Effect of anti-mosquito midgut antibodies on …nopr.niscair.res.in/bitstream/123456789/11386/1/IJEB 49(4...Indian Journal of Experimental Biology Vol. 49, April 2011, pp.245-253 Effect

Indian Journal of Experimental Biology

Vol. 49, April 2011, pp.245-253

Effect of anti-mosquito midgut antibodies on development of malaria

parasite, Plasmodium vivax and fecundity in vector mosquito

Anopheles culicifacies (Diptera: culicidae)

Manoj Chugh*, T Adak

†, Neelam Sehrawat & S K Gakhar

Centre for Biotechnology, Maharshi Dayanand University, Rohtak 124 001, India † National Institute of Malaria Research, Dwarka, Delhi 110 009, India

Received 28 May 2009; revised 1 February 2011

The effect of anti-mosquito-midgut antibodies on the development of the malaria parasite, P.vivax was studied by

feeding the vector mosquito, An. culicifacies with infected blood supplemented with serum from immunized rabbits. In

order to get antisera, rabbits were immunized with midgut proteins of three siblings species of Anopheles culicifacies,

reported to exhibit differential vectorial capacity. The mosquitoes that ingested anti-midgut antibodies along with infectious

parasites had significantly fewer oocysts compared to the control group of mosquitoes. The immunized rabbits generated

high titer of antibodies. Their cross reactivity amongst various tissues of the same species and with other sibling species was

also determined. Immunogenic polypeptides expressed in the midgut of glucose or blood fed An. culicifacies sibling species

were identified by Western blotting. One immunogenic polypeptide of 62 kDa was exclusively present in the midgut of

species A. Similarly, three polypeptides of 97, 94 and 58 kDa and one polypeptide of 23 kDa were present exclusively in

species B and C respectively. Immunoelectron microscopy revealed the localization of these antigens on baso-lateral

membrane and microvilli. The effects of anti-mosquito midgut antibodies on fecundity, longevity, mortality and

engorgement of mosquitoes were studied. Fecundity was also reduced significantly. These observations open an avenue for

research toward the development of a vector-based malaria parasite transmission-blocking vaccine.

Keywords: Anti-mosquito midgut antibodies, Anopheles culicifacies, Plasmodium vivax

The mosquito midgut represents one of the

most challenging environments for the survival

and development of Plasmodium1. The sporogonic

development of Plasmodium, from gamete to oocyst

formation, takes place in the lumen and epithelium of

the mosquito midgut. It therefore forms one of the

most attractive sites for novel antigenic targets so as

to draw malaria control strategies like transmission

blocking vaccine (TBV).

Among members of the Anopheles culicifacies complex, only species A, B and C have been colonized so far. Various host-parasite interaction studies involving species A, B and C and different Plasmodium species unequivocally demonstrated that species A and C are highly susceptible while species B is the least susceptible to human

2,3 and rodent

malaria parasites4. The natural variability in malaria

susceptibility because of differential genetic factors of mosquitoes is not yet been clearly understood. However, studies on An. culicifacies have shown

correlation between different types of mosquito immune responses and differential susceptibility to various parasitic infections

5. Vector internal organs

(concealed antigens) can induce artificial immune responses in various species viz., An. stephensi

2,6-9.

Anti-midgut antibodies have already been postulated to cause deleterious effects on the reproductive capacity of mosquitoes and they simultaneously block parasite development in the mosquito midgut

10,11.

Presence of some common antigenic peptides has already been indicated in different species of mosquitoes and also in various tissues (viz., salivary gland, midgut and haemolymph) of a given mosquito species

7,12. However, no such study

appears to have been undertaken on any sibling species complex of mosquitoes.

Various studies unequivocally incriminated An. culicifacies as the major vector, responsible for 70% of malaria cases in India. An. culicifacies exists as a complex of five sibling species provisionally designated as A, B

13, C

14, D

15 and E

16.

These sibling species are reported to have various biological differences viz., their distribution,

——————

*Correspondent author

E-mail: [email protected]

INDIAN J EXP BIOL, APRIL 2011

246

response to insecticides17

host preferences18

and vectorial capacity

19. An. culicifacies A and C are

primary vectors whereas, species B has very little role if at all, in the transmission of malaria

16.

The present study has been designed to determine

the effect of anti-mosquito midgut antibodies on the

development of malaria parasite and fecundity of the

vector mosquito.

Materials and Methods

Mosquito rearing—Cyclic colonies of An.

culicifacies Species A (Dhera Strain), B (Ladpur

strain) and C (Raurkela strain) were maintained in an

insectary at 28± 2°C and 70-80% RH and fitted with a

simulated dawn and dusk machine to maintain a

photoperiod of 14 h light and 10h dark. Adult

mosquitoes were kept in 30 cubic cm cloth cages and

were fed on 1% glucose solution and water soaked

resins. Females were allowed to feed on rabbit blood

for ovarian development. On the third day post-blood-

feeding, females were allowed to lay eggs on filter

paper lined in water filled plastic bowls. Larvae were

reared in enamel trays and were fed on yeast extract

and dog biscuits in the ratio 2:3 (w/w). The pupae

were transferred to fresh bowl and kept in cloth cages

for emergence to adult mosquitoes.

Antigen preparation and immunization—Midguts

from either glucose or blood fed An. culicifacies

sibling species mosquitoes were dissected out in ice

cold phosphate buffered saline (PBS) containing

2 mM phenyl methyl sulfonyl fluoride (PMSF).

Antisera were raised in New Zealand White rabbits by

injecting subcutaneously with 100 µg protein/per kg

body weight only once as described earlier10

. The

control rabbits were injected with PBS and Freund’s

adjuvant in the similar manner. Rabbits of either sex

and irrespective of age were procured from H.A.U.

Hisar after animal ethical clearance.

In vitro and in vivo ELISA—Antibody titer was

measured by two-fold serial dilution of antisera raised

against midgut proteins from either glucose or blood

fed mosquitoes from three sibling species of

An. culicifacies using in vitro ELISA10

. Immunizing

antigen (10 µg/ml) was used to coat the 96-wells

ELISA plate (Tarsons, India). The antigen coated

plate was incubated for 1 h at room temperature and

then at 4°C overnight. This was followed by blocking

for 1 h at room temperature with 5% dried skimmed

milk (DSM) in PBS. Washing was done five times

with PBS-Tween-20 (PBS-T) on the ELISA washer

(Thermo Labsystem, USA ) followed by incubation

with alkaline phosphatase conjugated goat anti-rabbit

IgG (1:5000) for 2 h. Anti-rabbit IgG was used

because IgG was the primary antibody generated after

infection. The immune complex was detected by

p-nitrophenyl phosphate substrate system. Absorbance

was read at 405 nm on ELISA reader (Thermo

Scientific, USA).

The cross reactivity of the midgut antibodies with

other tissues (salivary glands, hemolymph, ovary and

midgut from other members of sibling species

complex) was also examined by in vivo ELISA

as described previously4. For in vivo ELISA,

20 mosquitoes were selected randomly after 24 h

of blood feeding from immunized blood fed and non-

immunized blood fed mosquitoes as control.

SDS PAGE—Soluble proteins were separated by

SDS-PAGE using thick slab gels (1 mm) containing

10% acrylamide and a tris-glycine (pH 8.6) buffer

system10,20

. Equivalent amount of proteins from

all the tissues were loaded in duplicates. One-half

of the gel was silver stained21

and the other half

was transferred electrophoretically to 0.45 nm nitro-

cellulose sheet for Western blotting22

.

Immunoblotting—Nitrocellulose sheet with separated midgut proteins of glucose or blood fed mosquitoes from different members of An.

culicifacies species complex and other tissues (salivary glands, ovary and haemolymph of the same

species) was blocked with 5% (w/v) dried skimmed milk (DSM) in PBS. Nitrocellulose membrane was incubated with hyper-immunized serum (primary antibody 1:100) in PBS for 1 h, washed with PBS containing tween-20 (0.1%) and then incubated with alkaline phosphatase (ALP) conjugated goat anti-

rabbit antibodies (1:1000) in PBS for 1 h followed by washing. Antibody binding was visualized using BCIP-NBT substrate

10.

Immunized blood feeding, fecundity, mortality and

longevity of mosquitoes—Egg-laying pattern in An.

culicifacies mosquitoes fed on rabbits (immunized

with midgut of either glucose or blood fed mosquitoes belonging to three sibling species) was examined. Different sets of 8-10 h starved mosquitoes were allowed to feed on sensitized rabbit for eight consecutive weeks after the last booster, i.e every week new set of mosquitoes were fed. Each set

consisted of about 120 female mosquitoes. The procedure to evaluate the percentage reduction in fecundity, hatchability, mortality and longevity has been described in detail by Suneja et al

10. Two days

after feeding, 5 sub-sets were made from each group.

CHUGH et al.: ANTI – MOSQUITO MIDGUT ANTIBODIES OF An. CULICIFACIES

247

Total number of eggs laid, larvae hatched and the number of dead mosquitoes were observed. Ovaries of the females were also examined for the presence of any retained eggs after egg laying. Total number of egg production during first gonotrophic cycle was

then calculated by adding up oviposited and retained eggs of females fed on blood from the sensitized and control rabbits. Fecundity, hatchability, engorgement and increase in mortality were calculated as under:

Fecundity femalesofNo.Total

laideggsofNo.Total=

Mortality = femalesofNo.Total

femalesdeadofNo.Total

Engorgement = Weight of mosquito after blood meal – Weight of

mosquito before blood meal

Reduction in fecundity (%) =

female ollaid/Contr eggs of No.

Female izedlaid/immun eggs No.of-femaleollaid/ContreggsofNo. ×100

Reduction in hatchability (%) =

female ntrolhatched/co eggs of No.

female d /immunizehatched eggs of No.-femalentrolhatched/coeggsofNo. ×100

Increase in mortality (%) =

)(immunized died mosquito of No.

(control) died mosquito of )/No.(immunizeddiedmosquitoofNo. ×100

Parasite invasion blocking assay—Membrane feeding was used to examine the effect of anti-midgut antibodies on the development of the parasite in An. culicifacies. Female mosquitoes were fed artificially

23 on antiserum from the sensitized rabbits

mixed with the blood drawn from human hosts

infected with 0.05% gametocytes of P. vivax with the help of parafilm. The gametocytes of P. vivax were obtained from patients visiting National Malaria Research Institute, Delhi. The mixed blood was kept in glass mold maintained at 37°C with the help of circulating waterbath. Each experimental group of

100 mosquitoes (5 days old) was membrane fed on the anti-serum (100µg of IgGs) of immunized rabbit along with P. vivax infected blood. Similarly, control group of 100 mosquitoes (5-day old) was fed on serum of unsensitized rabbit added to the P. vivax infected blood. Unfed or partially fed females were

not considered. After 6 days, midguts were dissected out to count the number of oocysts. Similarly, after 12 days, salivary glands were dissected out to observe the presence of sporozoites. Percentage transmission blocking was determined as by Ponnundrai et al.

24 by

using the following formula. All experiments were

conducted in triplicates.

Transmission blocking (%) = (Mean oocyst

number in control-Mean oocyst number in mosquitoes

fed on immunized blood) × 100/Mean oocyst number

in control.

Mann-Whitney u test (one-tailed) was applied by

using the GRAPH-PAD prism software to evaluate

the difference in oocyst count and also to determine

significance of fecundity reduction between

experimental and control groups.

Immunoelectron microscopy—Localization of the

midgut proteins was examined by immunoelectron

microscopy as per Dinglasan et al25

. Briefly, unfed

midguts from 8- to 10-day-old An. culicifacies

mosquitoes were fixed in PBS containing 1%

paraformaldehyde and 0.1% glutaraldehyde for 24 h

at room temperature. Ultrathin sections of guts,

embedded in LR-White resin were mounted on nickel

grids and immunostained with PAbs raised against

midgut proteins of An. culicifacies. Antibody binding

was visualized by using a 10-nm-colloidal gold-

conjugated goat anti-mouse IgG. The grids were

counter stained with uranyl acetate and examined with

a Morgagni-268D Transmission electron microscope.

Results The antibody titers in the rabbits (six sets

immunized with the antigens of the midgut from An.

culicifacies species complex fed on either glucose or

blood) reached at least 1:107 during the third week

after immunizations, as revealed by in vitro ELISA.

This level reached at peak during the third week after

the last booster, then declined to the minimum level

during the sixth week (Fig. 1).

Anti-midgut antibodies were capable of binding to various tissues viz., salivary glands, haemolymph and ovary and also to the midgut of other sibling species (Fig. 2 a and b). However, the highest cross-reactivity was observed between the salivary glands and midgut

of the same species. These results exhibit that antibodies fed to the mosquitoes traverse through the midgut wall and reach other target tissues. The cross reactivity of these antibodies with various tissues and the midgut of other sibling species was also studied by Western blotting (Figs 3, 4 and 5). This

demonstrated that species A and C (primary vectors) are more closely related than species B (poor vector), as antibodies raised against midgut of species A reacted with midgut proteins of species C and vice-versa but antibodies raised against midgut proteins of species B (especially glucose fed) exhibited minimal

cross reactivity to the midgut of species A or C.


248

Species-specific polypeptides—Species specific

anti-mosquito midgut antibodies helped in recognizing

the following peptides of the midgut from both

glucose and blood fed mosquitoes. One 66 kDa

polypeptide was recognized in the midgut of all the

three sibling species; 92 kDa polypeptide was

recognized only in species A and C.

One polypeptide (62 kDa) was exclusively present

in the species A. Three polypeptides (97, 94 and

58 kDa) were found specifically in species B.

Similarly, 23 kDa peptide could be resolved only in

species C (Figs 3, 4 and 5).

Blood and glucose meal induced polypeptides—

After feeding the mosquitoes on blood, the following

immunogenic polypeptides were recognized in their

midgut. One polypeptide of 47 kDa could be

recognized in the midgut of all the three sibling

species. Two peptides of 80 and 70 kDa were

Fig. 1—Antibody titer in the serum raised against midgut proteins of glucose or blood fed mosquitoes from different species of An.

culicifacies complex, as measured by antibody capture ELISA. GF-Glucose fed; BF-Blood fed.

Fig. 2—In vivo binding and quantitative estimation of cross-

reactivity of antibodies raised against midgut proteins of (a)

blood-fed, (b) glucose-fed An. culicifacies mosquitoes with

different tissues. SG-Salivary glands; HL-Haemolymph; MG-

Midgut; OV-Ovary.

Fig. 3—Effect of antibodies raised against the midgut proteins of

(a) blood-fed, (b) glucose-fed An. culicifacies mosquitoes on the

number of eggs laid.


249

recognized only in the midgut of species A and C.

Similarly, 40kDa peptide was present in species C and

76 kDa peptide in species B. However, one

polypeptide (39 kDa) was observed only in the

glucose fed An. Culicifacies A (Fig. 3b).

Cross-reactivity in the midgut—Antibodies raised

against the midgut proteins of either glucose or

blood fed mosquitoes of species A could commonly

recognize two polypeptides (66 and 62 kDa) in the

midgut of the other two sibling species B and C

(Fig. 3a and b). However, 39 kDa polypeptide was

revealed in all the three species with antibodies raised

against the midgut of glucose fed mosquitoes of

species A (Fig. 3b). Similarly three polypeptides

(86, 52 and 47 kDa), were also recognized in the midgut

of all three species by the immune sera raised against

the midgut of blood fed An. Culicifacies A (Fig. 3a).

Antiserum against the midgut proteins of species A (glucose fed) could recognize 92 and 14 kDa polypeptides in the midgut of species- A and C. Similarly, the immune sera raised against the midgut proteins of blood fed species-A recognized two polypeptides i.e. 80 and 70 kDa in the species A and C.

Antibodies against the glucose fed midgut proteins

of An. culicifacies C revealed 92 and 66 kDa

Fig. 4—Tissue and species specific immunogenic polypeptides

recognized by antisera raised against the midgut proteins of (a)

blood-fed, (b) glucose-fed An. culicifacies A mosquitoes. SG-

Salivary glands; HL-Haemolymph; MG-Midgut; OV-Ovary.



blood-fed, (b) glucose-fed An. culicifacies B mosquitoes. SG-



250

polypeptides in the midgut of all the three sibling

species (Fig. 5b). Similarly three more polypeptides

(66, 47 and 40 kDa) could be resolved in all the three

sibling species when reacted with the antibodies

against the blood fed midgut of species C (Fig. 5a).

It was interesting to note that the antibodies raised

against the midgut of An. culicifacies B did not

exhibit cross reactivity to the midgut of species-A or

C (Fig. 4a).

Cross-reactivity among the various tissues was also

determined by Western blots and is shown in Table 1.

Effect on fecundity of mosquitoes—Significant

reduction in fecundity was observed in An.

culicifacies mosquitoes after they had ingested anti-

midgut antibodies as compared to the control.

Maximum reduction (40%, P<0.01) in fecundity was

observed in the mosquitoes fed on rabbit immunized

with midgut antigens of the glucose fed mosquitoes

during the third week after immunization (Fig. 6b).

Similarly, fecundity was reduced by 37% (P<0.05) in

mosquitoes after they had ingested polyclonal

antibodies raised against the midgut proteins of blood

fed mosquitoes (Fig. 6a). However, no statistically

significant difference was noticed in engorgement,

longevity and mortality of the females fed on

immunized and non-immunized serum.

Transmission blocking activity—Significant

reduction in parasite infection was observed in

An. culicifacies mosquitoes that ingested anti-midgut

polyclonal antibodies added to the P. vivax infected

blood, as compared to the control. Statistical analysis

showed that the reduction in infection rate and

number of oocysts was more in glucose fed than in

blood fed. The infection rate in An. culicifacies

reduced by about 27% and the mean number of

oocysts, per infected mosquito reduced by about 90%

(P<0.01) after they had ingested polyclonal antibodies

raised against midgut proteins of glucose fed

mosquitoes along with the blood infected with

malarial parasite (Table 2). Similarly, infection rate

Table 1—Cross reactive peptides amongst various tissues in An.

culicifacies species complex (glucose or blood fed)

Cross reactive polypeptides (kDa) in various

tissues recognized by Anti-midgut antibodies

Antibodies against

midgut of

Salivary glands Haemolymph Ovary

An. culicifacies A

(BF )

66, 62, 52,

47 and 43

66, 62, 60, 58,

52, 47 and 43

-

An. culicifacies A

(GF)

92, 43 and 39 50, 39 -

An. culicifacies B

(BF)

- 60, 58 and 56 47

An. culicifacies B

(GF)

66 - -

An. culicifacies C

(BF)

66, 60, 52

and 50

66, 60, 52 and 50 -

An. culicifacies C

(GF)

66 and 43 - -

BF-Blood fed; GF-Glucose fed



blood-fed, (b) glucose-fed An. culicifacies C mosquitoes. SG-



251

reduced by 25% and the mean number of oocysts per

infected mosquito by about 87% (P<0.01) after they

ingested antibodies raised against the midgut proteins

of blood fed mosquitoes (Table 2).

Immunolocalization—PAbs recognize midgut antigens that are specifically located along the brush border which lines the apical side of the gut epithelial cells facing the lumen as observed by immuno-electron microscopy. Besides the microvillar region, PAbs labeled the basement membrane of the epithelial cells (Fig. 7 a, b, c).

Discussion

The present results demonstrated that antibodies raised against midgut proteins not only blocked parasite (P. vivax) development but concomitantly reduced the fecundity of An. stephensi mosquitoes as reported earlier

11,10,27.

The reduced fecundity of An. culicifacies

mosquitoes fed on immunized rabbit suggested that

anti mosquito antibodies somehow interfere with the

normal process of oogenesis. However, it did not

seem to be related to any decline in the rate of

oviposition. A few female mosquitoes from each

feeding set were dissected after they had been fed on

sensitized or control rabbit. All seemed to have laid

their full complement of eggs. The mechanism of the

anti-mosquito response is not yet known but could be

one or combination of several factors i.e. fat body

synthesis of vitellogenins may be down regulated, the

uptake of circulatory vitellogenins may be inhibited or

the content of some of the developing follicles may be

reabsorbed10

. Alternatively, the injested anti-mosquito

antibodies may irritate the gut which in turn reduce

the blood meal or reduce availability of nutrients.

Moreover it is yet to identify the change in

vitellogenesis26

. It has also been observed that anti-

micro villi antibodies bind to the midgut wall and can

inhibit normal epithelial processes, such as enzyme or

peritrophic membrane secretion3.

Previous observations on Ae. aegypti and An.

stephensi mosquitoes, fed on similarly produced

anti-mosquito antibodies, showed that the effects on

mosquito mortality are small and variable2,10,27

.

However, the present investigations demonstrated that

the ingestion of rabbit anti-An. culicifacies antibodies

had no statistically significant effect on its mortality

and longevity. Differences in the results could also be

attributed to the amount of protein ingested,

differences in antibody titers, biological activities

(influenced by factor such as complement fixing

ability and stability to proteases in the midgut) and

specificity influenced by factors such as antigen being

derived from blood and sugar fed mosquitoes.

The present study also indicated that anti-mosquito

midgut antibodies when ingested with infected blood,

adversely affect the development of oocysts in the

midgut and/or translocation of sporozoites into the

salivary glands. These results are in agreement with

the findings of Suneja et al. 10

.

Fig. 7—Immunoelectron microscopy of unfed An. culicifacies

midgut. Transverse section of the midgut showing the association

of antibodies with the (a) basolateral membrane of epithelial cell

(b) extracellular microvilli (c) extracellular glycocalyx (35,000×).

Table 2—Effect of anti-mosquito antibodies raised against midgut of glucose and blood fed An. Culicifacies mosquitoes

on malaria parasite development

[Standard Deviation has been mentioned in parenthesis]

No. of mosquitoes

Fed/ group

Infected

mosquitoes (%)

Mean no. of

oocysts /mosquito

Transmission

blocking (%)

Control Anti-MG Control Anti- MG Control Anti- MG Anti-MG

Glucose fed 70 62 37 27 140 (± 2.67) 17.50 (± 4.4) 90.5*

Blood fed 59 56 40 30 125 (± 5.5) 16.33 (±3.17) 86.9*

* Significant at P< 0.001


252

The target molecule for the fecundity reduction and their relationship to the molecules mediating reduction in the infectivity of malarial parasite, if any, are yet to be determined. For the anti-mosquito antibodies to affect the parasite, they must either

inhibit a physiological process essential to the parasite or block the normal ookinete-midgut interaction that may or may not be cell-specific

27. The reduced

intensity of infections after ingestion of blood along with anti-midgut antibodies can be attributed to the inhibition of cellular interactions which are

possibly directed at glycoproteins on the mosquito midgut cell surface

12,29,30. Antibodies generated in the

present study recognized the antigens present at microvillar, glycocalyx and baso-lateral membrane. Oligosaccharides on gut microvillar glyco-conjugates have already been implicated as both receptors

for microbial attachment and as a protective barrier against pathogens in both vertebrates and invertebrates

6,1. Midgut microvilli (MMV)

glycoconjugates have been shown to play a role in establishment of parasite infections in the mosquitoes

11,19.

The disruption of the ookinete-to-oocyst transition

may therefore occur either at the level of attachment to the glycocalyx or to the microvillus itself or at the level of more downstream events following invasion, e.g. interference of cell signaling and/or cell functioning

9. The reduction in the number of oocysts

developed in the midgut may be due to two reasons.

First, antibodies against midgut may have similar target sites for the parasite to recognize and invade, therefore may competitively inhibit the parasite. Second, these antibodies may adhere and lead to allosteric alteration in the receptor recognition site in the midgut and hence deny ookinetes to recognize and

subsequently adhere to the target site10,25

. Three immunogenic polypeptides (97, 94 and

58 kDa) exclusively present in the poor vector

(An. culicifacies-B) in both glucose and blood fed

mosquitoes may be involved in defence reactions

directed against Plasmodium. Immunological

superiority of An. culicifacies B refractory phenotype

over susceptible strains has also been reported5.

Presence of 62 kDa peptide in species-A, 97, 94

and 58 kDa in species-B and 23 kDa peptide in

species-C may be used to target for designing the

species-specific immunodiagnostic probes.

Extensive cross-reactivity of anti-mosquito midgut

antibodies with various tissues viz., salivary glands,

haemolymph and ovaries of same species vis-à-vis

midgut of other sibling species, as observed by

Western blots and in vivo ELISA may be attributed

to the conserved antigens/epitopes in different tissues,

or non-specific binding by low affinity antibodies.

These experiments reiterated previous reports that

anti-midgut antibodies could traverse through the

midgut and reach other tissues31

.

Development of vector based malaria transmission

blocking vaccines (TBVs) remains one such

progmatic approach that can complement or replace

existing control methods.The results from the present

study assertively implicated the midgut as the likely

source for candidate antigens for malaria transmission

blocking vaccine, however, the final formulation

should be designed concocting multiple antigens from

multiple tissues.

Acknowledgement Thanks are due to technical staff, NIMR for

assistance, Prof. R. P. Maleyvar for review of the

manuscript, and CSIR, New Delhi, India for financial

assistance.

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