Cell Mediated Immune Response of Helicoverpa armigera Hubner and Spodoptera litura Fabricius to Fern Phytoecdysteron
The aim of this study to evaluat the immunological impact of the Crude Ecdysone Fractions (CEF) of 3 ferns such as Christella parasitica (L.) H.lev., Pteridium aquilinum (L.) Khun and Hemionitis arifolia (Burm) T. More. on two major polyphagous pests, Helicoverpa armigera Hubner and Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). The results revealed that the Haemosomic Index (HI) and the Total Haemocyte Count (THC) were higher in control than the experimental insects. Irrespective of the CEF treatment, HI increased gradually from the first day to the fourth day of observation. Similar trend was also observed for the THC. Among the three ferns, C. parasitica highly reduce the HI followed by H. arifolia and P. aquilinum. Six types of haemocytes viz., Prohaemocytes (PR), Plasmatocytes (PL), Granular cells (GR), Spherule cells (SP), Oenocytoids (OE) and Adipohaemocytes (AD) were observed in both pests. Among the six types of haemocytes observed, PL constituted highest percentage control categories of both H. armigera and S. litura population. In ferns treated categories, PR, SP and AD levels of both pests were increased. Among the six types of haemocytes, cellular mediated response was observed only on GR, SP, PR and PL cells, which either underwent lyses or aggregation. Lyses or aggregation were well pronounced in H. armigera than S. litura.
Helicoverpa armigera Hubner is one of the most important pests of filed crops worldwide. It is highly polyphagous and causes severe damage and loss to a wide range of food, fiber, oil, fodder, vegetable, horticultural, ornamental, aromatic and medicinal plants (Neoliya et al., 2007). It has developed resistance against most of the modern classes of insecticides like organophosphate, pyrethroid and carbomates (Yadav et al., 2006). Spodoptera litura (Fab.) is also one among economically important polyphagous pest causing damage to more than 50 crops (Venkateswarlu et al., 2006). This is the first lepidopteran to develop insecticide resistance in India during 1965. Development of insecticide resistance in this pest has been faster in the last two decades (Arms et al., 1997; Kranthi et al., 2002; Radhika et al., 2006). Very recently Srivastava and Srinivas Reddy (2006) reported that this pest could be controlled using neem based insecticides. Misuse and excessive use of synthetic pesticides and also the hazards associated with these chemicals forced the pest management practitioners to explore alternative and ecofriendly means of crop protection. Phytochemical pesticides gained considerable impportance throughout the world, as they are environmentally safe and much safer to higher animals including humans.
Ferns are members of a large and diverse group of plants commonly referred to as the lower plants. Ecdysones have also been isolated from ferns and are believed to deter feeding by mimicking insect molting hormones (Markham et al., 2006; Meepagala et al., 2006). Ecdysone analogues or phytoecdysone were identified from ferns phytoecdysones are being suggested as suitable biopesticide for the pest control since insects may not be able to develop resistance to these compounds (Selvarj, 2002; Selvarj and Sahayarij, 2005; Selvarj et al., 2005). The insect immune system is different from vertebrates immune system as they lack an antigen-antibody complex, though they are capable of responding very effectively against the various foreign invaders. Tiwari et al. (1999), Tiwari and Shukla (2000) and Neoliya et al. (2007) reported the hematological changes of insects in response to the exogenous administration of ecdysone has been reported. Impact of metyrapone and nucleopolyhedro virus on the morphology of hemocytes of Spodoptera littoralis (Boisd.) and Helicoverpa armigera (Hubner) has studied by Gelbic et al. (2006) and Kalia et al. (2001) respectively. However no information was available for the immunological response of H. armigera and S. litura against phytoecdysone except Sharma et al., (2003). Moreover, Anitha and Subramanyam (1998) studied the juvenoid induced of S. litura. Therefore we evaluated the effect of fern phytoecdysone on total count (THC) and differential count of haemocytes (DHC), Haemosomic Index (HI) and cell mediated of H. armigera and S. litura.
MATERIALS AND METHODS
Fern Collection Andphytoecdysone Extracts Preparation
C. parasitica, P. aquilinum and H. arifolia were collected
from Kothiyar Hills and identified by Dr. V.S. Manickam, Center for Biodiversity
and Biotechnology, St. Xaviers College, Palayamkottai. The voucher specimens
have been preserved in our laboratory. Laboratory experiments were conducted
in Crop Protection Research Centre, St. Xaviers College in 2002. The plant
materials were washed thrice in tap water and once with distilled water. They
were shade dried for two weeks. The dried plant materials were ground with domestic
grinder and stored in refrigerator for further use. The phytoecdysone fraction
(PEF) was extracted using the standard method of Horn and Bergamasco (1985).
From the PEFs, 0.1% solutions prepared with ethanol and used for the experiment.
Collection and Maintenance of Pests
Different larva of H. armigera and S. litura were collected
from the groundnut fields of Tirunelveli and Kanyakumari districts, Tamil Nadu
and were reared on groundnut leaves under laboratory conditions on fresh groundnut
leaves (29±1°C; 65-70% RH and 11L and 13D photoperiod). S. litura
were reared in plastic trough (21.0x28.0x9.0 cm) whereas H. armigera
larvae were reared individually in plastic containers (100 mL volume) to avoid
cannibalism. Laboratory emerged ten days old larva (225-250 mg for S. litura
and 200-225 mg for H. armigera) was used for the experiment.
Five grams of fresh groundnut leaves (TMV 7 Variety) were taken and were
immersed in PEF (10 mL) separately for experiments and ethanol for control for
5 min. The immersed leaves were then shade dried for 10 minutes and provided
to the laboratory emerged 6 h. starved 10 day old 4th instars S. litura
and H. armigera larvae separately in plastic containers (250 mL capacity).
Sixty larvae were maintained for each treatment as well as for control categories
separately. The containers were covered with muslin cloth and the larvae were
allowed to feed the PEF (0.1%) treated groundnut leaves continuously for four
days. Cleaning and feeding were done for every 24 h. Ten replication was maintained
for each plant separately. After every 24 h of treatment 6 larvae were selected
randomly from each categories and their initial weight was recorded. Forelegs
of the larvae were amputated using fine sterilized scissors and the haemolymph
was collected. Again the larval final weight was recorded. By using the initial
and final weight of the above haemosomic index (HI) was calculated using the
The haemolymph collected from the insect was directly drawn into the WBC pipette
up to 0.5 marking and mixing immediately diluted with an acidified physiological
saline (NaCl-4.65 g, KCl-0.5 g, CaCl2-0.11 g, Gentian violet -0.005
g and acetic acid -0.125 mL and made up into 100 mL with distilled water) mixed
thoroughly. THCs were conducted with a standard haemocytometer (Naubauers)
and the haemocytes population per mm3 of haemolymph was calculated
by multiplying the average haemocytes per count with a depth and a dilution
For haemocyte profile, one or two drops of 4% formal in were placed on a clean micro slide. Then place one or two drops of haemolymph on to the formal in solution. They were mixed well and a thin film was smeared on the slide and then the film was air dried for few minutes. The slides were stained with Giemsas stain for about 10-15 min, allowed them to dry and observe under the microscope. The haemocytes of different kinds (DHC) were counted and expressed in percent (Sharma et al., 2003). Microscopic studies of the haemocytes were conducted on control and all fern treated larvae after 96 h of treatment. Correlation and multiple regressions analyses were performed between control to fern extracts and also within the extracts.
Haemosomic index of both the experimental and control categories were increased as the exposure time increased (Table 1 and 2). The results also revealed that the HI was lower in the experimental animals than the control larvae at different intervals of the exposure. Even though the experimental categories showed a gradual increase, they did not achieve the increase as in control. Among the experimental categories of H. armigera, highest and lowest HI was recorded in P. aquilinum and C. parasitica treated categories, respectively (Table 1). In control S. litura larvae, HI was 40, 41, 46 and 53% for 24, 48, 72 and 96 h, respectively.
Total Haemocytes Count (THC) of H. armigera and S. litura is
presented in Table 2. From the results, it is very clear that
the THC increased with the increase of exposure period. Both H. armigera
and S. litura THC population was higher and lower in CP and PA respectively
than the control. THC in HA is either equal for S. litura or higher for
H. armigera than control category.
||Haemosomic index (in %) of S. litura
and H. armigerain in relation to phytoecdysone fraction of C.
parasitica (CP), P. aquilinum (PA), H. arifolia (HA)
and Control (CO)
||Total haemocytes count THC (104/cu
mm3) of H. armigera and S. litura treated with
crude phytoecdysone fraction of C. parasitica, P. aquilinum
and H. arifolia
The correlation analyses between CP to PA were significant at 5% level (r =
0.97; p<0.05). The correlation was slightly reduced between CP and HA (r
= 0.95; p<0.05) treated H. armogera. Correlation coefficient value
between the control and CP was 0.99 and 0.95 for H. armigera and S.
litura, respectively. For the both the pests, we obtained perfect Correlation
coefficient value (r = 1) between P. aquilinum and H. arifolia.
Multiple regression analysis between exposure time and ferns treatments showed
that the result was insignificant for H. armigera (F = 395.10; p = 0.0025;
p<0.0046) than S. litura (F = 44.88; p = 0.021; p<0.0118).
We found haemocytes to be composed of prohaemocytes (PR), plasmatocytes (PL), granular cells (GR), spherule cells (SP), oenocytoids (OE) and adipohaemocytes (AD) in both H. armigera and S. litura (Table 3 and 4). Proportion of PL population was higher in control H. armigera, followed by SP, GR, PR, OE and AD. In experimental categories, highest PR count was recorded for P. aquilinum treatment followed by H. arifolia and C. parasitica at 24 h. The PL count was decreased in all the three ferns PEF treated groundnut leaves fed H. armigera (Table 3). For instance, highest reduction was observed in P. aquilinum followed by H. arifolia (Table 3) and C. parasitica. Even though PL count of the control gradually decreased form 24-72 h. of exposure, its population increased at 96 h. of observation. The reduction in PL count was more pronounced in the experimental categories and it was extended up to the 72 h of observation. Similar trend was also observed in GR. However in C. parasitica treated category, the GR count was gradually decreased up to 96 h. Same trend was observed for OE with an exception of P. aquilinum at 24 h SP cell count of the control showed marked increase from 24 h up to 96 h. of observation. This increasing trend was more pronounced in the experimental categories. For instance, the order of increase was 31.2, 31.4, 35.5 and 39.0% from 24-96 h for P. aquilinum.
The results of S. litura DHC showed that after 24 h of exposure, percentage
of individual haemocyte type increased in all the experimental categories except
GR. Maximum GR was recorded in C. parasitica followed by P. aquilinum
treated categories. Whereas, SP and PR cells counts were increased steadily
in H. arifolia treated category when compared to the control. At 48 h
of exposure, PL count was decreased in P. aquilinum and H. arifolia
treated categories. When compared to the control PR, SP, AD and OE counts
were increased up to 48 h observation, a reduction was observed in GR count.
The highest reduction of GR was recorded in P. aquilinum followed by
H. arifolia. The highest PR count was recorded in P. aquilinum
followed by H. arifolia. The SP cell count was higher in H. arifolia
followed by P. aquilinum. AD count was 1.8% in H. arifolia and
the highest OE was recorded in P. aquilinum and H. arifolia treated
categories. After 72 h of exposure a steady increase of both PR and SP were
recorded in P. aquilinum treated category while the highest percentage
of PL and GR were recorded in C. parasitica treated category. In the
experimental categories the OE population at 96 h was maintained as observed
at 72 h exposure.
||Differential haemocytes count THC (104/cu
mm3) of H. armigera treated with crude phytoecdysone fraction
of control (CO), C. parasitical, P. aquilinum and H.
||Differential haemocytes count DHC of
S. litura treated with crude phytoecdysone fraction of Control (CO),
C. parasitica, P. aquilinum and H. arifolia
But in the control an increase up to 3% was observed. The AD counts of P.
aquilinum and H. arifolia treated categories recorded a decline but
an opposite trend was observed in the control and C. parasitica treated
categories. The PR count in the control was increased from 72-96 h. An opposite
trend has been observed in the experimental categories. The increased of PL
and GR counts were compensated by the decline of SP and AD cells (Table
Among the haemocytes, cytotoxic responses were observed in PL, GR, PH and SP
cells. They showed either lyses or aggregation behaviour (Fig.
1 and 2). Among the two pests, both the cell lyses and
cell aggregation was more pronounced in H. armigera (Fig.
1) than S. litura (Fig. 2). In H. armigera
the above said impacts were mainly caused by P. aqulinum (Fig.
1b, f, I and m). P.
aqulinum induce all types of cells such as, PL, SP and PH. In addition to
the cell lyses and cell aggregation H. arifolia caused pseudopodia formation
in GR (Fig. 1e). In S. litura maximum cell aggregation
was more pronounced in H. arifolia treatment than P. aquilinum.
All the three ferns caused SP cell lyses. It was more pronounced in P. aquilinum
followed by C. parasitica and H. arifolia (Fig.
||Haemyocytes of control H. armigera larvae plasmatocytes
(A) and its aggregation caused by P. aquilinum (B) H. arifolia
(C) granulocytes normal (D) and H. arifolia pseudopodia (E) cytolysis
caused by P. aquilinum (F) and C. parasitica (G) normal
spherule calls (H) and its cells lysis caused by C. parasitica
(I) P. aquilinum (J) and H. arifolia (K), proheamocytes
(L) normal and its cell lysis P. aquilinum (M) H. arifolia
(N) and (O)
||Haemyocytes of control S. litura larvae Granulocytes
(A) and its cell aggregation and lysis caused by C. paracitica (B and C)
P. aquiilinum (D) H. arifolia (E) ruptured spherile cells
treated with P. aquiilinum (F) showing disintegration of lipid droplet
Haemocytes appears to be an important indicator for the growth and metamorphosis of insects. Chapman (1998) stated that the haemosomic index and the total haemocytes count increases as the concentration of ecdysone increases in the haemolymph. It helps the animal to digest the dead cells and tissues during the process of metamorphosis and to cast the molt skin during the process of molting (Karlson, 1996; Kalia et al., 2001). The results of this study also support their view. The process of molting could be triggered by the exogenous administration of ecdysone hormonal analogues like fern PEFs. It may interfere with the normal developmental process and the animals prepare to molt but they failed to complete the task (Kalia et al., 2001; Hadapad et al., 2001; Falleiros et al., 2003; Gelbic et al., 2006).
We observed a distinct decrease HI of H. armigera and S. litura when they were fed with PEF treated leaf throughout the experimental period. These changes may be due to the interactions of beta ecdysone in CP and HA and alpha and beta ecdysone in PA (Selvarj et al., 2005). Furthermore, this might be the result of feeding deterrence as well as the unpalatability of the food or due to the prevention from feeding and locomotion (Kubo and Hanke, 1986). In lepidopteran caterpillars, water comprises 50% of the haemolymph volume. It reflects the importance of hydrostatic function of haemolymph (Chapman, 1998). He further added that the increased volume of haemolymph is associated with an increased activity of an antidiuretic hormone. However the reduction in HI of the experimental insects might be due to starvation too. Previously Wigglesworth (1972) reported that the haemocytes transport ecdysone to the epidermis. But though the larva consumed ecdysone through food, it could not be reached into the epidermis. However Kubo and Hanke (1986) and Keyserlingk (1986) reported that precocious molting occurred in the treatment categories of their experiment and hence they were in doubt that whether the ecdysone is transported through the haemocytes or through some other carrier proteins.
Treatment of PEF of CP in 10 days old H. armigera and S. litura exhibited decreased in the THC. It was well pronounced (8%) in H. armigera than in S. litura (1%). These observations are in conformity with those described for neem gold on the THC of S. litura (Sharma et al., 2003). These findings clearly bring forth the efficacy of the CP in drastically lowering down the heamocyte counts. In this experiment P. aquilinum treated S. litura and H. armigera recorded lowest haemocyte count at 24 h exposure. After 24 h the haemocyte population was increased. This suppression at the initial stage might be due to the high deterrence of these plants. Sharma et al. (2003) found that the neem gold treatment decreased THC after 48 h. Followed by an increase might be due to the consumption of the treated food due to prolonged starvation. Tiwari et al., (1999) and Tiwari and Shukla (2000) also recorded similar results in Papilio demoleus larvae treated with 20-hydroxy ecdysone.
There is often a disagreement with the nomenclature and classification of haemocytes due to variability in morphological characteristics of haemocytes. Based upon the morphology we recorded 6 types of haemocytes in both the insects (PR, PL, SP, GR, AD and OE). Kalia et al. (2001) reported all these six types of haemocytes in H. armigera. However, previously only five types of haemocytes such as PR, SP, PL, CO (coagulocyte) and OE were observed in the same species. Structural organizations and morphology of these cells are similar in H. armigera and S. litura. The production and differentiation of haemocytes have been shown to be regulated either by prothorasic glands (PTG) or by changes in hormone levels which influence the mitotic index and size of the haemocyte population either directly or indirectly (Tiwari et al., 1999). DHCs in the larvae under the influence of PEFs of ferns reveled that changes in the differential counts after phytoecdysone treatment and these changes simulated acceleration in the aging process. Pech and Strand (1996) reported that both plasmatocytes and granulocytes are the two sub-classes of heamocytes adhere to foreign surface and involved in encapsulation. Present study reveled that population of both plasmatocytes and granulocytes decreased after 24 h of fern treatment indicates; these cells are not involved in encapsulation process. Similar reduction in plasmatocytes and granulocytes number was also observed by Sharma et al. (2003); Gelbic et al. (2006). Tiwari and Shukla (2000) added that GR was played an important role in encapsulation reaction ultimately it decrease the level of GR. Along with the GR, PL also involved in the immune response of the insects.
Rajkumar et al. (2000) and Sharma et al. (2003) reported that the plant biopesticide treatment generally decrease the PR, PL, SP population and increase the GR and OE population. In the present experiment, the highest response was observed in PEF of P. aquilinum treated category. This response coincides with the immunological response during ecdycial events. But no clear-cut pathological response was observed in this experiment, as observed by Kalia et al. (2001) in H. armigera treated with NPV. However incidence of cytolysis was observed in case of GR and SP and clumping behaviour in PL. Gupta (1994) stated that both PL and GR are involved in the cell-mediated immune response to invading foreign materials and suppression of such process. In addition GR and SP are also involved in this cellular defense, which is evident from its higher degree of cytolytic response. Williams (1956) stated that since the ecdysones are the hormonal factors of insects, the pests might not develop any defensive strategies against these compounds. We also agree with the statement of Williams and we believe that the increasing haemocyte count in the experimental categories might be the result of the immuno stimulant effect triggered by the exogenous administration of fern phytoecdysone as suggested by Fomovskaia (1992) and Tiwari et al. (1999). It can also be assumed that the cuticular disruption as observed in the fern phytoecdysone treated insects (Selvarj, 2002) might lead to microbial infection through the wound. So that the haemocytes showed some kind of cell mediated immune response. The structural details of the affected haemocytes further revealed destruction of plasma membrane in many PL there by the PL count drastically decreased. Furthermore, PL and GR are the major immune responding cells in the insect so phytoectysone first to attack the PL and GR and their population either decreased undergoes lyses sometime GR showed budding or encapsulation. The rapid lyses of GR may be initiated by vacuolization and loss of compactness of organelles leading to degranulation and a degenerative transformation (Sharma et al., 2003). From the results it is very clear that if an insect take molting hormone mimicking phytoecdysone through their food, it may change the heamolymph as well as their constituents both by number and structure, the use of these substances for integrated pest control seems to have good potential.
The authors are thankful to Rev. Fr. A. Antonysamy, S.J. Principal and Prof. M. Thomas Punithan, Head Dept. of Zoology, St. Xavier College, Palayamkottai for institutional facilities. The senior author (KSR) is thankful to DST, Govt. of India, New Delhi for financial support (Ref. No. SP/SO/C51/99) for this work.
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