Detection of Insect Immunity Substances (Lectins) in the Midgut Extracts from Larvae and Adult Red Palm Weevil Rhynchophorus ferrugineus (Olivier) in Al-Ahsa, Saudi Arabia
The hemagglutination activities of midgut (MG) fractions of red palm weevil, Rhynchophorus ferrugineus against mammalian erythrocytes (RBC) from man (ABO), rabbit, horse and sheep were studied. The highest titers were seen with rabbit RBC (p<0.05) followed by human group B, human group O, horse, human group A, human group AB and sheep, respectively. Insect age and nutritional status significantly influenced the agglutinating activities. Some of the unfed insects were having low activities (p>0.01) than the well fed insects (p<0.05). The agglutination activities were linearly correlated with the insect age. The agglutination activities were higher in old insects than the young insects (p<0.05). Further studies are needed to investigate the lectins in the hemolymph of the red palm weevil, Rhynchophorus ferrugineus beside demonstrating their agglutination activities against the natural entomopathogenes organisms of this economically important insect for use in its biological control.
to cite this article:
M.H. Abdally, M.M. Abo-Elsaad, A.A. Al-Shaggag, M.M. Al-Bagshy and A.A. Al-Shawaf, 2010. Detection of Insect Immunity Substances (Lectins) in the Midgut Extracts from Larvae and Adult Red Palm Weevil Rhynchophorus ferrugineus (Olivier) in Al-Ahsa, Saudi Arabia. Pakistan Journal of Biological Sciences, 13: 223-228.
The Red Palm Weevil (RPW), Rhynchophorus ferrugineus (Curculionidae:
Coleoptera) is an economically important tissue boring pest of date palm in
many parts of the world (Kaakeh, 2005) with its native
home in Southern Asia and Melanesia and is a serious pest of coconuts. In the
Middle-East, it gained importance in the mid 1980s when, it was first
reported from UAE in 1985 and spread to the entire region including the kingdom
of Saudi Arabia thereafter (Kaakeh, 2005; El-Ezaby,
1997). Currently, pheromone traps (monitors) are used as new tactics in
an Integrated Pest Management (IPM) of the red palm weevil (Abraham
et al., 1998). These management tactics were used in Saudi Arabia
and other Middle East countries (Vidyasgar et al.,
2000; Abbas et al., 2006). These methods
may give good results if employed with the knowledge of refractoriness (defense
mechanisms) of this insect and well studied (from all sides including lectins)
against the microbial entomopathogenes e.g., Metarhizium anisopliae
(Kershaw et al., 1999) and Bacillus thuringiensis
(Mohan and Gujar, 2002).
Generally, all the insects (including the non-blood sucking) display diverse
arrays of defense mechanisms to counteract the wide spectrum of potential pathogens
and harm substances in the surrounding environment (Lackie,
1988). In fact, both cellular (haemocyte-mediated) and non-cellular or humoral
(haemolymph and midgut lections) defense responses were reported in blood sucking
insects against naturally-occurring pathogenic organisms such as bacteria and
parasites (Ingram and Molyneux, 1988; Abdally,
1996). These defense mechanisms were studied also in non blood sucking insects
like American cockroaches (Periplanata americana) and locusts (Schistocerca
gregaria) by detecting the lectins (agglutinins) using agglutination activities
of these substances against some mammalian RBCs. These lectins were then considered
to be a part of those insects with immune response (Molyneux
et al., 1986; Whitcomb et al., 1974;
Lackie, 1981). In another study (of the same insects),
haemagglutination activity was tested against nine types of RBC. Of these nine,
human (ABO) and rabbit RBC showed the highest titres (Hapner,
Previous studies found lectins in vector (blood sucking) and non-vector (non
blood sucking) insects and they may play a very important role in resistance
against pathogenic organisms (Ingram and Molyneux, 1988;
Abdally, 1996) beside their digestion functions (Osir
et al., 1995; Lehane, 1976). These substances
can be detected only by agglutinating the Red Blood Cells (RBCs) (Ingram
and Molyneux, 1988; Abdally, 1996). Earlier, Croft
et al. (1982), Ingram and Molyneux (1988)
and Welburn et al. (1989) used relatively heat-labile,
human ABO(H) blood group against samples from testes flies Glossina morsitans
morsitans, G. palpalis gambiensis and G. tachinoides. They
reported that non-specific lectins or lectin-like agglutinins were detected
in their midguts and haemolymph of Glossina species (titres ranged from
2-9-2-16 in haemolymph and from 2-3-2-7
in midgut). In the present study agglutination activity was determined against
human (ABO), rabbit, sheep and horse RBCs according to Ingram
and Molyneux (1988) and Abdally (1996) in order to
detect lectins (the immunity substances) in the midgut (MG) of the red palm
weevil with a long term objective of refining this pest by identifying weak
links in the immunity system for contributing in its biological and Integrated
Pest Managements (IPM).
According to our knowledge and survey of literature, this study is an introductory type of investigation (first in the world) of the lectins (hemagglutinins) in the midgut (MG) of the Red Palm Weevil (Rhynchophorus ferrugineus) of different ages, sexes and nutritional status.
MATERIALS AND METHODS
The study was carried at Department of Microbiology and Parasitology, College of Veterinary Medicine and Animal Resources, P.O. Box 1757, Al-Ahsa 31982, King Faisal University, Al-Ahsa, Kingdom of Saudi Arabia during 2006-2008.
Maintenance of the weevils: Weevils were obtained from colonies maintained in the laboratory at 25-28°C in dark place at Al-Hasa National Date Palm Research Center, Saudi Arabia. Larvae were reared on a mixture of sugar canes and palm tissues in plastic containers.
MG extraction: The samples were collected from L1, L2,
L3 larvae (fed and unfed) and from adult insects of 1, 5, 10, 25
and 30 days old (fed and unfed). Then, the midguts were carefully removed from
4 larvae and 4 adults dissected insects (of each sex) at each sampling time.
The tissues were placed in 1 mL of cold Phosphate Buffer Saline (PBS) with a
pH of 7.4 and were kept in an ice bath until the completion of dissection (every
stage alone). In these experiments, MG were homogenized and used in agglutination
assays because, these tissues had the strongest agglutination activity without
causing haemolysis of the RBC (pilot experiment). Tissues were washed twice
in cold PBS and then homogenized in chilled PBS using a 1 mL homogeniser (UNIFORM
Jencons, England). The Gut homogenates were stored in l mL aliquots and left
at 4°C for 3-4 h, after which the samples were resuspended, frozen and thawed
three times at -20°C. The samples were then kept overnight at 4°C to
permit the release of agglutinins (lectins) from tissues. The gut extracts were
then centrifuged at 6000 rpm for 5 min after which the supernatant was dispensed
into another aliquot (1 mL), labeled and stored at -20°C until required.
Then, the concentration of this homogenate was adjusted to 4 MG mL-1
by following the methods of Ingram and Molyneux (1988)
and Abdally (1996).
Preparation of RBC suspension
Obtaining the blood samples: The required volumes of blood (2-5 mL) were
taken aseptically from stocks of fresh rabbit and human (A, AB, B and O) blood.
Rabbit blood was obtained via syringe (containing anti-coagulant) from the ear
vein. While, horse and sheep blood were obtained from the jugular vein. Human
blood was obtained from King Fahd Hospital, Hofuf. The samples were tested for
the presence of rouleaux formation and intra-erythrocytic haemoglobin crystals.
Rouleaux formation is found in the thicker regions of blood films and wet preparations
of whole blood. It appears as a face to face superimposition of the cells into
stacks of various lengths and can be mistaken for agglutination, while the intra-erythrocytic
haemoglobin crystals can disrupt the spreading of the blood film and makes the
red cells appear as they are agglutinated (Hawkey and Dennett,
1989). The blood samples, which showed abnormalities, were rejected. The
erythrocyte suspensions were prepared by treating the samples according to the
method described by Kabat (1971) and Abdally
Adjustment of RBC suspension: Each blood sample was centrifuged (10-15
min) and the supernatant was discarded. The pellets were washed in 5-10 volumes
of isotonic PBS with a pH of 7.4 for 3-5 times (until the supernatant appeared
very clear). The washed erythrocytes were then resuspended in approximately
19 volumes of PBS with a pH of 7.4, in order to achieve an approximate 5% suspension.
Then, 1 mL of the suspension was lysed with 14 mL of 0.1% aqueous solution of
anhydrous sodium carbonate. The Optical Density (OD) of the clear lysate was
measured against a water blank using a Beckman spectrophotometer/ Ultraspec
II with a lcm cuvette at wavelength of 541 nm. An OD of 0.7 corresponds approximately
to 1x109 RBC mL-1 of 5% cell suspension. The original
suspension was then adjusted to 5% using the Kabat's formula:
These standardized erythrocyte suspensions were labeled and well-stoppered
flasks and kept in the refrigerator at 4°C until required. Later on, 0.1%
suspensions were prepared at the time of each assay according to procedure of
Running the assays
Dilution of the homogenates: Double dilutions of Midgut Homogenates (MG)
were performed with PBS in microtitre plates (96 wells). In order to make sure
that the assays were done correctly, the first experiment was done by using
the lectin concanavalin A (Con A) from Ccmvalla ensiformis, obtained
from Sigma. The Con A was used as a positive control by following the methodology
of Msangi (1988) and Abdally (1996).
It was reported that Con A was found to agglutinate 2% fresh human erythrocytes
in 0.1 M PBS with a pH of 6.8 at 25 mg mL-1 (Sigma). However in the
laboratory, it was found to agglutinate 0.1% suspensions of rabbit erythrocytes
and human (ABO), sheep and horse erythrocytes in 0.1 M PBS with a pH of 7.4.
The haemagglutination activities of MG samples were compared to Con A (positive
control). Assays were carried out in 96 well plates (Dynatech). The first well
from every sample row was used for the neat sample. In these assays dilution
started from 2-1 (well 2) to 2-12 (well 12) for each sample.
Five microliter of PBS was added to all the wells in the rows except well 1
in rows A, B, D, E, G and H in which 10 μL of the sample (neat) was added.
Doubling the dilutions of the samples was then performed by the transference
of 5 μL of the neat sample from well 1 to the diluent (PBS) in well 2.
The solution was mixed thoroughly. After changing the tip, 5 μL of this
diluted solution was transferred to the next 5 μL of the diluent (PBS)
in well 3 and the process was repeated up until well 12. This process was performed
for all samples. Every sample of HL and MG was tested in a single plate alone.
The plates were covered and incubated at 25-26°C for 2 h after which the
haemagglutination activity was determined.
Detection the degree of the agglutination activities: Equal volumes
of the adjusted RBCs were added to all wells. The plates were then incubated
at 25-26°C for two hours and examined for haemagglutination. The degree
of agglutination was usually assessed using a seven score relative scale according
to Cunningham and Vickerman (1992), Ingram
and Molyneux (1988) and Abdally (1996). 100% agglutination
(3+), 75% (3+/2), 50%(2+/l), 25%(2+/+), IO-2O%(1+), trace (tr) and no agglutination
(0). The dilutions which just failed to give visible agglutination were regarded
as the end points (titres). They were expressed as -Iog2 2-n.
The controls comprised PBS alone against each sample and all the experiments
were duplicated. The results were treated by using the Analysis of Variance
RESULTS AND DISCUSSION
The results are expressed in Table 1 (for larval stages), Table 2 (adult males fed and unfed) and Table 3 (adult females fed and unfed).
Most susceptible RBC types: The results indicated (Table 1-3) that the rabbit RBCs are the most sensitive cells (susceptible indicators) as these gave the highest titers (p<0.05) followed by human RBCs group B, human RBCs group O, horse RBCs, human RBCs group A and AB and the sheep RBCs (the lowest titers).
Effect of age, sex and nutrition
Larval stages: There were no significant differences in the activities of
L1, L2 and L3 larvae (fed and unfed) of each stage against each RBC indicator
(Table 1). However, it was found that the activity increased
in the advanced larval stages. This is clearly shown in case of rabbit RBCs
where, L1(3), L2(5) and L3(5) and human RBCs group B where L1 (2), L2 (4) and
L3 (4). While, assays against the other RBC types almost gave less titers.
Adult insects: The results in Table 1 and 2
clearly indicated, especially with rabbit and human B RBCs, that the old insects
gave higher activities (p<0.05) than the young insects (p>0.01). Similarly,
the activities of males (p<0.05) were higher than the females (p>0.01).
It was also observed that the activities showed increases when the insects were
well fed (p<0.05).
agglutination activity titration (-log 2-n) of MG of Red Palm
Weevil (Rhynchophorus ferrugineus) larval stages (fed and unfed)
against mammalian RBC used in the investigation
Larval stage (both fed and unfed larval stages gave the same results)
effects of age and nutritional status on agglutination activity titration
(-log 2-n) of MG from adult male Red Palm Weevil (Rhynchophorus
ferrugineus) fed and unfed against mammalian RBC used in the investigation
inflwenc of age and nutrion on the agglutination activity titration (-log2-n)
of MG from female Red Palm Weevil (Rhynchophorus ferrugineus) against
mammalian RBC used in the investigation
This behavior is clear especially in the results of the assay against the rabbit
RBCs (indicators) (Table 2, 3).
The results of this study showed that there are substances in the midgut (MG)
of the Red Palm Weevil (RPW) which are responsible for the agglutination of
the Red Blood Cells (RBCs) and these might be lectins. The rabbit RBCs were
the most agglutinating indicators (p<0.05). These RBCs were similar to those
found in stable flies S. calcitrans haemolymph (HL) midgut (MG) (Abdally,
1996). It has been observed in various insects that if the HL and MG lectins
were from the same insect, they would have the same RBCs indicators and played
significant roles in that insect immunity (Abdally, 1996).
Therefore, for this reason, the rabbit RBCs were strongly recommended for use
only in investigating the HL lectins of the Red Palm Weevil (RPW), Rhynchophorus
ferrugineus. The strength of the hemagglutination activities of an insect
lectins might be influenced by its food types and the tissue extractions. This
was seen clearly in case of the blood sucking stable fly, S. calcitrans,
where the maximum agglutination activity was observed with homogenates taken
only from the reservoir zone of the midgut (MG), while the whole midgut (MG)
extracts containing the opaque and the lipoid zones caused less agglutination
than the reserve zone. This is besides the hemolysis the tested RBCs (Abdally,
1996). This was observed also with tsetse flies Glossina morsitans morsitans,
G. palpalis gambiensis and G. tachinoides (Ingram
and Molyneux, 1988). However, in the present work, the activity of whole
MG tissues of RPW was higher than the blood parasitic flies. This could be attributed
to the food types of RPW. In fact, the RPW feeds on tissues of palms containing
a lot of sugars and they need this agglutinins to accumulate in order to be
digested, while blood sucking flies need them to prevent them from pathogenic
organisms found in ingested blood as innate immunity (Boulanger
et al., 2002), beside helping them in digestion (Abdally,
1996). This is not surprising as there were other reports of hemolysis occurring
with the use of whole midgut extracts (Spates and Deloach,
1980; Spates, 1981). Lehane
(1976) reported that the opaque zone midgut cells of S. calcitrans were
responsible for the production and release of proteolytic digestive enzymes
into the gut lumen via Membrane Bound Vesicles (MBV). Later, Blakemore
et al. (1993) reported that the trypsin enzyme in the opaque zone
was stimulated by cyclic AMP but not cyclic GMP. Therefore, it was well known
that the peritrophic membrane (pm) functions as a physical barrier (a primary
barrier) which separates blood contents and parasites from the midgut epithelial
cells (Dunn, 1986; Lehane, 1991).
However, it was reported that trypanosomes can penetrate the peritrophic membrane
9-11 days after ingestion of infective blood (Ellis and
Evans, 1977). This indicates that the peritrophic membrane is not a physical
barrier to the penetration of trypanosomes to the haemocoel of the infected
insects (Lehane and Msangi, 1991). Generally, the lectins
are known to play an important role as humoral defence factors in the immune
mechanisms of insects (Dunn, 1986; Lackie,
1988). They were reported in the haemolymph of G. m. morsitans and
had anti-parasitic agglutination activity against T. brucei, T.
vivax and T. congolense in vitro (East et
al., 1983). The agglutination activity was reported also in the haemolymph
mid and hind guts of G. m. morsitans and G. austeni against T.
brucei and RBCs of several animal species. In some studies, the insect lectins
(agglutinins) were found to have a selective reactivity against D(+)-glucosamine
(Ibrahim et al., 1984, Mohamed
et al., 1992). Hemagglutination activity was reported in gut and
hemolymph extracts of G. morsitans, G. palpalis gambiensis and
G. tachinoides (Ingram and Molyneux, 1988) and
hemolymph of G. fuscipes fuscipes (Ingram and Molyneux,
1990) against human erythrocytes (RBC) of ABO (H) blood groups. These findings
supported our investigation on the immunity of the RPW MG lectins against all
the tested RBCs where the highest titers were, seen in case of the rabbit RBCs,
2-5 -2-12 (p<0.05). Therefore, the rabbit RBCs should
be used alone for detection of RPW HL and Mg lectins besides detecting their
specific carbohydrate inhibitors. There is another thing which is very important
in the digestion and defence of insects which should be investigated in this
weevil. It is the peritrophic membrane (pm). Abdally (1996)
investigated this in stable flies following the same method and found results
similar to those of Lehane and Msangi (1991), who studied
extracts of whole gut, peritrophic membrane and midgut remains in order to investigate
the hemagglutination activity towards human erythrocytes of AB Rh -ve blood
group. They found that there was no activity in the extracts of whole gut of
newly-emerged G. m. morsitans flies and only a trace of agglutination
was observed in flies aged 3-8 days post-emergence (p.e.) (titres neat -2°)
whilst insects aged 14 days p.e. had the activity of 50% (2+) and titres ranged
neat -2-8. The same RBC type was found to be agglutinated by midgut
(MG) extracts of G. m. morsitans (titre 2-7), G. palpalis
gambiensis (titres 2-3) (Ingram and Molyneux,
1988). However, larvae and young adult (1-10) RPW showed some agglutination
activities (p>0.01) almost against all the RBCs used. This might be due to
the weevils food types which are obtained from plant tissues (e.g., Rice,
sugar canes, Palm trees etc.) that are used as typical sources of carbohydrates
for men and animals. In fact this will be more clear if the investigation of
the pretrophic membrane of the weevil gut and the most specific inhibitor sugars
of its agglutinins (lectins) of MG and HL were done. Because, it will elaborate
the defense mechanisms of this economical important insect especially if these
studies are carried with the biological insecticides containing the entomopathogenes
(the natural enemies of the insects) beside the chemical insecticides in order
to determine their integrated effects against the RPW. It will also support
the Integrated Management (IM) of the Red Palm Weevil in Saudi Arabia and other
Middle East Countries.
This cooperative study was carried by The Date Palm Research Center (Center
of Excellence) of King Faisal University (KFU) and Al-Hasa National Date Palm
Research Center, Ministry of Agriculture under the Memorandum of Understanding
between KFU and The ministry. Thanks are extended to the Hematology Department,
King Fahd Hospital, Hofuf for providing the healthy human blood types. The authors
appreciate the support and encouragement received from Mr. Adnan Al-Afaleg,
Director, Al-Hasa National Date Palm Research Centre. Thanks are also extended
to Prof. Dr. S. Al-Eid, Director, Date Palm Research Center (Center of Excellence),
King Faisal University and Dr. Abdul Kader Sallam and their colleagues for their
supports. Authors also appreciate the useful information and cooperation received
from Dr. Jameel Al-Khayri (Member of Shura Council), Prof. Dr A. AL-Ajlaan (KFU)
and Dr. J. R. Faleiro (Entomology ICAR Research Complex For Goa, India). Finally,
they wish to express their great appreciation to their colleagues Prof. Dr.
M. Abdul-Azeeze and Dr. M.J. Al-Hajar (KFU) for their help in statistical analysis
and useful comments for improving this manuscript.
1: Abbas, M.S.T., S.B. Hanounik, A.S. Shahdad and S.A. Al-Bagham, 2006. Aggregation pheromone traps, a major component of IPM strategy for the red palm weevil, Rhynchophorus ferrugineus in date palms (Colleopetra: Curculionidae). J. Pest Sci., 79: 69-73.
2: Abdally, M.H., 1996. A study of the midgut (reservoir zone) and heamolymph lectins of the stable fly, Stomoxys calcitrans. Ph.D. Thesis, University of Wales.
3: Abraham, V.A., M. Al-Shuaibi, J.R. Faleiro, R.A. Abozuhairah and P.S.P.V. Vdyasagar, 1998. An integrated approach for the management of red palm weevil rhynchophorus ferrugineus oliv. A key pest of date palm in the Middle East. Agric. Sci., 3: 77-83.
4: Blakemore, D., M.J. Lehane and S. Williams, 1993. Cyclic AMP can promote the secretion of digestive enzymes in Stomoxys calcitrans. Insect Biochem. Mol. Biol., 23: 331-335.
5: Boulanger, N., R.J.L. Munks, V. Hamilton, F. Vovelle, R. Brun, M.J. Lehane and P. Bulet, 2002. Anovel antimicrobial peptide with antiparasitic activity in the blood-sucking insect Stomoxys calcitrans (Epithelhal Innate Immunity). J. Biolog. Chem., 277: 345-356.
6: Croft, S.L., J.S. East and D.H. Molyneux, 1982. Anti-trypanosomal factor in the haemolymph of Glossina. Acta Trop., 39: 293-302.
7: Cunningham, M.P. and K. Vickerman, 1992. Antigenic analysis of the Trypanosoma brucei group using the agglutination reaction. Trans. R. Soc. Trop. Med. Hygiene, 65: 48-59.
8: Dunn, P.E., 1986. Biochemical aspects of insect immunology. Ann. Rev. Entomol., 32: 321-339.
9: East, J., D.H. Moiyneux, Maudlin and P. Dukes, 1983. Effect of Glossina haemolymph on salivarian trypanosomes in vitro. Ann. Trop. Med. Parasitol., 77: 97-99.
10: El-Ezaby, F., 1997. A biological in vitro study on the red indian date palm weevil. Arab J. Plant Prot., 15: 84-87.
11: Ellis, D.S. and D.A. Evans, 1977. Passage of Trypanosoma brucei rhodesiense through the peritrophic membrane of Glossina morsitans morsitans. Nature, 267: 834-835.
CrossRef | PubMed |
12: Hapner, K.D., 1983. Haemagglutination activity in the haemolymph of individual Acrididae (grasshopper) specimens. J. Insect Physiol., 29: 101-106.
13: Hawkey, C.M. and T.B. Dennett, 1989. A Colour Atlas of Comparative Veterinary Haematology: Normal and Abnormal Blood Cells in Mammals, Birds and Reptiles. Wolfe Publishing Ltd., UK.
14: Ibrahim, E.A., G.A. Ingram and D.H. Molyneux, 1984. Haemagglutinins and parasite agglutinins in haemolymph and gut of Glossina. Tropenmedezin unde Parasitol., 35: 151-156.
PubMed | Direct Link |
15: Ingram, G.A. and D.H. Molyneux, 1988. Sugar specificities of anti-human ABO(H) blood group erythrocyte agglutinins (lectins) and haemolytic activity in Haemolymph and gat extracts of three Glossina species. Insect Biochem., 18: 269-279.
16: Ingram, G.A. and D.H. Molyneux, 1990. Lectins (haemagglutinins) in the haemolymph of Glossina fuscipes fuscipes: Isolation, partial characterization, selected physico-chemical properties and carbohydrate-binding specificities. Insect Biochem., 20: 13-27.
17: Kaakeh, W., 2005. Longevity, fecundity and fertility of the red palm weevil, Rynchophorus ferrugineus Oliver (Coleoptera: Curculionidae) on natural and artificial diets. Emirates J. Agric. Sci., 17: 45-52.
18: Kabat, E.A., 1971. Experimental Immunochemistry. 2nd Edn., Thomas, Springfield, Illinois.
19: Kershaw, M.J., E.R. Moorhouse, R. Bateman, S.E. Reynolds and A.K. Charnley, 1999. The role of destruxins in the pathogenicity of Metarhizium anisopliae for three species of insect. J. Invertebrate Pathol., 74: 213-223.
CrossRef | Direct Link |
20: Lackie, A.M., 1981. The specificity of the serum agglutinins of Periplaneta Americana and Schistocerca gregaria and its relationship to the insect's immune response. J. Insect Physiol., 27: 139-143.
21: Lackie, A.M., 1988. Immune mechanisms in insects. Parasitol. Today, 4: 98-105.
22: Lehane, M.J., 1976. Digestive enzyme secretion in Stomoxys calcitrans (Diptera: Muscidae). Cell Tissue Res., 170: 275-287.
23: Lehane, M.J., 1991. Biology of Blood-Sucking Insects. 1st Edn., Berne Convention. Harper Collins Academic, London, UK.
24: Lehane, M.J. and A. Msangi, 1991. Lectins and peritrophic membrane development in the gut of Glossina morsitans morsitans and a discussion of their role in protecting the fly against trypanosome infection. Med. Vet. Entomol., 5: 495-501.
25: Mohamed, H.A., G.A. Ingram and D.H. Molyneux, 1992. Carbohydrate-binding specificities of anti-erythrocyte lectins (haemagglutinins) in Anopheles gambiae gut extracts and haemolymph. Med. Vet. Entomol., 6: 217-224.
26: Mohan, M. and G.T. Gujar, 2002. Geographical variations in larval susceptibility of the diamondback moth, Plutella xylstella (Lepidoptera: Plutellidae) to Bacillus thuriniensis spore-crystal mixture and purified crystal proteins and associated resistance development in India. Bull. Entomol. Res., 92: 488-498.
27: Molyneux, D.H., G. Takle, E.A.R. Ibrahim and G.A. Ingram, 1986. Insect immunity to Trypanosomatidae. Symp. Zool. Soc. London, 56: 117-144.
28: Msangi, A.R., 1988. Lectin activity in the gut of Glossina morsitans morsitans Wiedemann. M.Sc. Thesis, UCNW., Bangor.
29: Osir, E.O., L. Abubakar and M.O. Imbuga, 1995. Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly Gjossina longipennis. Parasitol. Res., 81: 276-281.
30: Spates, G.E., 1981. Proteolytic and haemolytic activity in the midgut of the stable fly Stomoxys calcitrans (L.): Partial purification of the haemolysin. Insect Biochem., Ll: l43-147.
31: Spates, G.E. and J.R. Deloach, 1980. Hemolysin of the stable fly, Stomoxys calcitrans. Insect Biochem., 67B: 121-125.
32: Vidyasgar, P.S.P. H. Mohammed, R.A. Abozuhairah, O.A. Al-Mohana and A.A. Saihati, 2000. Impact of mass pheromone trapping on red palm weevil adult population and infestation level in date palm gardens of Saudi Arabia. The Planter, 76: 891-899.
33: Welburn, S.C., I. Maudlin and D.S. Ellis, 1989. Rate of trypanosome killing by lectins in midguts of different species and strains of Glossina. Med. Vet. Entomol., 3: 77-82.
34: Whitcomb, R.F., M. Shapiro and R.R. Grandos, 1974. Insect Defence Mechanisms Against Microorganisms and Parasitoids. In: The Physiology of Insecta, Rockstein, M., (Ed.). Vol. 5, Academic Press, New York, pp: 448-537.