiensis kurstaki Berliner into the haemocoel. At the same time, changes in the total and differential haemocyte counts were investigated. Also, several pathological consequences were observed in the haemocytes (Plasmatocytes, coagulocytes and granulocytes) including variation in the cell volume, vacuolization in the cytoplasm, distortion of the cell membrane and pycnosis in the nuclei.

Key words: Schistocerca gregaria, Bacillus thuringiensis, haemolymph, haemocytes.

INTRODUCTION

Insect haemolymph is influenced at least on the level of its physical properties such as volume, density and pH, or on its biochemical composition by several factors, among them are: diet, temperature and disease (Carrel et al., 1990), the physiological condition of the insect (Chapman, 1973), and the developmental stage (Jones, 1977). Total haemocyte counts (THCs) and differential haemocyte counts (DHCs) in different insect species have also received a considerable attention. Much works have been done on changes in the haemocyte picture following injury and haemorrhage, in insects attacked by parasites and particularly during ecdysis (Salt, 1970). These counts have a great significance in insect immunity due to their share in defense reactions.

The high interest in biological means of controlling insects intensifies the need for investigating the response of insect to disease organisms and foreign proteins. The haemolymph and the haemocytes,

V

J. Egypt. Acad. Soc. Environ. Develop. (A. Entomology), Vol. 2, No. (1): 95 – 115 (2002) ISSN 1110 – 8746

in particular, offer a readily accessible criterion of this response. Also, due to the succeeded use of biological control agents especially Bacillus thuringiensis kurstaki Berliner (B. t.) formulations in the Egyptian fields against Coleoptera, Lepidoptera, and Diptera. The present study was under taken to evaluate the impact of this pathogen against the most destructive orthopteran pest, Schistocerca gregaria (Forskal). For work to be meaningful, the normal laboratory insect should be examined throughout its life. The expected results may lead to understand of the physiology of infected insects, and hence the improvement of using entomopathogenic agents in biological control measures or in integrated pest management programs.

MATERIALS AND METHODS

Insects:

The desert locust, S. gregaria (Forskal) was originated from Aswan and established in the Locust and Grasshopper Research Department, Plant Protection Research Institute, Agricultural Research Center, Cairo, Egypt. Methods used for rearing and maintaining locusts were those reported by Huxham et al. (1989). The locusts were held at 30 ± 2°C, a photoperiod of 16:8 (Light: Dark) and the relative humidity varied between 60 and 80%. The diet was offered daily as a dry mixture of: 2 bran: 2 dried whole milk: 2 wheat: 1 dried brewer's yeast (part by volume) recommended by Howden and Hunter-Jones (1958) plus small quantities of fresh clover leaves. All experiments outlined below were carried out with fifth nymphal instar and adults (both sexes), all being within 2 - 4 days after ecdysis.

Bacteria:

The bacterium, B. thuringiensis kurstaki Berliner as wettable powder formulation (AGERIN, 3200 IU/mg) was produced by The Agricultural Genetic Engineering Research Institute, Ministry of Agriculture, Egypt, and was grown aerobically at 28 ± 2°C in nutrient broth tubes for 48 hr. Inoculates of the grown bacteria were cultured on nutrient agar plates at 28 ± 2°C for another 48 hr. After growth, pure isolates were selected, cultured on nutrient agar slants and incubated at 28 ± 2°C for 48 hr, and then kept in me refrigerator at 4°C until used.

Prior to use, pure isolate previously prepared was transferred to a nutrient agar medium and incubated at 28 ± 2°C for 24 hr. The

96 Emad M. S. Barakat et al.

grown bacteria were harvested by suspending in a physiological saline (0.5% NaCl) and centrifuged at 6000 rpm for 30 min. The sediment bacteria were washed several times with a sterile saline solution and centrifuged again at the same rate till the saline solution becomes completely clear. The bacterial suspension was adjusted to a sublethal concentration of 2.33 X 103 cells/ml.

Injection technique:

Injections of insects were made with a 10 µl Hamilton microsyringe fitted with a 26-gauge needle according to Miranpuri and Khachatourians (1993). Prior to injection, the locusts were chilled on ice for 20 min and the site of injection was swabbed with 70% ethanol. Injection was made through petroleum jelly, which helped to seal the wound and prevent excessive haemolymph loss following injection. Ten µl of the bacterial concentration were injected into each insect in the last coxal corium. If any insect bled or the bacterial suspension was lost during or after injection, the insect was discarded. Control insects were injected only with equivalent volumes of 0.5% saline. Another negative control group of insects were not injected.

Collection of haemolymph:

Normal (uninjected) and injected locusts after 6, 12, 24 and 48 hr along with saline-injected controls were weighted individually, and then submerged in hot water bath at 60°C for 2-5 min then, the locusts were allowed to dry on paper towel. The heat-killed insects were amputated at the hind coxa with fine scissors. Gentle pressure was applied to the thorax until a drop of haemolymph appeared at the point of amputation. Haemolymph from two individual insects was never combined.

Haemolymph volume:

The haemolymph volume was determined for normal and bacteria-injected insects, along with their controls following the method described by Yeager and Munson (1950) and modified by Lee (1961). The tested insects were weighted individually and injected with 10 µl of 0.2% amaranth red dye (20 mg/ml of 0.5% NaCl). After allowing 5 min for the dye to mix thoroughly with the blood, 10 µl of blood were extracted and diluted to 1ml with 0.5% saline solution. A blank solution was made up from 10 µl of blood, taken before injection and diluted to 1

CHANGES IN THE HAEMOLYMPH OF THE DESERT LOCUST, 97 SCHISTOCERCA GREGARIA AFTER INJECTION WITH BACILLUS

THURINGIENSIS

ml. The optical density was measured spectrophotometrically by (Novaspec, Pharmacy Biotech.) at 515 nm using 1ml cuvette against the standard solution (prepared by diluting 10 µl of the dye solution in 1 ml of saline solution) to construct a standard calibration curve. The blood volume (V) was calculated from the following equation:

V = (dg1/g2) - a.

Where g1 weight of dye injected, g2 weight of dye in the sample, d volume of sample and, a volume of saline injected with the dye. The measurements were replicated 10 times at each of the different time intervals for each determination.

Haemolymph density:

The haemolymph densities of normal and injected insects were determined at 28 ± 2ºC for different time intervals after bacterial injection following the method described by Carrel et al. (1990). The heat-fixed locusts were amputated in the hind leg with a scissors. The oozed blood was withdrawn directly into micro-capillary tubes calibrated at 1 µl and pre-weighed using an electronic balance (BRAINWEIGH B100, OHAUS Scale, CORP. USA). The filled tube was quickly re-weighed. The haemolymph density was expressed as mg/µl. The measurements were replicated 10 times at each of the different time intervals for each determination.

Haemolymph pH:

The haemolymph pH was determined for normal and injected locusts according to the method described by Heimpel (1955). The hind leg of the locust was amputated with a fine scissors. The bulb of the microelectrode (Model 671, pH meter, Extech, USA) was brought into contact with a drop of oozed haemolymph. Determination of the haemolymph pH was completed within a maximum of 15 sec, to reduce the possibility of altering the pH value by absorption of carbon dioxide. All measurements were accomplished at 28 ± 2ºC and samples were replicated 10 times at each time intervals.

Differential haemocyte counts (DHCs):

Examining samples of haemolymph from 10 insects in a given stage made differential haemocyte counts. Whenever possible a minimum of 100 cells/insect stage was investigated. Two methods were


used: (1) Fresh preparation, the haemolymph was collected on a pre-cleaned glass slide directly into a drop of paraffin oil. A drop of 0.5% saline solution was then added and the cells were allowed to adhere to the glass slide for 3-5 minutes. (2) Stained preparations, according to Arnold and Hinks (1979) haemolymph smeared on clean glass slides, allowing to dry for 1 min, and fixed for 2 min with drops of absolute methyl alcohol. Fixed cells were stained with Giemsa’s solution (diluted 1:20 in distilled water) for 20 minutes, and washed several times with distilled water, and then dipped in tap water. The stained smears were air-dried and mounted in Canada balsam with cover slips. The haemocytes were viewed under oil immersion with a Leitz Wetzlar microscope (1600X) and 100 cells per slide were examined. Cell-shape, diameter, nuclear-cytoplasmic ratio and cytoplasmic inclusions were used for the classification of haemocytes using the classification scheme of Brehelin and Zachary (1986). The percentage of haemocyte types may be calculated by the formula:

Number of each haemocyte type _____________________________________ X100

Total number of haemocytes examined

The measurements were replicated 10 times for each stage and time interval.

Total haemocyte counts (THCs):

The haemolymph was collected into Thoma-white blood cell diluting pipette to the mark (0.5). Diluting solution (NaCl - 4.65 g, KC1-0.15 g, CaCl2 - 0.11 g, crystal violet - 0.05 g and acetic acid - 1.25 ml/liter distilled water) was taken up to the mark (11) on the pipette (dilution is 20). The first 3 drops were discarded to avoid errors. The mixture was dispensed to both chambers of the counting slide. After about 3 minutes, the total numbers of cells recognized in 64 squares of the four comers were counted. Duplicate counts were made with each sample to check counting error. The count was repeated for at least 10 times. If either the cells clumped or uneven distribution (a difference of more than 20 cells between any two squares) of haemocytes occurred in the chamber, the preparation was discarded. The number of haemocytes per cubic millimeter was calculated according to the formula of Jones (1962).


THURINGIENSIS

Number of haemocyte counted per chamber X dilution X depth factor _________________________________________________________

Number of l mm squares counted

The depth factor is usually 10.

Data analysis:

Data were expressed as mean ± standard error (SE), and compared using Student's "t test" for paired samples. The level of significance for each experiment was set at P < 0.05.

RESULTS

Effect of B. t. injection on some physical parameters of the haemolymph:

Haemolymph volume:

The haemolymph volume in uninjected fifth nymphal instar was 232.07 ± 10.17 µl/insect. In saline-injected insects, there was a slight increase (P > 0.05) in haemolymph volume till 48 hr compared to the uninjected insects. After bacterial treatment, the haemolymph volume showed no significant changes at all time intervals compared to the saline-injected insects (Fig. 1,A).

The haemolymph volume of uninjected adult was 237.00 ± 9.66 µl/insect. In saline-injected insects, the haemolymph volume showed insignificant difference (P > 0.05) till the end of the experiment compared to the uninjected insects. Bacterial injected insects suffered a significant decrease (P < 0.05) in the haemolymph volume compared to saline-injected insects at all periods post-injection (Fig. 1, B).

Haemolymph density:

The estimated mean value of the haemolymph density of uninjected fifth nymphal instar was 0.98 ± 0.03 mg/µl. Blood density of saline-injected insects showed insignificant difference (P > 0.05) at 6, 24 and 48 hr post-injection. However, a significant increase (P < 0.05) of haemolymph density of saline-injected insects was observed only at 12 hr post-injection compared to the uninjected insects. In bacterial injected insects, the haemolymph density increased significantly (P < 0.05) at 6, 12 and 24 hr, and then increased slightly (P > 0.05) at 48 hr after treatment compared to the saline-injected insects (Fig. 2, A).


The estimated mean value of the haemolymph density of uninjected adults was 1.04 ± 0.02 mg/µl. The haemolymph density of control insects showed non-significant difference (P > 0.05) at all post-injection periods compared to the uninjected insects. However, after bacterial injection, the haemolymph density increased significantly (P < 0.05) at all periods after injection compared to the saline-injected insects (Fig. 2, B).

Haemolymph pH:

In uninjected fifth nymphal instar, the mean haemolymph pH value was 7.10 ± 0.03 (i. e. slightly alkaline haemolymph). Although, there were significant decreases (P < 0.05) in the haemolymph pH of saline-injected insects at 6 hr and significant increase (P < 0.05) at 6, 12 and 24 hr in the haemolymph pH of treated insects (Fig. 3, A), the haemolymph pH was still slightly alkaline.

The haemolymph mean pH value of uninjected adult 7.13 ± 0.03 was found to be slightly alkaline as in fifth nymphal instar. The haemolymph mean pH values were significantly decreased (P < 0.05) at 6 and 12 hr post-injection in saline-injected adult compared to uninjected insects, while, significantly increased (P < 0.05) at 12 hr in treated adult compared to saline-injected controls (Fig. 3, B).

Effect of B. t. injection on the haemocytes:

Description of the haemocytes:

Haemocytes were distinguished on the basis of morphological characteristics and staining affinity. Three main haemocyte types have been found in fifth nymphal instar and adult S. gregaria (Fig. 4). These are: plasmatocytes (PLs), coagulocytes (COs), and granulocytes (GRs).

Plasmatocytes (PLs):

These cells were polymorphic (Fig. 4, Al), ovoid (Fig. 4, A2) or spindle in shape (Fig. 4, A3, A5). These cells contained large amount of basophilic cytoplasm (faintly stained) and loaded with fine granules. The majority of these cells range between 16 - 33 µn in diameter with mean value of 22.1 ± 1.394 µm. The nucleus was large and centric (Fig. 4, A5, A6) or eccentric (Fig. 4, Al, A2, A7); round (Fig. 4, Al, A2, A4, A6) or ovoid (Fig. 4, A3, A5. A7) with a diameter ranged between 7 -


THURINGIENSIS

16 µm and mean value of 10.1 ± 0.9 µm, occupying 40.0 - 55.0% of the cell volume.

Coagulocytes (COs):

These cells were round (Fig. 4, Bl) or ovoid (Fig. 4, B2) in shape with small nuclei and pale hyaline cytoplasm containing scattered granules. They had cartwheel appearance. The mean cell diameter was 15.0 ± 0.7 µm (ranged between 10 - 18 µm). The nuclei were round (Fig, 4, B3) or ovoid (Fig. 4, Bl, B2); and may be centric (Fig. 4, B2, B3) or eccentric (Fig. 4, Bl, B4) with a mean value of 9.7 ± 0.5 µm (ranged between 8 - 13 µm). The nucleus occupies about 58.3 - 75.0% of the cell volume.

Granulocytcs (GRs):

These cells were round (Fig. 4, C4, C5), ovoid (Fig. 4, Cl) or fusiform-shaped cells (Fig. 4, C2, C3). The diameter ranged between 10-16 µm with a mean value of 14.7 ± 0.6 µm. The cytoplasm was basophilic (deeply stained) which contained large number of acidophilic granules that vary in size within the same cell. The nuclei of the GRs were of variable shape, spherical (Fig. 4, Cl) or ovoid (Fig. 4, C3); and may be centric (Fig. 4, C4) or eccentric (Fig. 4, C5). The mean diameter was 9.2 ± 0.5 µm with a range of 7-12 µm. The nucleus occupied 58.3 - 66.6% of the cell volume.

Pathological effects of B. t. on the haemocytes:

Due to B. t. injection into the haemocoel of locusts, certain pathological consequences were observed in the denned haemocytes (Fig. 5). The PLs showed great variation in the cell volume (Fig. 5, Al), vacuolization in the cytoplasm (Fig. 5, A2) and distortion of the cell membrane (Fig. 5, A3). Some nuclei appeared bilobed (Fig. 5, A4, A5) or binucleateded (Fig. 5, A7) or even lost part of their chromatin materials and showed vacuoles in their nucleopiasm (Fig. 5, A6). In the COs, the nucleus appeared bilobed (Fig. 5, Bl, B3) and the cytoplasm may be eroded, ruptured, and the cytoplasmic contents were extruded (Fig. 5, B2). The GRs have been affected severely in the cytoplasm and nucleus. Pycnosis appeared in the nuclei, and cells were characterized by highly granulated and deeply stained nucleoproteins (Fig. 5, Cl). Some cells became lysed (Fig. 5, C2).


Differential haemocyte counts (DHCs):

In uninjected fifth nymphal instar, the percentages of PLs, COs and GRs were 50.40, 25.80 and 23.8%, respectively. In saline-injected insects, the percentages of PLs were significantly higher (P < 0.05) at 6, 12 and 24 hr post-injection than those of uninjected insects, but not significant at 48 hr post-injection (Fig. 6, Al). The COs showed an initial marked decrease (P < 0.05) at 6 hr compared to uninjected insects, followed by a non-significant differences (P > 0.05) at 12, 24 and 48 hr post-injection compared to the uninjected inescts (Fig. 6, A2). There was a significant increase (P < 0.05) at 6, 12, and significant decrease at 24 hr post-injection in the percentage of GRs (Fig. 6, A3) as well as non-significant difference at 48 hr post-injection comparable to the uninjected insects. In bacteria-injected insects, the percentage of PLs showed significant increase (P < 0.05) at 6 and 48 hr compared to the saline-injected insects, although at 12 and 24 hr post-injection the PLs were significantly reduced (P < 0.05) (Fig. 6, Al). All the percentages of COs from bacteria-injected locusts were significantly higher (P < 0.05) than those of saline-injected insects (Fig. 6, A2). The GRs showed significant decreases (P < 0.05) at 6, 24 and 48 hr, and non-significant difference at 12 hr compared to the saline-injected insects (Fig. 6, A3).

The percentages of PLs, COs and GRs in uninjected adults were 52.60, 25.21 and 22.20, respectively. In saline-injected adults, there were no significant differences in the percentages of PLs (Fig. 6, Bl) and GRs (Fig. 6, B3) at all post-injection periods compared to the uninjected insects. The COs of saline-injected adults increased significantly (P < 0.05) at 6 hr followed by non-significant difference at 12, 24, and 48 hr compared to the uninjected insects (Fig. 6, B2). The injection of bacteria induces an initial singificant increase (P < 0.05) in the percentages of PLs at 6 and 12 hr following injection compared to the saline-injected insects, this increase was followed by non-significant change in PLs at 24 and 48 hr (Fig. 6, Bl). There was a significant decrease (P < 0.05) in COs at 6 and 12 hr followed by a significant increase (P < 0.05) at 24 and 48 hr compared to the saline-injected insects (Fig. 6, B2). Percentage of GRs showed a significant decrease (P < 0.05) at all periods post-injection compared to saline-injected insects (Fig. 6, B3).


THURINGIENSIS

Total haemocyte counts (THCs):

The mean THCs of uninjected fifth nymphal instar and adult S. gregaria were 10030 ± 281 and 9525 ± 256 cells/mm3, respectively (Fig. 7).

No significant differences in the THC of saline-injected fifth nymphal instar were observed at 6, 24 and 48 hr post-injection compared to the uninjected insects, although at 12 hr post-injection the THCs were significantly higher (P < 0.05) (Fig. 7, A). After bacterial injection, a sudden significant increase (P < 0.05) at all periods was observed compared to the saline-injected insects (Fig. 7, A).

No significant difference in the THC between the saline-injected and unmjected adult S. gregaria was shown (Fig. 7, B). The THCs from bacteria-injected adults were significantly lower (P < 0.05) at 6 and 12 hr post-injection, however, at 24 and 48 hr post-injection, there were no significant differences in THCs between the bacterial-injected and saline-injected insects (Fig. 7, B).

DISCUSSION

The present investigation determined the basic pattern of the blood cell types and changes in the haemolymph and the haemocyte's picture in the desert locust, S. gregaria following challenge with B. t. The mean values of haemolymph volume of the uninjected fifth nymphal instar and adult stages are close to the findings of Lee (1961) on S. gregaria and Hill and Goldsworthy (1968) on L. migratoria nymphs.

The observed insignificant changes in blood volume in the fifth nymphal instar injected with bacteria at different time intervals may be attributed to the use of sublethal dose of bacteria. These results agree with the findings of Shapiro (1968) on the wax moth, G. mellonella larvae, who found that no significant differences in the blood volumes of control and wounded insects during the course of the entire experimental periods.

The estimated significant decrease of the blood volume in the adult stage during the course of infection may be attributed to water loss from blood and tissues as a result of bacterial infection. The present results agree with the findings of Webley (1951) on L. migratoria. Extreme loss of water in the cells and tissues and subsequent loss of


water from the haemolymph was reported by Bucher (1957) on Malacosoma pluvial larvae that infected with spore-forming bacteria.

The significant increase of haemolymph density was observed by the injection of bacteria into the fifth nymphal instar and adult S. gregaria at almost all post-injection periods. This may be due to the increase in THC and the decrease of blood volume as well as the increase of bacterial metabolites. These results are in agreement with those of Wemer and Jones (1969) on G. mellonella larvae injected with Sarcina and Staphylococcus and Barakat (1997) on the same insect injected with Bacillus cereus. The authors demonstrated that the injection of bacteria into the haemocoel of insect induced a significant increase in the haemolymph density.

The significant increase in the haemolymph pH in both fifth nymphal instar and adult S. gregaria after bacterial injection may be due to the induction of bacterial metabolites. Our results were in consistence with the findings of Angus and Heimpel (1956) on the larvae of Bombyx mori infected with Bacillus sotto.

Three types of haemocytes were described in the fifth nymphal instar and adult S. gregaria: PIasmatocytes (PLs), coagulocytes (COs) and granulocytes (GRs). Description was based on the basis of cytological parameters such as cell shape, size, nuclear-cytoplasmic ratio and cytoplasmic inclusions as well as staining affinity.

The observed pathological conditions in the infected haemocytes, characterized as (1) changes in the plasma membrane (erosion and extrusions of their cytoplasmic contents), (2) vacuolization and degeneration of the cytoplasm and (3) nuclear changes (pycnosis, karyorrhexis and granulosis of the nuclei), may be induced by the action of B. t. exotoxins as previously indicated by the work of Miselyunene (1976) on the caterpillar of cabbage butterfly; El-Kattan (1995) on the larvae of India meal moth, Plodia interpunctella.

The present results, on DHCs showed that in the uninjected fifth nymphal instar and adult stages, PLs represent about 50% of the total cells number. COs and GRs represent the other 50% of the total cell numbers. After bacteria-injection in the fifth nymphal instar, the proportion of PLs increased at 6 and 48 hr, but fall at 12 and 24 hr post-injection. The proportion of COs increased at all periods post-injection. The GRs decreased at 6, 24 and 48 hr after bacterial injection. The


THURINGIENSIS

initial increase in number of PLs following bacterial injection may be attributed to the increase of THC due to the activation of cell division and the release of sessile haemocytes after wounding. Similar explanation was given by Shapiro (1968) on G. mellonella larvae and Gupta (1985) on different insect species. The late decrease in PLs and GRs observed in B. t. injected insects was most likely due to the initial events of recognition of foreignness, involvement in phagocytosis, cell clumping and wound healing. This opinion is supported by Barakat (1997) on G. mellonella larvae after injection with B. cereus. The increase in COs may be attributed to the increase of mitotic index and THC. These results agree with the findings of Abu El-Magd et al. (1994) on Spodoptera littoralis larvae and contradict the results of Abu El-Magd (1992) on fifth nymphal instar S. gregaria, because of different agent (laminarin) and dose (25 µl), besides the differences in rearing conditions, diet and using unfixed haemolymph.

Following bacterial injection into the adults, the PLs increased at 6 and 12 hr, and then decreased, the COs decreased at 6 and 12 hr and GRs decreased at all post-injection periods. The increase in PLs immediately after bacterial injection was attributed to the release of sessile haemocytes after wounding, and the decrease in COs and GRs may be attributed to their involvement in phagocytosis, nodule formation and wound healing. This concept is supported by the observations of Laigo and Paschke (1966) on caterpillars, Trichoplus sp. infected with Nosema. They recorded decrease in the number of phagocytic cells at least temporary due to phagocytosis. Our results were also agreed with those of Lia-Fook (1968) on Rhodnius prollixus after wounding; Horohov and Dunn (1982) on Manduca sexta larvae following injection with two species of bacteria and Ayaad et al. (2001) on Parasarcophaga surcoufi larvae infected with a nematode.

The immediate decrease of COs at 6 and 12 hr post-injection may be due to the great demand of them into the site of injection to form a temporary closure or may be rupture to initiate the coagulation in nodule formation. There is an evidence indicating that the COs may be the first cell to contact the foreign surface and may then lyse releasing granule contents (Lackie et al., 1985). But the late increase in this cell type at 24 and 48 hr post-injection may be due to the decline of nodulation and the increase of THC.

The observed remarkable increase in THC in the fifth nymphal


instar S. gregaria after bacterial injection may be due to the release of sessile haemocytes and the activation of mitotic activity of the haemocytes, and/or to the increase of COs. These results are similar to the findings of dark and Harvy (1965); Shapiro (1968) on G. mellonella larvae; Horohov and Dunn (1982) on M. sexta larvae. Guzo and Stoltz (1987) on the tussock moth, Orgyia leucostigma, noted that the nodulation of smaller objects such as yeast cells were accompanied by a rapid and sustained increase in THC. Meanwhile, the present results disagree with the findings of Hoffinann et al. (1974) on L. migratoria, who found immediate decrease in THC following injection of B. t. This may be due to species difference.

Bacterial-injection into the adults caused marked drop in THC at 6 and 12 hr indicating that B. t. have serious consequences on haemocytes. This reduction may be attributed to the decrease of COs and GRs. in addition to the involvement of the haemocytes in phagocytosis and nodule formation, which always accompanied with the death of defensive haemocytes or may be due to the action of the released toxins. The contrast effect of treatment on THCs of fifth instar and adult may be attributed to the stage difference. Our results agree, in this respect, with the results of some authors working on lepidopterous larvae after injection with live B. t. (Wittig, 1966; Gagen and Ratcliffe, 1976, Faye, 1978; Abu El-Magd et al., 1994). Similar observations were also reported by Hoffmann et al. (1974) on L. migratoria. Barakat (2001) noted the same effect on adult worker Apis mellifera injected with Pseudomonus aeruginosa. Contrarily, these results disagree with Abu El-Magd (1992) on L. migratoria due to species and technique differences.

ACKNOWLEDGEMENT

We wish to thank Dr. El-Kattan, N.A.I. for his useful suggestion and assistance in preparing the final version of this article.

REFERENCES

Abu El-Magd, A. A. (1992). Modifications of the haemogramme and of some cellular defense reactions of the desert locust, Schistocerca gregaria 5th nymphs after activation of the prophenoloxidase system. J. Egypt Ger. Soc. Zool., 5(A), 23-36.


THURINGIENSIS

Abu El-Magd, A. A.; Harb, M. F.; El-Katatny, M. S. (1994). In vivo studies on cellular and humoral reactions of Spodoptera littoralis larvae to Bacillus thuringiensis bacteria and spore-6-endotoxins. Bull. Fac. Sci., Assuit Univ., 2(2-E), 201-214.

Angus, I. A. and Heimpel, A. M. (1956). An effect of Bacillus sotto on the larvae of Bombyx mori. Can. Entomologist., 88, 138-139.

Arnold, J.W. and Hinks, C.F. (1979). Insect haemocytes under light microscopy: technique. In: Insect Haemocytes (A.P. Gupta, ed.). Cambridge University Press, Cambridge.

Ayaad, T. H.; Dorrah, M. A. Shaurub, E. H. and El-Sadawy, H. A. (2001). Effects of the entomopathogenic nematode, Heterohabditis bacteriophora HP 88 and Azadirachtin on the immune defense response and prophenoloxidase of 108 Parasarcophaga surcoufi larvae (Diptera: Sarcophagidae). J. Egypt Soc. Parasitol., 31(1), 295-325.

Barakat, E. M. S. (1997). A comparative study on the immune response of the wax moth, Galleria mellonella (L.) to some biotic and abiotic materials. Ph. D. Thesis, Ain Shams Univ.

Barakat, E. M. S. (2001). Haemocytic changes in honey bee. Apis mellifera (L.) following injection of bacteria. Ain Shams Sci. Bull., 38, 500-517.

Brehelin, M. and Zachary, D. (1986). Insect haemocytes: A new classification to rule out the controversy. In: Immunity in invertebrates. (Brehelin, M. Ed.). Pp. 36-46; Speringer-Verlag.

Bucher, G. E. (1957). Disease of the larvae of tent caterpillars caused by a spore forming bacterium. Can. J. MicrobioL, 3, 695-709.

Carrel, J. E.; Wood, J. M.; Yang, Z.; Mecairel, M, H. and Hindman, E. E. (1990). Diet, body water, and haemolymph content in the Blister beetle, Lytta polita (Coleoptera: Meloidae). Environ. Entomol., 79(5), 1283-1288.

Chapman, R. F. (1973). The insects' structure and function. ELBS and the English Universities Press, Ltd., London.

Clark, R. M. and Harvy, W. R. (1965). Cellular membrane formation by plasmatocytes of diapausing Cecropia pupae. J. Insect Physiol., II, 161-175.


El-Kattan, N. A. I. (1995). Physiololgical studies on the Indian meal moth, Plodia interpunctella HB. (Pyralidae: Lepidoptera) infected with microbial entomopathogen. Ph.D. Thesis, Ain Shams Univ.

Faye, I. (1978). Insect immunity: early fate of bacteria injected in Satumiid pupae. J. Invertebr. Pathol., 31, 19-26.

Gagen, S. J. and Ratcliffe, N. A. (1976). Studies on the in vivo cellular reactions and fate of injured bacteria in Galleria mellonella and Pieris brassicae larvae. J. Invertebr. Pathol., 25(1), 17-24.

Gupta, A. P. (1985). Cellular elements in the haemolymph. In: Comparative insect physiology, biochemistry and pharmacology. Pp. 401-451 (G. A. Kerkut and L. I. Gilert, eds.). Pergamon Press, Oxford-New York.

Guzo, D. and D. B. Stoltz. (1987). Observations on cellular immunity and parasitism in the tussock moth. J. Insect Physiol., 33(1), 19-31.

Heimpel, A. M. (1955). The pH in the gut and blood of the larch sawfly, Pristiphora erichosonii (HTG.), and other insects with reference to the pathogenicity of Bacillus cereus. Can. J. Zool., 33, 99-106.

Hill, L. and Goldworthy, G. J. (1968). Growth, feeding and activity, and the utilization of reserves in larvae of Locusta. J. Insect Physiol., 14,1085-1098.

Hoffmann, D.; Brehelin, M. and Hoffmann, J. A. (1974). Modification of the haemogramme and of the haemopioetic tissue of male adults of Locusta migratoria (Orthoptera) after injection of Bacillus thuringiensis. J. Invertebr. Pathol., 24, 238-247.

Horohov, D. W. and Dunn, P. E. (1982). Changes in the circulating haemocytes population of Manduca sexta larva following injection of bacteria. J. Invertebr. Pathol., 40, 327-339.

Howden, G. F. and Hunter-Jones, P. (1958). An artificial diet for the laboratory rearing of locusts. Nature, 128, 1527-1528.

Huxham, I. M.; Samuel, K. D. Z.; Heale, J. B. and MacCorkindale, N. J. (1989). In vivo and in vitro assays for pathogenicity of wild type and mutant strains of Metarhizium anisopliae for three


THURINGIENSIS

insect species. J. Invertebr. Pathol., 53, 143-151.

Jones, J. C. (1962). Current concepts concerning insect haemocytes. Amer. Zool., 2, 209-246.

Jones, J. C. (1977). The circulatory system oflnsecta, Charles C. Thomas, Springfield, 111.

Lackie, A. M.; Takle, G. and Tetley, L. (1985). Haemocytic encapsulation in the desert locust, Schistocerca gregaria and the American cockroach, Periplaneta americana. Cell Tiss. Res., 240, 343-351.

Laigo, F. M. and Paschke, J. D. (1966). Variations in the total haemocyte counts as induced by a nosemosis in the cabbage looper, Trichoplnsia ni. J. Invertebr. Pathol., 8,175-179.

Lee, R. M. (1961). The variation of blood volume with age in the desert locust, Schistocerca gregaria (Fosk.). J. Insect Physiol., 6, 36-51.

Lia-Fook, J. (1968). The fine structure of wound repair in an insect, Rhodnius prollixus, J. Morphol., 124, 37-78.

Miranpuri, G.S. and Khachatourians, G.G. (1993). Haemocyte surface changes in the migratory grasshopper, Melanoplus sanguinipes in response to wounding and infection with Beuveria bassiana. J. Entomol. Exp., 68, 157-164.

Miselyunene, I. S. (1976). Changes in the morphology and relationship of different types of haemolymph cells in cabbage butterfly caterpillars infected with endobacterin. Tsitologiya., 75(10), 1220-1225.

Salt, G. (1970). The cellular defense reactions of insects. Cambridge Monograph in Experimental Biology, No. 16. Cambridge University Press, Cambridge.

Shapiro, M. (1968). Changes in the haemocyte population of the wax moth, Galleria mellonella, during the wound healing. J. Insect Physiol., 14, 1725-1733.

Webley, D. P. (1951). Blood cell counts in the African migratory locust, Lousta migratoria migratorioides Reiche and Faimaire. Proc. Roy. Ent. Soc. London., A 26, 25-37.


Werner, R. A. and Jones, J. C. (1969). Phagocytic haemocytes in unfixed Galleria mellonella larvae. J. Insect Physiol., 15(3) 425-437.

Wittig, G. (1966). Phagocytosis by blood cells in healthy and diseased caterpillars. II-A consideration of the methods of making haemocyte counts. J. Invertebr. Pathol., 5, 461-477.

Yeager, J. F. and Munson, S. C. (1950). Blood volume of the roach, Periplaneta americana determined by several methods. Arthropoda., 1, 255-256.


THURINGIENSIS

A B

0

50

100

150

200

250

300

Uninjected 6 12 24 48

Hours post-injection

Volume (µl/insect)

ControlTreated

0

50

100

150

200

250

300



Volume (µl/insect)

ControlTreated

Figure 1: Haemolymph volume (µl/insect) of fifth nymphal instar (A) and adult (B) of S. gregaria determined at different time intervals post-injection with B.

thuringiensis using Amaranth dye method.

A B

0

0.2

0.4

0.6

0.8

1

1.2

1.4



Density (mg/µl)

ControlTreated

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8



Density (mg/µl)

ControlTreated

Figure 2: Haemolymph density (mg/µl) of fifth nymphal instar (A) and adult (B) of S. gregaria determined at different time intervals post-injection with B.

thuringiensis.

A B

6

6.2

6.4

6.6

6.8

7

7.2

7.4



pH value

ControlTreated

6

6.2

6.4

6.6

6.8

7

7.2

7.4



pH value

ControlTreated

Figure 3: Haemolymph pH of fifth nymphal instar (A) and adult (B) of S. gregaria determined at different time intervals post-injection with B.

thuringiensis.


Figure 4: Photomicrographs of haemocytes of normal S. gregaria.

(A1, 2, 3, 6) PLs, from fifth nymphal instar; (A4, 5, 7) PLs, from adult; (B1, 2) COs, from adult; (B3, 4) COs, from fifth nymphal instar; (C1, 2, 3) GRs, from fifth nymphal instar; (C4, 5) GRs, from

adult. Bar = 10 µm.

Figure 3: Photomicrographs of infected circulating haemocytes with B. t. of

fifth nymphal instar and adult S. gregaria. (A1, 4, 7) PLs, from fifth nymphal instar; (A2, 3, 5, 6) PLs, from adult; (B1) CO, from fifth nymphal instar; (B2) CO, from adult; C1, GR, from adult; (C2) GR, from fifth nymphal instar. Bar = 10 µm.


THURINGIENSIS

A B Plasmatocytes

0

10

20

30

40

50

60

70



% of cell number

ControlTreated

Plasmatocytes

0

10

20

30

40

50

60

70

80



% of cell number

ControlTreated

Coagulocytes

0

5

10

15

20

25

30

35



% of cell number

ControlTreated

Coagulocytes

0

5

10

15

20

25

30

35



% of cell number

ControlTreated

Granulocytes

0

5

10

15

20

25

30



% of cell number

ControlTreated

Granulocytes

0

5

10

15

20

25

30



% of cell number

ControlTreated

Figure 6: Differential haemocyte counts (DHCs) (cell/mm3) of fifth nymphal instar (A) and adult (B) of S. gregaria determined at different time intervals

post-injection with B. thuringiensis.

A B

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000



Number of haemocytes (cells/mm3)

ControlTreated

0

2000

4000

6000

8000

10000

12000



Number of haemocytes (cells/mm3)

ControlTreated

Figure 7: Total haemocyte counts (THCs) (cell/mm3) of fifth nymphal instar (A) and adult (B) of S. gregaria determined at different time intervals post-injection

with B. thuringiensis.


نتيجة ، شيستوسيرآا جريجارياالتغييرات الناشئة فى دم الجراد الصحراوى

باسيلس ثيورنجينسزالحقن ببكتيريا

1مجدى جبريل شحاته – 2وسام صالح الدين على مشرف – 1عماد محمود سعيد برآات

. مصر –القاهرة –العباسية –آلية العلوم جامعة عين شمس –قسم علم الحشرات – 1

. مصر – طنطا – طنطاآلية العلوم جامعة –سم علم الحيوان ق - 2

ه ه ، و أس دم ، و آثافت م ال ى حج ة ف رات ملحوظ دوث تغيي ة ح رت الدراس أظه

ى حشرة الجراد الصحراوى دروجينى ف االهي يرآا جريجاري رات ) فورسكال( شيستوس د فت بع

دموى آورستاآى باسيلس ثيورنجينسزببكتيريا زمنية مختلفة من الحقن بيرلينر فى االتجويف ال

دم ، . ا ال وعى لخالي ى و الن د الكل رات فى الع ا لوحظت وفى نفس الوقت تمت دراسة التغيي آم

ة (عدة تغييرات مرضية فى الخاليا الدموية ا الحبيبي تجلط و الخالي ا ال ة و خالي ) الخاليا البالزمي

يتوبالز وى و شملت تغييرًا فى حجم الخلية ، و ظهور فجوات فى الس ًا فى الغشاء الخل م ، و تلف

. النواة

) 2002( المجلد الثانى العدد األول –الجمعية األآاديمية المصرية لتنمية البيئة 115 )علم الحشرات –أ (

CHANGES IN THE HAEMOLYMPH OF THE DESERT LOCUST, SCHISTOCERCA GREGARIA AFTER INJECTION WITH BACILLUS...

Documents

Transcript of CHANGES IN THE HAEMOLYMPH OF THE DESERT LOCUST, SCHISTOCERCA GREGARIA AFTER INJECTION WITH BACILLUS...