Indian Journal of Experimental Biology Vol. 39, November 2001 , pp. 1 123- 1 129

Changes in haemolymph constituents of American bollworm,

Helicoverpa armigera (HUbner), infected with nuc1eopolyhedrovirus

V Kalia, S Chaudhari & G T Gujar Division of Entomology, Indian Agricultural Research Institute, New Delhi 1 1 0012, India

E-mail : vkalia_1998@yahoo.com; v_kalia98@hotmail.com Received 6 May 2000; revised 28 June 2001

Six types of haemocytes viz., prohaemocytes, plasmatocytes (round, fusiform, vermiform and spindle shaped), granular cells, spherule cells, oenocytoids and adipohaemocytes were found in the haemolymph of larvae of American bollworm H. armigera. The total and differential haemocyte counts (THC and DHC) in H. armigera haemolymph were affected by nucIeopolyhedrovirus (NPV) treatment. There was a general decrease in THC in response to NPV treatment in both young and old larvae. However the decrease was more apparent in 5 and 8 day old larvae than in 10 day old larvae. The differential haemocytes showed less of granular cells and more of spherule cells and prohaemocytes in the old larvae. Plasmatocytes and granular cells in 10 day old larvae initially phagocytosed polyhedra; however, disintegrated after 3 to 4 hr. The haemolymph of NPV treated larvae melanized slowly particularly in old larvae. Phenoloxidase (PO) activity decreased positively with granular cells and oenocytoids in 10 day old treated larvae. Cellular fraction had high level of PO activity, which was transferred to plasma in response to NPV infection in the older larvae. The role of NPV pathogenesis vis-iI-vis immunity in insect is discussed.

Insects are being continuously exposed to micro­organisms of all kinds and have developed an ability to resist them. This ability is associated with cellular and humoral components of insect immune system. Insect haemocytes provide an immediate response to the onslaught of microbes. This is followed by secretion of humoral factors, mostly proteins to destroy remaining microbes. Insect haemocytes have been studied in respect of their morphology and function to some extent , ,2 . However, very little is known of their role vis-a-vis viral infection. Shapiro et aP . reported adverse effect of nucleopolyhedrovirus (NPV) on the haemocytes of HeLiothis zea. Besides haemocytes, phenoloxidases (PO) recognise presence of microbial infection in the body at its earliest, and help in melanization of the target4.

American bollworm, HeLicoverpa armigera, is an important polyphagous pest of agricultural crops. Essawy et ai.5 characterized its haemocytes by light and electron microscopy. However, little information is available on effect of NPV infection on haemolymph constituents of H. armigera6• The present study has been undertaken to report characterization of haemocytes in larvae of H. armigera and the effect of NPV on haemocytes and PO activity in haemolymph of the test insect.

Materials and Methods H. armigera larvae used were collected from the

field and reared in the laboratory on semi-synthetic diet7 with minor modifications at 27° ± 1 °C and 60 ± 10% RH. For characterization of haemocytes, a small drop of haemolymph was drawn from last instar larvae directly on the slide and covered with a coverslip quickly and examined immediately by phase contrast microscopy at 1 ,000 x magnification. The haemocytes smears were prepared and stained by Giemsa in some cases. They were identified essentially as per Gupta2 .

To study histological changes in haemocytes of different stages of larvae, 5, 8 and 10 day old larvae were grouped and each group was divided into two subgroups of control and NPV treatment. The test larvae were fed on surface contaminated diet piece of one cm3 treated with 0.2 ml of 2 x 105 occlusion bodies (OB) mr' stock solution, whereas in control, larvae were fed on diet treated with distilled water and allowed to feed for 24 hr, after which they were transferred to fresh untreated diet. Three replicates of 10 larvae each were treated. Haemolymph was drawn from larva of each subgroup everyday till mortality or pupation for total haemocyte count (THC), differential haemocyte count (DHC) and PO studies.

1 1 24 INDIAN J EXP B IOL, NOVEMBER 2001

THC was performed as described by Miranpuri et a18• Fresh haemo1ymph (20 iii) was diluted in 1 80 iii anticoagulant buffer (98mM sodium hydroxide, 146mM sodium chloride, 1 7mM ethylene diamine tetra acetic acid, 4 1mM citric acid, pH 4.S). Haemocytes were counted from atleast 8 squares of each slide preparation using a Neubar haemocytometer of 0. 1 mm depth and THC were calculated by multiplying average counts per square of 0. 1 mm2 with a factor of 1 0 and also a dilution factor. The THC are a mean of 10 replicates.

The haemolymph samples from control and treated larvae were collected separately at various times post­inoculation. Haemolymph (6 iii) was diluted with 30 iiI of 1 :4 mixture of anticoagulant buffer and Dulbecco' s phosphate buffer saline (DPBS- 1 .SmM dipotassium hydrogen phosphate anhydrous, 8mM sodium dihydrogen phosphate monohydrate, 0.9mM calcium chloride, 2.7mM potassium chloride, O.SmM magnesium chloride, 0.3mM sodium chloride, pH 7.2). The samples were observed immediately at 400 x and 1000 x magnification in a phase-contrast microscopy8. The haemocytes of different kinds were counted and expressed in per cent for DHC.

For PO activity 200 iii haemolymph was mixed with 300 iii neutralized saturated ammonium sulphate solution. This mixture was incubated for 30 min. A portion of this mixture was retained as whole haemolymph. The rest was centrifuged at 10,000 g for 20 min. The pellet and supernatant were separated to be used as cellular and plasma fractions respectively. The pellet was resuspended in I OmM sodium phosphate buffer (PH 8). The assay was initiated by the addition of 100 iiI sample to a reaction mixture [Tris (hydroxy methyl) aminomethane- hydrochloride, pH 7.S (700 iiI) and 20mM L-3, 4-dihydroxyphenyl­alanine (L-DOPA)-400 iiI] and absorbance was measured at 490 nm every min. and PO activity expressed in units as per Anderson et a19•

NPY (S iiI of 2 x 105 OB.mrl dilution) was injected in the healthy Sth instar larvae. The haemolymph was collected at regular intervals from NPV injected larvae and observed for an evidence of NPV phagocytosis by haemocytes.

Results

Characterization of haemocytes Six types of haemocytes, viz. prohaemocytes,

plasmatocytes, granular cells, spherule cells, oenocytoids and adipohaemocytes were found in

larvae of H. armigera on the basis of identification key (Fig. 1 ) .

Prohaemocytes (PR): They are small round and spherical in shape. Each cell has a large centrally located nucleus, which is surrounded by a small amount of cytoplasm. In smears stained with Giemsa, nucleus is acidophilic stained pink almost fi lling the cell and very thin peripheral basophilic bluish cytoplasm around the nucleus (Fig. l A).

Plasmatocytes (PL): The PL are polymorphic (round, vermiform, fusiform, and spindle shaped) and variable in size, usually ovoid or spindle shaped with a basophilic blue cytoplasm (Fig. 10). The nucleus can be round or elongated and is often centrally located. Sometimes numerous and large vacuoles are present in PL.

Granular cells (GR): They show dense granules in the cytoplasm. GR degenerate very quickly on contact with glass slide. Neither the GR spread like PL on glass surface nor show cytoplasmic extension. Nucleus is comparatively small, compact and centrally located. In smears stained with Giemsa, the cytoplasm appears pale blue with faintly pink coloured granules, and nucleus is stained pink (Fig. l B). The degranulated GR are infrequently classed as coagulocytes (CO).

Spherule cells (SP) : SP are irregular in shape with variable sizes. The cytoplasm contains many small spherules or few large spherules. Nucleus is rather small, central or eccentric. In smears stained with Giemsa, the spherules are weakly basophilic and nucleus stained pink (Fig. 1 C). The cell with large and numerous spherules has nucleus almost concealed.

Oenocytoids (OE): The OE are large cells, with nucleus generally small in size but bigger than GR and SP, and usually eccentric. In smears stained with Giemsa, cytoplasm is basophilic (Fig. 1 E).

Adipohaemocytes (AD): The AD are large round cells. Fat droplets generally fill the cell. Cytoplasm may also contain other non-lipid granules (Fig. I F) .

Effects of NPV on total haemocyte count (THC) Five day old larvae: The mean number of THC

increased significantly in control larvae up to 4 day, whereas in treated larvae, THC increased significantly up to 2 day post-infection (pi) and then decreased significantly up to 4 day pi (Fig. 2). There was no significant difference between control and treatment up to 2 day after which the THC of treated insects were significantly less than that of control.

Eight day old larvae: The mean number of THC increased significantly in both treated and control

, �

KAllA et at.: HAEMOL YMPH CONSTITUENTS OF HELICOVERPA FOLLOWING NPV 1 125

Fig. I -Photomicrographs of different types of haemocytes of H. annigera by phase contrast microscopy [A-Prohaemocyte, B-Granular cell, C-Spherule cell, D-Plasmatocyte, E-Oenocytoid, F-Adipohaemocyte (x 1000) . Inset with Giemsa stain (x 400)

40000

30000

} � 2oooo 5 day old II11V88

10000

" 'O" �" " "A" " ' " · · · 0 ···· · ·0 1 0 day old larvae

__ S day oontrol

· · 0" 5 day treated

-'- S day control . . 0- . B day

treated -.- 10day

control . . A· · 10 day

treated O +-----r----,�--�-----r----�----T_----�--�

S 6 7 8 9 10 Age of larvae (days)

1 1 12 13

Fig. 2 -Total haemocyte counts/mm3 of haemolymph in the larva of H. annigera in response to NPV infection

larvae up to one day but after that there was a decline in THe. The decrease of THC was nonsignificant up to 4 day in control larvae whereas in treated larvae decrease was significant upto 5 day pi (Fig. 2). The difference between control and treatment was not significant up to two day after which the difference was significant.

Ten day old larvae: There was a decline in THC in treated as well as control larvae. However the decrease was more in treated ones after one day pi. The difference between control and treatment was not significant upto one day after which the difference was significant at 2 day pi. But after three day pi there was no significant difference (Fig. 2).

1 1 26 INDIAN J EXP BIOL, NOVEMBER 2oo1

Effect of NPV on differential haemocyte count (DHC) Five day old larvae: PR increased significantly in

response to NPV after three day pi as compared to control. PL decreased in response to NPV treatment on day 3 of treatment in comparison to control. In control larvae there was no significant change in number of PL and PR with increase in time. The number of spindle shaped PL decreased drastically in treated larvae after two day pi . In GR, no significant difference was found between control and treated larvae throughout the experimental period i.e. till four day pi . However in control as larval age advanced, GR decreased, whereas in NPV treatment GR remained constant till day 2 and decreased significantly on day 3 and remained constant on day 4 as larvae aged (Table 1 ) .

SP increased two day after treatment with NPV and remained constant till day 4 of NPV treatment. The control larvae showed an increase in SP on day 3 and remained at par on day 4.

No significant change in AD was observed as larvae advanced in age in treatment and control. Similar trend was also seen in OE except for increase in NPV treatment on day 3 as compared to control .

Ten day old larvae: PR increased ir. NPV treatment on day 1 and remained constant till day 3 pi. PR

remained constant throughout experimental period in control. But declined as treated insects advanced in age from day 1 to day 3 . NPV treatment reduced PL significantly on day 2 as compared to control. PL remained constant as larvae aged both in control and treatment (Table 1 ).

GR declined in NPV treatment on day 3 as compared to control. They remained constant till day 3 in control . However, decline in GR with respect to age started from day 1 in treatment. A steady increase in SP occurred in both NPV -treatment and control. SP increased from day 1 till day 3 in treatment.

There was no change in AD either with respect to time or treatment. OE declined from day 2 in control and treatment as age advanced. However, NPV treatment showed less of OE from day 2 as compared to control.

Effect of NPV on phenoloxidase activity In general, the PO activity of plasma was increased

with age. PO activity of haemocytes increased up to two day in response to NPV treatment in 5 day old larvae, but declined in 1 0 day old larvae, whereas the PO activity of plasma fraction increased in both 5 as well as 1 0 day old larvae. However, the magnitude of PO activity was higher in 1 0 day old larvae (Fig. 3).

Table 1 -Differential haemocyte count (%) in larvae of Helicoverpa armigera in response to nucleopolyhedro virus treatment

Post infection period (days)

0

2

3

4

0

2

3

Prohaemoc�tes Plasmatoc�tes Granular cells Sl2herule cells o\dil2ohaemoc�tes C T C T C T C T C T

Differential haemocyte count (%)* in 5 day old larvae in response to NPV treatment

4.8aA 5 .0 aA 37.3 aA

(0.84) (0.79) (2. 19) 5.6 ·A 5 .2 aA 38.2 aA

(0.82) (0.57) (3.58) 4.6 aA 4.7 aA 40.4 aA

(0.96) ( 1 .04 ) (4.29) 4.4 aA 6.4 bB 39.6 aA

(0.74) (0.82) (2.43 ) 4.2 aA 7.0 bB 38. 1 aA

(0.57) (0.79) (2. 1 6)

38.4 aA

( 1 .67) 37.2 aA

(2.36) 36.3 aA

(2. 1 9) 30. 1 bB

(2. 1 3) 29.8 bB

( 1 .30)

39.7 .A

( 1 .64 ) 37.3 bA

(2.28) 37.3bA

(2.28) 27.5 cA

(2.67) 26.2 cA

( 1 .26)

38.5 ·A

( 1 . 1 7 ) 37.8 aA

(0.9 1 ) 36. 1 aA

( 1 .56) 28.8 bA

( 1 .53 ) 27.0 bA

( 1 .54)

1 6.6 ·A

( 1 .98) 1 6.7 .A

(3 .72) 1 5.3 aA

(4.39) 26.0 bA

( 1 .22) 28.7 bA

(2.82)

1 6.5 ·A

( 1 .50) 1 7.7 aA

(2.79) 20.8 aB

(2.78) 3 1 .9 bB

(2.82) 34.2 bB

( 1 .44)

0.6 aA 0.5 ·A

(0.42) (0.6 1 ) 0.6 aA 0.7 .A

(0.65) (0.27) 0.5 aA 0.7 'A

(0.35) (0.55) 0.6 aA 0.7 aA

(0.60) (0.45) 0.9 aA 0.8 aA

(0.65) (0.27)

Differential haemocyte count (%)* in 10 day old larvae in response to NPV treatment

2.8 aA 2.9 aA

(0.57) (0.42) 3 .0 aA 5 .0 bB

(0.79 (0.6 1 ) 3.2 aA 4.6 bB

(0.57) (0.65) 2.6 aA 3 .6 cB

(0.42) (0.42)

36.4 aA

( 1 . 19) 30.9 bA

( 1 .75) 32.7 bA

( 1 .92) 32.9 bA

( 1 .24)

35.8 aA

( 1 .64) 32.2 bA

(2.02) 29.8 bB

( 1 .6 1 ) 29.8 bB

( 1 .6 1 )

26.3 ·A

( 1 .39) 24.7 aA

( 1 .68) 23.0 aA

( 1 .69) 2 1 .6 aA

(2.48)

26.6aA 29.2 aA 29.6 'A 1 .7 ·A 1 .2 .A

( 1 . 14) ( 1 .52) (2.53) (0.45) (0.45) 23.6 bA 35.7 bA 34.0 bB 1 .5 aA 1 .2 aA

( 1 .85) (2.02) (2.72) (0.7 1 ) (0.27) 2 1 .8 bA 36.3 bA 40.8 oB 1 .4 .A 0.9 ·A

( 1 . 15 ) (2. 1 4) ( 1 .82) (0.54) (0.42) 1 9.8 oB 37.2 bA 44.4 dB 1 .8 aA 0.9 aA

( 1 .82) (3 .40) (2.28) (0.27) (0.42)

Oenoc�toides C T

1 .0 aA 1 . 1 aA

(0.6 1 ) (0.42) 1 .6 aA 1 .4 aA

( 1 . 14) (0.74) 1 .9 aA 1 .5 aA

(0.42) (0.50) 1 .9 aA 2. 1 bA

(0.74) (0.65) 1 .9 aA 1 .2 aA

(0.4 1 ) (0.57)

4. 1 "A 3 .4 aA

(0.74) (0.55) 4.2 aA 4.0 aA

(0.57) (0.79) 3 .4 bA 2. l oB

(0.42) (0.74) 3.3 bA 1 .5 bR

( 1 .8) (0.50)

*Means of 5 replication, C-control, T-treatment, Figures in parentheses indicate SO values. Means within a column and across column (between treatment and control of particular type of haemocyte) followed by same lowercase and capital leiters respectively do not differ significantly (P<0.05)

KALlA et at.: HAEMOL YMPH CONSTITUENTS OF HELICOVERPA FOLLOWING NPY 1 127

60 5 day old larvae

50 'c

10 day old larvae

. 0 --,l- Control cellular

fraction _____ Control plasma

fraction 2-� 40 .:; 13 Ql QJ 30 II) Ql "0 'x 0 (5 20 c: QJ .r= !l. 10

:

. 0 . . . . . .. 8

o ::>

· . -0 . . Treatment cellular fraction

· • {] . . Treatment plasma fraction

--,l- Control cellular fraction

_____ Control plasma fraction

· . -0 . . Treatment cellular fraction

· . 0 . . Treatment plasma fraction b

5 6 7 8 9 1 0 1 1 1 2 1 3 Larval age (Days)

Fig. 3 -Phenoloxidase activity of haemolymph in the larva of H. anlligera in response to NPY infection.

The haemolymph of NPV - treated larvae showed higher PO activity than the control larvae particularly in the late stage of the larvae. The haemolymph of NPV-treated larvae showed about 1 .5-fold PO activity over that of control in both the sets of experiments involving 5 and 10 day old larvae. The plasma PO activity was quite high in NPV -treated larvae as compared to control . However, the cellular PO activity showed the drastic reversals. NPV treated 5 day old larvae showed higher PO activity than control. However, the cellular PO activity of 1 0 day old larvae was less than that of control. There was negative correlation between the relative proportion of GR and OE in THC and the total PO activity. The similar trend was also found in NPV -treated insects. The higher PO activity in NPV treated insects over that of control may have come from the sources of synthesis other than GR and OE. Phagocytosis of OB by haemocytes

The haemocytes viz., PL and GR phagocytosized almost all OB within 30 min of injection of 10 day old larvae (Fig. 4C and D). But after 3 to 4 hr the immunocytes were full of vacuoles and eventually brokedown (Fig. 4E).

Discussion There is often a disagreement on nomenclature and

classification of haemocytes due to variability in morphological characteristics of haemocytes per se and artifacts in morphology due to use of different techniques. In the present study, 6 types of haemocytes viz. , PR, PL, SP, GR, OE and AD were characterized in larvae of H. armigera. Essawy et ai.5

however reported only 5 types of haemocytes (PR, PL, SP, CO, and OE) in H. armigera larvae by phase

contrast microscopy and electron microscopy. Although CO were considered as a separate entity in the earlier studies on lepidopteran haemocytes 10, Gupta2 found them to be essentially degranulated GR. We also observed degranulation in GR in due course of time and preferred use of GR in place of CO. Interestingly AD found in the present study were not reported earlier by Essawy et az5.

Changes in haemocyte morphology and function during an infection have been extensively studied as part of investigation on insect immunity l l . l 2 . THC pattern increased with larval growth upto 10th day and decreased slowly as prepupation was initiated. Similar results were also reported in Pseudaletia unipuncta1 3, Heliothis virescensl4 and Euxoa declo rata 15, but differed with those in H. zea wherein THC remained constant from six to 10 day old larval age3.

THC increased with respect to age from 5 day old to 9 day old larvae and later decreased till prepupationlpupation. In NPV treatment also the same trend was observed temporally. NPV infection decreased THC only after two days of infection. Similar decrease in THC was also reported in H. zea, H. ar11ligera, Galleria mellonella and Spodoptera iilura larvae in response to infection with the polyhedrosis virus3•6,9. 16.20.

PL, GR and SP constituted bulk to the extent of 92% of THC and appeared to play a significant role in their dynamics with respect to treatment with NPV. The decline in immunocytes especially appeared to be compensated by increase in SP as a result of NPV treatment. PR showed significant increase in the NPV treatment of 1 0 day old larvae and hence may have a significant role being a progenitor in compensating mechanisms.

1 1 28 INDIAN J EXP BIOL, NOVEMBER 200l

Fig. 4-Haemocytes from NPV diseased H. armigera larvae­nucleus filled with polyhedra (arrow) (A-Plasmatocyte; B ­Granular cell). Haemocytes (C-Plasmatocyte; D-Granular cell) of H. annigera larvae injected with NPV- cells completely filled with polyhedra (arrow). E-Disintegrated haemocytes of H. amligera larva of 4 hr after injection with NPV showing vacuoles in cells (arrows) [x 400].

Wittigl7 also observed similar compensating changes in DHC in P. unipunctata infected with virus; GR 'initially increased then decreased while the SP and PR increased. In G. mellon ella larvae, infection with NPV caused significant decrease in PL and increase in GR and SpI B. The significant increase of SP was also reported after 4 days of treatment in 6 day old NPV treated larvae of H. zea3•

The present study showed that in general PR, PL and GR decreased in number from 5 to 10 day old

larvae whereas the number of SP and AD increased as the larvae pupated. However the population of OE decreased before reaching prepupation. PR decreased and GR increased during larval growth prior to pupation in Prodenia eridanial9, G. mellonella l B and E. declorata15• AD increase, similar to present findings, was reported in G. mellonella2 1 •

Melanization is one of the important events in cellular defence mediated through phenoloxidase cascade. The higher activity of PO in NPV-treated insects may have been inducted with the onset of NPV -infection. It is to protect the insect with the help of melanin formation against NPV. Pathogenesis of haemocytes brought about changes in PO activity, which is an index of melanization. The higher PO activity in plasma over that of cell fraction especially for day 9 of larval age suggested disintegration of haemocytes and release of PO into plasma. This is correlated with decrease THC counts before pupation.

Insects haemocytes are capable of reducing the number of foreign bodies in the haemocoel by phagocytosis, encapsulation or by nodule formation22. In the present study when polyhedral suspension was injected into 10 day old larvae the PL and GR phagocytosed almost all polyhedra within 1 5 to 30 min of injection. But after 3 to 4 hr the immunocytes became vacuolated and disintegrated cells. Similar results were reported on phagocytosis of baculovirus virion in P. unipunctata13 and phagocytosis of OB in Spodoptera littoralii3• The inability of haemocytes to destroy virus on phagocytosis may be related to the high rate of viral multiplication and pathogenesis.

Anderson et al.9 reported reduction in THC with activation of PO reaction. Normally high THC in the last larval instar is an indicator of higher activity of insect to be immune to infections, since during this stage insect larva is highly mobile and interacting with diversity of microbial fauna. Kisler et aL.23 also reported haemocytic ability to phagocytose NPV in S. littoralis similar to present findings. NPV treatment at high dose affected both cellular and humoral immunity. Similar results were also been reported by Ourth and Renis24. Interactive effect of insect immunity with that of pathogenicity of NPV will depend upon an initial dose of infection, THC and DHC contents of haemolymph.

Acknowledgement VK is grateful to IARI for the award of a Senior

Research Fellowship. Thanks are due to Dr. K. Narayanan, Project Directorate of Biological Control, Bangalore for constructive comments.

KALlA et al.: HAEMOL YMPH CONSTITUENTS OF HEUCOVERPA FOLLOWING NPV 1 129

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