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Parasitism by Chelonus blackburni (Hymenoptera) affects food consumption and development of Helicoverpa armigera (Lepidoptera) and cellular architecture of the midgut
Journal of Asia-Pacific Entomology 19 (2016) 65–70
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Parasitism by Chelonus blackburni (Hymenoptera) affects food consumption and development of Helicoverpa armigera (Lepidoptera) and cellular architecture of the midgut Yogita Sanap a, Vishal V. Dawkar b, Ashok P. Giri b, Avalokiteswar Sen c, Radhakrishna S. Pandit a,⁎ a b c
Department of Zoology, Savitribai Phule Pune University, Ganeshkhind, Pune, MS, India Plant Molecular Biology Unit, Division of Biochemical Sciences, CSIR–National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, MS 411008, India Laboratory of Entomology, Division of Organic Chemistry, CSIR–National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, MS 411008, India
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Article history: Received 3 September 2015 Revised 16 October 2015 Accepted 2 November 2015 Available online 26 November 2015 Keywords: Biocontrol Helicoverpa armigera Chelonus blackburni Histology Total hemocyte counts
a b s t r a c t Biological control agents are vital components of an integrated pest management strategy, and this is frequently referred to as natural control. Natural enemies of insect pests include predators, parasitoids, and pathogens. Among them, a parasitoid, Chelonus blackburni (Cameron), was found to be the best biological control agent for the polyphagous pest, Helicoverpa armigera (Hübner). C. blackburni alters the feeding performance of H. armigera larvae upon parasitism and as a result severely affects growth and development. Moreover, it shortens the feeding period of H. armigera and increases mortality. Furthermore, total hemocyte count (THC) was significantly decreased in parasitized larvae than control. Parasitized H. armigera had 26% less number of blood cells compared to healthy larvae. Histological studies showed that the structure of midgut of H. armigera is drastically affected by C. blackburni leading to reduced food consumption, which ultimately led to larval death. The present study provides an insight to changes involved in H. armigera due to parasitism by C. blackburni, a parasite that could be used as an effective biocontrol agent to manage H. armigera. © 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Introduction The cotton bollworm, Helicoverpa armigera (Hübner), is an important polyphagous pest feeding on more than 181 cultivated and wild species spread across 45 families, including economically important crops such as cotton, pigeon pea, chickpea, and sorghum as well as horticultural and ornamental crops (Manjunath et al., 1989; Sarate et al., 2012). The continuous availability of host crops, often planted as monocultures, and the lack of crop rotation greatly contribute to the maintenance of high population levels of H. armigera and the consequent damage to crops. Synthetic pesticides have primarily been used to control this pest over the past several decades; however, widespread and indiscriminate use of these groups of chemicals has resulted in the development of resistant strains inviting still increasing doses of synthetic pesticides, particularly in developing countries. Such large-scale uses of synthetic pesticides have ecological implications, including environmental pollution and crop residues (Smagghe et al., 1999; Mishra et al., 2015). Parasitoids are an important component in integrated pest management (IPM) programs. It has generated a great deal of interest due to ⁎ Corresponding author at: Department of Zoology, Savitribai Phule Pune University, Pune, MS 411008, India. Tel.: +91 20 25601436; fax: +91 20 25690617. E-mail address: [email protected] (R.S. Pandit).
their ability to suppress pest populations and thus help diminish the damage caused by agricultural pests. Often, parasitism may also change the host feeding behavior (Lewis and Burton, 1970; Powell, 1989; Morales et al., 2007). Common feeding patterns and the amount of food consumed differ between infected and normal larvae, and usually, it has been observed that parasitized larvae eat very modest amounts of food (Parker and Pinnell, 1973; Beckage and Riddiford, 1978; Couchman and King, 1979; Duodu and Antoh, 1984; Danyk et al., 2005; Morales et al., 2007). Among the promising parasitoids, the braconid Chelonus blackburni (Cameron) is a key parasitoid used in biological control or IPM for controlling H. armigera. It is a solitary endoparasitoid that completely depends upon its host larva for food and shelter to complete its development from embryo to the third instar larva. Subsequently, it emerges from the dead host and spins a silken cocoon in the vicinity (Grossniklaus-Burgin et al., 1994). With this background and efforts to look at alternative insect pest control measures, we studied the effect of parasitization by C. blackburni on food consumption and development in H. armigera. This study shows that parasitized larvae did not grow normally as reflected by their reduced food consumption. Analysis of hemolymph from parasitized and unparasitized H. armigera larvae showed significant differences in total hemocyte counts as well as in the numbers of the different hemocytes. In addition, there were drastic changes in cell aggregation and shapes of the different hemocytes. Furthermore, histological studies on the midgut
http://dx.doi.org/10.1016/j.aspen.2015.11.005 1226-8615/© 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
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of parasitized larvae of H. armigera show the destruction of the peritrophic membrane besides extensive damage to the midgut epithelial cells. Materials and methods
morphological characters as described by Gupta (1985). Further, to determine the DHC, cell categories were counted in 200 cells chosen from random areas of the stained hemolymph smear by a laboratory cell counter.
Rearing of H. armigera and parasitism by C. blackburni
Histology
Larvae of H. armigera were collected from fields of Mahatma Phule Krishi Vidyapeeth, Rahuri, India. They were maintained on a chickpea based artificial diet (AD) (Nagarkatti and Prakash, 1974) in the laboratory under controlled conditions (temperature: 27 °C; humidity: 60% and photoperiod: 16:8). To ensure greater genetic homogeneity among test populations, insects were maintained on AD for three generations. H. armigera egg sheets (~ 300 eggs) were exposed to adults of C. blackburni in a parasitoid oviposition chamber for 24 h. After hatching, neonates from each group (control and parasitized) were taken for further experiments. Food consumption and weight of larvae were recorded every 2 days. The experiment was terminated on day 22 since by that time unparasitized larvae of H. armigera attain pupal stage while the parasitized larvae die as indicated by the spinning of silk cocoons in the vicinity. The numbers of well-formed cocoons were recorded. All the above experiments were replicated thrice.
Parasitized and unparasitized H. armigera larvae were anesthetized with chloroform and dissected under a microscope to isolate the midgut. The collected specimens were immediately put in alcoholic Boüins and kept for 2 h at room temperature (~ 24 °C), following which they were dehydrated in a graded series of alcohols and finally cleared in xylene. The material was embedded in paraffin wax, and 6 μm sections were obtained with the help of a microtome and stained with hematoxylin–eosin. Morphological analysis was carried out using an OLYMPUS BX-49 light microscope.
Hemolymph collection and hematology
Results
Prior to hemolymph collection, the insects were chilled for 15 min at 4 °C. Hemolymph samples were obtained by puncturing the larval abdomen with a sterile needle. The outflowing hemolymph was immediately transferred into sterile and chilled Eppendorf tubes containing a few crystals of phenylthiourea (PTU) to prevent melanization and stored at −80 °C until used (Andrejko and Mizerska-Dudka, 2012). For hematological experiments, hemolymph (10 μL) was stained with Giemsa (4%). Free hemocytes were counted using a hemocytometer with improved double Neubauer ruling under a phase contrast microscope.
Effect of parasitism by C. blackburni on H. armigera
Differential hemocyte counts (DHC) For smear slide preparation, a small drop of larval hemolymph was obtained by clipping a proleg. The drop was then smeared into a thin film, air-dried, and fixed in absolute methanol for 4 min before staining for 2-3 min in Giemsa (4%), air-dried, and subsequently washed with distilled water. The rinsed slide was dried and then mounted in DPX. Observations were made under a phase contrast microscope at 40X magnification and images were acquired with a digital camera. The different types of hemocytes were identified using established
Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparisons test. Data points were considered significant at p b 0.05, p b 0.01, and p b 0.0001.
Feeding in larvae of H. armigera parasitized by C. blackburni is greatly reduced with the larvae upon reaching the fourth instar develop into a pre-pupa from which a freshly molted 3rd instar larva of C. blackburni emerges. In comparison, unparasitized larvae continue their development until they reach the pupal stage. In unparasitized larvae, normal food consumption and normal growth and development of larvae were observed. Consequently, there were significant differences in growth as reflected in the amount of food consumed and larval mass between unparasitized and parasitized larvae (Fig. 1A and B; p b 0.01 and p b 0.005, respectively). Food consumption increased gradually in unparasitized larvae of H. armigera. However, in parasitized larvae, initially, the food consumption increased gradually up to day 12 but later decreased from day 14 (Fig. 1A). The mean food consumed by parasitized larvae on day 12 was 89 ± 0.5 mg which was significantly less than the food consumed by unparasitized larvae (202 ±10.46 mg; p b 0.01). Interestingly, the amount of food consumed by parasitized larvae was higher than unparasitized larvae till day 12 (89.5 ± 5.01 mg), which reduced to 53.0 ± 4.73 mg on day 14. This was probably due to
Fig. 1. Development and food consumption of unparasitized and parasitized Helicoverpa armigera by Chelonus blackburni. (A) Larval mass gain by unparasitized (black bars) and parasitized (hollow bars) H. armigera. Graph shows average mass from each set of 15 larvae. Larvae were critically weighed on every fifth day. (B) Food consumption data of larvae. Standard error means are indicated. * and ** indicate that values are significantly different from each other at p b 0.01 and p b 0.005, respectively.
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up to three times. No weight gain was observed in parasitized larvae from day 12 onward.
Unparasitized Parasitized
Hematology
Area (µm2)
40 30 20 10 0
Fig. 2. Morphometric analysis of different types of hemocytes found in the hemolymph of unparasitized and parasitized Helicoverpa armigera larvae. Standard error means are indicated.
the larvae having entered the pre-pupal stage. The overall food consumed by parasitized H. armigera larvae up to day 14 was only 34.21% of the total food consumed by unparasitized larvae. Studies related to the effect of parasitism on weight gain by larvae of H. armigera indicate that although there were no significant differences in larval body mass of H. armigera till day 8, however, on subsequent days, differences were significant between the control and parasitized larvae of H. armigera, respectively (p b 0.01 and p b 0.005) (Fig. 1B). Unparasitized larvae of H. armigera grew slowly during the initial 10 days, until they molt into the 3rd instar, following which their weight and food consumption increased rapidly. On day 12, the weight of unparasitized larvae was two times more than that of parasitized larvae, and once they had molted into 4th instar on day 14, the difference increased
A
B
The effect of parasitism by C. blackburni on larvae of H. armigera revealed prominent changes in the total hemocyte count (THC). There was a significant decrease in THC in parasitized larvae of H. armigera compared to unparasitized larvae. The number of cells counted per microliter at the end of day 12 was significantly lower in parasitized larvae (2440 ± 104) than in unparasitized larvae [(9143 ± 410); (p b 0.0001; F = 1.12, df = 4, t = 44.28); (Fig. 2)]. Six primary types of hemocytes, viz. prohemocytes (PR), plasmatocytes (PL), granulocytes (GR), sperulocytes (SP), adipohemocytes (AD), and oenocytoids (OE), were observed in hemolymph of parasitized and unparasitized larvae of H. armigera (Fig. 3). In case of PR, the cells were either round, oval, or elliptical in unparasitized larvae while in parasitized larvae, the PR was abnormal, and the cytoplasm occupied most of cell forming a very thin layer around the nucleus. PLs are highly polymorphic cells with their shapes varying from being spindle shaped to one with a very pointed end. In unparasitized larvae, these cells were oval with a large centrally located nucleus; however, this type of shape was not observed in parasitized larvae. GRs are also variable in shape and size and are easily distinguished by the presence of vacuoles in the cytoplasm, but this was not observed in the parasitized larvae of H. armigera. SPs, ADs, and OEs show radical differences in parasitized larval hemolymph. There was a drastic difference in DHC of parasitized and control larvae of H. armigera (Table 1). The larvae of H. armigera parasitized by C. blackburni reveal a significant increase in percentages of PRs and GRs, while in unparasitized larvae, the percentage of PLs and SPs significantly increased (p b 0.001; F = 4 and t = 44.72). However, there were no significant differences in percentage of ADs and OEs between parasitized and unparasitized larvae. In parasitized larvae of H. armigera, the amount of PLs was
A
1. Prohemocyte A
B
2. Plasmatocyte A
B
5. Adipohaemocyte
B
4. Sperulocyte
3. Granulocyte A
B
A
B
6. Oenocytoid
Fig. 3. Morphology of haemocytes viz. prohemocyte, plasmatocyte, granulocyte, sperulocyte, adipohemocyte, and oenocytoid of unparasitized (A) and parasitized (B) Helicoverpa armigera after Giemsa staining.
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Table 1 The percentage of differential hemocyte count (DHC) in the parasitized and unparasitized larvae of Helicoverpa armigera. Cellular type (%) H. armigera
PR
PL
GR
SP
AD
OE
Unparasitized Parasitized
46⁎ 55.39
15.36⁎ 12.85
14.60⁎ 21.42
26.44⁎ 8.57
1.91⁎ 1.18
0.33⁎ 0.23
⁎ Values are significantly different from each other at p b 0.001; F = 4 and t = 44.721 with 4 degrees of freedom.
lower but cell aggregation and spreading of cells were observed. Cell aggregation was observed in hemolymph of parasitized larvae and morphology of PL differed in hemolymph of unparasitized and parasitized larvae of H. armigera (Fig. 4). Morphometric analysis of the different hemocytes types found in the hemolymph of unparasitized and parasitized H. armigera larvae is shown in the Tables 2A and B, respectively. Histological study of larval midgut of H. armigera midgut parasitized by C. blackburni In the present investigation, the midgut of fourth instar unparasitized larvae of H. armigera had many histological differences compared to parasitized larvae. In unparasitized larvae, the diameter of midgut was larger than that observed in parasitized insects and is lined with a prominent peritrophic membrane (Fig. 5A). The columnar epithelial cells with a granular cytoplasm are seen with the nuclei located centrally or distally. The basal layer of the epithelial cells rested on the basement membrane while the tip of the cell was toward the lumen (Fig. 5A). Large spongy flask-shaped cells, also referred to as goblet cells, are seen interspersed between the columnar cells and open into the lumen of the midgut (Fig. 5A). Regenerative cells are observed in small groups at the base of the epithelial cells. In contrast, the peritrophic membrane is not observed in parasitized larvae and, in general, shows the destruction of epithelial cells (Fig. 5B). There is a complete dissolution of midgut layers with the columnar epithelial cells detached completely (Fig. 5B). The nuclei of the columnar epithelial cells migrate toward the apex and eventually disintegrate. In addition, no demarcation of goblet and regenerative cells were seen (Fig. 5B). The columnar epithelial cells become loose, damaged, shrunk, and are distorted completely with the cytoplasmic residues scattered with no distinct cell boundaries. Disruption of microvilli of the columnar cells and formation of vacuoles in the lumen were also observed. Discussion In recent years, crop protection based on appropriate use of ecofriendly biological control agents has been recognized as a valuable
A
tool in sustainable and stable pest management programs. In light of this understanding, extensive work has been done on various species of parasitoids implicated as effective biocontrol agents (Mustafizur, 1970; Thompson, 1982; Powell, 1989; Ohnuma and Kainoh, 1992; Bell et al., 2000; Shi et al., 2002). The present study was initiated to understand the effects of parasitization by the endoparasitic wasp, C. blackburni on larvae of H. armigera with focus on food consumption, growth, and development. Being an egg-larval parasitoid, C. blackburni oviposit on the eggs of H. armigera and then develop a complex and unique association with the growing host larvae deriving nutrition from the host hemolymph. As a result, parasitized larvae consume less food affecting its growth and development. This may be attributed partly to the space occupied by the developing parasitoid larvae within the host hemocoel restricting the possibility of increase in size of the host. Consequently, this affects growth as observed in the present study with the unparasitized larva increasing in size and weight during the last 3 instars while the parasitized larvae had much lower weight gain and food consumption rates. Jackson et al. (1978) reported that the first 2 instars of C. blackburni feed on the hemolymph of the pink bollworm, Pectinophora gossypiella, while the 3rd instar damages the larval gut leading to less uptake of food. Parasitization affecting growth and development of the host insect has been reported in several host-parasite interactions, viz., Apantales rubecula (Mustafizur, 1970), Hyposoter exiguae (Thompson, 1982), Microplitis demolitor and M.croceipes (Powell, 1989), Ascogaster retinulates (Ohnuma and Kainoh, 1992), and Hyposoter didymator and Chelonus iniatus (Morales et al., 2007). It has been suggested that parasitization results in developmental arrest of the host to divert nutrients to support parasite development. Hemocytes play a vital role in defense mechanisms and are important indicators for growth and metamorphosis of insects. Physiological mechanisms of phagocytosis, encapsulation, and other related defense mechanisms primarily depend upon the availability of circulating immune cells (Sanjayan et al., 1996). The total number of circulating hemocytes in parasitized larvae of H. armigera was significantly reduced due to directly induced immune suppression. This is reflected in the reduction in numbers of PLs and SPs, which plays a major role in cellular immune responses. In parasitized larvae, the PLs tend to aggregate forming multiple layers as a result of which cell morphology changes. Six types of hemocytes were observed in hemolymph of 3rd instar of H. armigera, which were severely affected due to parasitization. The decrease in THC in parasitized insects indicates a severe and drastic decrease in the proportion of available metabolically active cells and reduces the ability of insect's defense system. Physiological mechanisms of phagocytosis, encapsulation, and other related defense mechanisms primarily depend upon the availability of circulatory immune cells (Sanjayan et al., 1996) and declined number of THC in insects showed an adverse effect on its growth and development (Ahmad, 1995).
B
Fig. 4. Cell aggregation and morphology of plasmatocytes (PL) in hemolymph of unparasitized (A) and parasitized Helicoverpa armigera (B).
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Table 2 Morphometric analysis of the different hemocytes types found in the hemolymph of unparasitized (A) and parasitized (B) H. armigera larvae.
A C.D. (μm) N.D. (μm)
PR
PL (spherical)
PL (oval)
GR (large)
GR (small)
SP
AD
OE
Max Min Max Min
3.42 ± 0.88 1.84 ± 0.47 2.64 ± 0.68 2.35 ± 0.60 5.43 ± 0.l3 4.90 ± 0.32 0.52 ± 0.04
3.83 ± 0.98 1.66 ± 0.43 2.77 ± 0.71 1.85 ± 0.47 5.93 ± 3.95 4.20 ± 0.28 1.73 ± 3.67
2.57 ± 0.81 1.51 ± 0.48 1.64 ± 0.51 2.35 ± 0.74 3.26 ± 0.32 3.12 ± 0.31 0.13 ± 0.01
4.35 ± 1.30 3.68 ± 1.10 2.31 ± 0.73 2.66 ± 0.84 12.68 ± 1.81 4.85 ± 0.48 7.82 ± 1.33
0.70 ± 0.24 1.78 ± 0.63 0.82 ± 0.29 1.26 ± 0.44 1.21 ± 0.15 0.85 ± 0.10 0.35 ± 0.05
4.32 ± 1.1 4.20 ± 1.0 1.93 ± 0.4 1.86 ± 0.48 14.25 ± 0.95 2.83 ± 0.18 11.42 ± 0.77
5.31 ± 1.30 3.11 ± 0.80 1.12 ± 0.29 1.11 ± 0.28 13.93 ± 0.92 0.98 ± 0.006 12.94 ± 0.91
17.10 ± 7.64 14.27 ± 6.38 4.45 ± 1.99 4.30 ± 1.92 193.11 ± 38.35 15.02 ± 3.00 178.09 ± 35.35
Max Min Max Min
1.77 ± 0.45 1.90 ± 0.49 1.88 ± 0.48 1.49 ± 0.38 2.65 ± 0.20 2.24 ± 0.14 0.40 ± 0.06
1.78 ± 0.46 3.99 ± 1.03 1.80 ± 0.46 2.36 ± 0.61 6.56 ± 0.32 3.40 ± 0.22 3.16 ± 0.1
1.60 ± 0.50 2.12 ± 0.67 1.77 ± 0.56 1.59 ± 0.50 2.71 ± 0.27 2.23 ± 0.22 0.48 ± 0.005
4.52 ± 1.40 2.99 ± 0.94 1.50 ± 0.47 1.35 ± 0.42 12.72 ± 0.23 4.85 ± 0.16 7.87 ± 0.04
1.18 ± 0.41 0.89 ± 0.31 1.30 ± 0.46 1.13 ± 0.40 1.21 ± 0.14 0.85 ± 0.10 0.36 ± 0.04
2.86 ± 0.73 4.13 ± 0.37 1.45 ± 0.37 1.24 ± 0.32 9.60 ± 0.95 1.43 ± 0.1 8.17 ± 0.85
2.01 ± 0.52 1.89 ± 0.49 1.07 ± 0.27 1.04 ± 0.26 3.00 ± 0.20 0.88 ± 0.05 2.12 ± 0.15
5.83 ± 2.61 3.30 ± 1.47 3.76 ± 1.68 3.76 ± 1.68 16.34 ± 3.26 11.09 ± 2.21 5.25 ± 1.05
C.A. (μm2) N.A. (μm2) Cy.A. (μm2) B C.D. (μm) N.D. (μm) C.A. (μm2) N.A. (μm2) Cy.A. (μm2)
Values are presented as mean of experiments ± SEM. C.D. = cellular diameter, N.D. = nuclear diameter, C.A. = cellular area, N.A. = nuclear area, Cy.A. = cytoplasmic area.
Hence, hemocyte can be used as an indicator for change and hemocyte based information may be used to taking preventative measures to save the economically important insects and control the insect pests. Histological studies on the midgut of parasitized larvae of H. armigera show the destruction of the peritrophic membrane besides extensive damage to the midgut epithelial cells. The hypertrophied columnar cells release granular material into the lumen as well as disruption of microvilli prevent the active transport of K+ ions from the hemolymph to the gut lumen (Anderson and Harvey, 1966; Moffett, 1979; Harvey et al., 1983; Zeiske et al., 2000), thereby resulting in poor uptake of nutrients by the parasitized larvae (Barbeta et al., 2008). As a result of parasitization, similar damage to columnar cells have been observed in Anticarsia gemmatalis (Matos et al., 1999), S. litura (Im et al., 1988), Autographa
californica (Keddie et al., 1989), Lymantria dispar (Adams et al., 1994), S. exigua (Flipsen et al., 1995; Knebel-Morsdorf et al., 1996), Agrotis segetum (Oballe et al., 1996), Zethenia rufescentaria (Lin et al., 1999), and H. armigera (Kumari and Singh, 2013). Circulating hemocytes (blood cells) play a vital role in defense mechanisms against microbes and parasitoids in the hemocoel. Hemocytes are vital components of the insect immune system and are biochemically very sensitive having multiple functions such as nodule formation, phagocytosis, encapsulation, synthesis, and transport of nutrients and hormones for proper growth and wound healing (Figueiredo et al., 2006). In the last 250 years, several studies on hemocytes and their role in insect immunity have been described (Swammerdam, 1758; Cuenot, 1897; Wigglesworth, 1939; Jones, 1959; Gupta, 1985; Pandey et al., 2003;
A
RC
ML
ME
BM
PM S C
L
GC
BM BB
ME
CC PM
L BM
B
L
RC L
BM
RC
Fig. 5. Light microscopy of unparasitized (A) and parasitized (B) midgut section of Helicoverpa armigera. Various parts viz. Midgut epithelium (ME), lumen (L), peritrophic membrane (PM), basement membrane (BM) columnar cells (CC), goblet cells (GC). *Cavity of Globet cell, brush border (BB) of the columnar cells, are as labeled. Muscular layer (ML) stem cells (SC) located along the basement membrane (BM) between columnar and goblet cells (GC) have been shown.
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Pandey and Tiwari, 2011, 2012). In the present study, we observe that C. blackburni have a unique and complex association with their host which develops in the hemocoel and feed on hemolymph of host larvae thereby deriving nutrients from the host. This results in damage to the midgut cells and consequent damage to the digestive system of H. armigera eventually leading to death. Conclusions In the present study, we undertook a hematological and histological approach to investigate the effect of C. blackburni on growth and development in the larvae of H. armigera. The results suggest that C. blackburni retards growth and development of H. armigera with drastic reduction in food consumption besides affecting the midgut cells that eventually leads to death. This study provides an insight where important changes take place due to parasitism in H. armigera larvae and suggest that C. blackburni could be useful as an effective biocontrol agent with other known pest controlling methods. Acknowledgments YS thanks to UGC for Research Fellowship and VVD is thankful to the Science and Engineering Research Board, Department of Science and Technology, Government of India, New Delhi, for funding under Young Scientist Scheme (SB/YS/LS-260/2013). Technical assistance of Miss Jyoti Chintalchere and Mr. Sachin Lakare is also acknowledged. References Adams, J.R., Sheppard, C.A., Shapiro, M., Tompkins, G.J., 1994. Light and electron microscopic investigations on the histopathology of the midgut of gypsy moth larvae infected with LdMNPV plus a fluorescent brightener. J. Invertebr. Pathol. 64, 156–159. Ahmad, S., 1995. Oxidative stress from environmental pollutants. Arch. Insect Biochem. 29, 135–157. Anderson, N.E., Harvey, W.R., 1966. Active transport in the Cecropia midgut II. Fine structure of the midgut epithelium. J. Cell Biol. 31, 107–134. Andrejko, M., Mizerska-Dudka, M., 2012. Effect of Pseudomonas aeruginosa elastase B on level and activity of immune proteins/peptides of Galleria mellonella hemolymph. J. Insect Sci. 12, 88. Barbeta, B., Marshal, A.T., Gillon, A., Craik, D., Anderson, M., 2008. Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proc. Natl. Acad. Sci. U. S. A. 105, 1221–1225. Beckage, N.E., Riddiford, L.M., 1978. Developmental interactions between the tobacco hornworm Manduca sexta and its braconid parasite Apanteles congregatus. Entomol. Exp. Appl. 23, 139–151. Bell, H.A., Marris, G.C., Bell, J., Edwards, J.P., 2000. The biology of Meteorus gyrator (Hymenoptera: Braconidae), a solitary endoparasitoid of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae). Bull. Entomol. Res. 90, 299–308. Couchman, J.R., King, P.E., 1979. Effect of the parasitoid Diaeretiella rapae on the feeding rate of its host Brevicoryne brassicae. Entomol. Exp. Appl. 25, 9–15. Cuenot, L., 1897. Les globules sanguins et les organes lymphoides des invertebres (Revue critique et nouvelles recherches). Arch. Ann. Microbiol. 1, 153–192. Danyk, T., Mackauer, M., Johnson, D., 2005. Reduced food consumption in the grasshopper Melanoplus sanguinipes (Orthoptera: Acrididae) parasitized by Blaesoxipha atlanis (Diptera: Sarcophagidae). Can. Entomol. 137, 356–366. Duodu, Y.A., Antoh, F.F., 1984. Effects of parasitism by Apanteles sagax (Hym.: Braconidae) on growth, food consumption and food utilization in Sylepta degogata larvae (Lep.: Pyralidae). Entomophaga 29, 63–71. Figueiredo, M.B., Castro, D.P., Nogueira, N.F.S., Garcia, E.S., Azambuja, P., 2006. Cellular immune response in Rhodnius prolixus: Role of ecdysone in haemocyte phagocytosis. J. Insect Physiol. 52, 711–716. Flipsen, J.T.M., Martens, J.W.M., Van Oers, M.M., Vlak, J.M., Vanlet, W.M., 1995. Passage of the Autographa californica nucleopolyhedrosis virus through the midgut epithelium of Spodoptera exigua larvae. Virology 208, 328–335. Grossniklaus-Burgin, C., Wyler, T., Pfister, R., Lanzrein, B., 1994. Biology and morphology of the parasitoid Chelonus inanitus (Braconidae, Hymenoptera) and effects on the development of its host Spodoptera littoralis (Noctuidae, Lepidoptera). Invertebr. Reprod. Dev. 25, 143–158. Gupta, A.P., 1985. Cellular Elements in the Hemolymph. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology Biochemistry Pharmacology. Pergamon Press, New York, pp. 400–451. Harvey, W.R., Cioffi, M., Dow, J., Wolferberger, M.G., 1983. Potassium ions transport ATPase in insect epithelia. J. Exp. Biol. 106, 91–117. Im, D.J., Shephard, B.M., Aguda, R.M., 1988. Pathogenicity and histopathology of a nuclear polyhedrosis virus of Spodoptera litura (Fab.). Insect Sci. Appl. 9, 539–542.
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Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Parasitism by Chelonus blackburni (Hymenoptera) affects food consumption and development of Helicoverpa armigera (Lepidoptera) and cellular architecture of the midgut Yogita Sanap a, Vishal V. Dawkar b, Ashok P. Giri b, Avalokiteswar Sen c, Radhakrishna S. Pandit a,⁎ a b c
Department of Zoology, Savitribai Phule Pune University, Ganeshkhind, Pune, MS, India Plant Molecular Biology Unit, Division of Biochemical Sciences, CSIR–National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, MS 411008, India Laboratory of Entomology, Division of Organic Chemistry, CSIR–National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, MS 411008, India
a r t i c l e
i n f o
Article history: Received 3 September 2015 Revised 16 October 2015 Accepted 2 November 2015 Available online 26 November 2015 Keywords: Biocontrol Helicoverpa armigera Chelonus blackburni Histology Total hemocyte counts
a b s t r a c t Biological control agents are vital components of an integrated pest management strategy, and this is frequently referred to as natural control. Natural enemies of insect pests include predators, parasitoids, and pathogens. Among them, a parasitoid, Chelonus blackburni (Cameron), was found to be the best biological control agent for the polyphagous pest, Helicoverpa armigera (Hübner). C. blackburni alters the feeding performance of H. armigera larvae upon parasitism and as a result severely affects growth and development. Moreover, it shortens the feeding period of H. armigera and increases mortality. Furthermore, total hemocyte count (THC) was significantly decreased in parasitized larvae than control. Parasitized H. armigera had 26% less number of blood cells compared to healthy larvae. Histological studies showed that the structure of midgut of H. armigera is drastically affected by C. blackburni leading to reduced food consumption, which ultimately led to larval death. The present study provides an insight to changes involved in H. armigera due to parasitism by C. blackburni, a parasite that could be used as an effective biocontrol agent to manage H. armigera. © 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Introduction The cotton bollworm, Helicoverpa armigera (Hübner), is an important polyphagous pest feeding on more than 181 cultivated and wild species spread across 45 families, including economically important crops such as cotton, pigeon pea, chickpea, and sorghum as well as horticultural and ornamental crops (Manjunath et al., 1989; Sarate et al., 2012). The continuous availability of host crops, often planted as monocultures, and the lack of crop rotation greatly contribute to the maintenance of high population levels of H. armigera and the consequent damage to crops. Synthetic pesticides have primarily been used to control this pest over the past several decades; however, widespread and indiscriminate use of these groups of chemicals has resulted in the development of resistant strains inviting still increasing doses of synthetic pesticides, particularly in developing countries. Such large-scale uses of synthetic pesticides have ecological implications, including environmental pollution and crop residues (Smagghe et al., 1999; Mishra et al., 2015). Parasitoids are an important component in integrated pest management (IPM) programs. It has generated a great deal of interest due to ⁎ Corresponding author at: Department of Zoology, Savitribai Phule Pune University, Pune, MS 411008, India. Tel.: +91 20 25601436; fax: +91 20 25690617. E-mail address: [email protected] (R.S. Pandit).
their ability to suppress pest populations and thus help diminish the damage caused by agricultural pests. Often, parasitism may also change the host feeding behavior (Lewis and Burton, 1970; Powell, 1989; Morales et al., 2007). Common feeding patterns and the amount of food consumed differ between infected and normal larvae, and usually, it has been observed that parasitized larvae eat very modest amounts of food (Parker and Pinnell, 1973; Beckage and Riddiford, 1978; Couchman and King, 1979; Duodu and Antoh, 1984; Danyk et al., 2005; Morales et al., 2007). Among the promising parasitoids, the braconid Chelonus blackburni (Cameron) is a key parasitoid used in biological control or IPM for controlling H. armigera. It is a solitary endoparasitoid that completely depends upon its host larva for food and shelter to complete its development from embryo to the third instar larva. Subsequently, it emerges from the dead host and spins a silken cocoon in the vicinity (Grossniklaus-Burgin et al., 1994). With this background and efforts to look at alternative insect pest control measures, we studied the effect of parasitization by C. blackburni on food consumption and development in H. armigera. This study shows that parasitized larvae did not grow normally as reflected by their reduced food consumption. Analysis of hemolymph from parasitized and unparasitized H. armigera larvae showed significant differences in total hemocyte counts as well as in the numbers of the different hemocytes. In addition, there were drastic changes in cell aggregation and shapes of the different hemocytes. Furthermore, histological studies on the midgut
http://dx.doi.org/10.1016/j.aspen.2015.11.005 1226-8615/© 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
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of parasitized larvae of H. armigera show the destruction of the peritrophic membrane besides extensive damage to the midgut epithelial cells. Materials and methods
morphological characters as described by Gupta (1985). Further, to determine the DHC, cell categories were counted in 200 cells chosen from random areas of the stained hemolymph smear by a laboratory cell counter.
Rearing of H. armigera and parasitism by C. blackburni
Histology
Larvae of H. armigera were collected from fields of Mahatma Phule Krishi Vidyapeeth, Rahuri, India. They were maintained on a chickpea based artificial diet (AD) (Nagarkatti and Prakash, 1974) in the laboratory under controlled conditions (temperature: 27 °C; humidity: 60% and photoperiod: 16:8). To ensure greater genetic homogeneity among test populations, insects were maintained on AD for three generations. H. armigera egg sheets (~ 300 eggs) were exposed to adults of C. blackburni in a parasitoid oviposition chamber for 24 h. After hatching, neonates from each group (control and parasitized) were taken for further experiments. Food consumption and weight of larvae were recorded every 2 days. The experiment was terminated on day 22 since by that time unparasitized larvae of H. armigera attain pupal stage while the parasitized larvae die as indicated by the spinning of silk cocoons in the vicinity. The numbers of well-formed cocoons were recorded. All the above experiments were replicated thrice.
Parasitized and unparasitized H. armigera larvae were anesthetized with chloroform and dissected under a microscope to isolate the midgut. The collected specimens were immediately put in alcoholic Boüins and kept for 2 h at room temperature (~ 24 °C), following which they were dehydrated in a graded series of alcohols and finally cleared in xylene. The material was embedded in paraffin wax, and 6 μm sections were obtained with the help of a microtome and stained with hematoxylin–eosin. Morphological analysis was carried out using an OLYMPUS BX-49 light microscope.
Hemolymph collection and hematology
Results
Prior to hemolymph collection, the insects were chilled for 15 min at 4 °C. Hemolymph samples were obtained by puncturing the larval abdomen with a sterile needle. The outflowing hemolymph was immediately transferred into sterile and chilled Eppendorf tubes containing a few crystals of phenylthiourea (PTU) to prevent melanization and stored at −80 °C until used (Andrejko and Mizerska-Dudka, 2012). For hematological experiments, hemolymph (10 μL) was stained with Giemsa (4%). Free hemocytes were counted using a hemocytometer with improved double Neubauer ruling under a phase contrast microscope.
Effect of parasitism by C. blackburni on H. armigera
Differential hemocyte counts (DHC) For smear slide preparation, a small drop of larval hemolymph was obtained by clipping a proleg. The drop was then smeared into a thin film, air-dried, and fixed in absolute methanol for 4 min before staining for 2-3 min in Giemsa (4%), air-dried, and subsequently washed with distilled water. The rinsed slide was dried and then mounted in DPX. Observations were made under a phase contrast microscope at 40X magnification and images were acquired with a digital camera. The different types of hemocytes were identified using established
Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparisons test. Data points were considered significant at p b 0.05, p b 0.01, and p b 0.0001.
Feeding in larvae of H. armigera parasitized by C. blackburni is greatly reduced with the larvae upon reaching the fourth instar develop into a pre-pupa from which a freshly molted 3rd instar larva of C. blackburni emerges. In comparison, unparasitized larvae continue their development until they reach the pupal stage. In unparasitized larvae, normal food consumption and normal growth and development of larvae were observed. Consequently, there were significant differences in growth as reflected in the amount of food consumed and larval mass between unparasitized and parasitized larvae (Fig. 1A and B; p b 0.01 and p b 0.005, respectively). Food consumption increased gradually in unparasitized larvae of H. armigera. However, in parasitized larvae, initially, the food consumption increased gradually up to day 12 but later decreased from day 14 (Fig. 1A). The mean food consumed by parasitized larvae on day 12 was 89 ± 0.5 mg which was significantly less than the food consumed by unparasitized larvae (202 ±10.46 mg; p b 0.01). Interestingly, the amount of food consumed by parasitized larvae was higher than unparasitized larvae till day 12 (89.5 ± 5.01 mg), which reduced to 53.0 ± 4.73 mg on day 14. This was probably due to
Fig. 1. Development and food consumption of unparasitized and parasitized Helicoverpa armigera by Chelonus blackburni. (A) Larval mass gain by unparasitized (black bars) and parasitized (hollow bars) H. armigera. Graph shows average mass from each set of 15 larvae. Larvae were critically weighed on every fifth day. (B) Food consumption data of larvae. Standard error means are indicated. * and ** indicate that values are significantly different from each other at p b 0.01 and p b 0.005, respectively.
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up to three times. No weight gain was observed in parasitized larvae from day 12 onward.
Unparasitized Parasitized
Hematology
Area (µm2)
40 30 20 10 0
Fig. 2. Morphometric analysis of different types of hemocytes found in the hemolymph of unparasitized and parasitized Helicoverpa armigera larvae. Standard error means are indicated.
the larvae having entered the pre-pupal stage. The overall food consumed by parasitized H. armigera larvae up to day 14 was only 34.21% of the total food consumed by unparasitized larvae. Studies related to the effect of parasitism on weight gain by larvae of H. armigera indicate that although there were no significant differences in larval body mass of H. armigera till day 8, however, on subsequent days, differences were significant between the control and parasitized larvae of H. armigera, respectively (p b 0.01 and p b 0.005) (Fig. 1B). Unparasitized larvae of H. armigera grew slowly during the initial 10 days, until they molt into the 3rd instar, following which their weight and food consumption increased rapidly. On day 12, the weight of unparasitized larvae was two times more than that of parasitized larvae, and once they had molted into 4th instar on day 14, the difference increased
A
B
The effect of parasitism by C. blackburni on larvae of H. armigera revealed prominent changes in the total hemocyte count (THC). There was a significant decrease in THC in parasitized larvae of H. armigera compared to unparasitized larvae. The number of cells counted per microliter at the end of day 12 was significantly lower in parasitized larvae (2440 ± 104) than in unparasitized larvae [(9143 ± 410); (p b 0.0001; F = 1.12, df = 4, t = 44.28); (Fig. 2)]. Six primary types of hemocytes, viz. prohemocytes (PR), plasmatocytes (PL), granulocytes (GR), sperulocytes (SP), adipohemocytes (AD), and oenocytoids (OE), were observed in hemolymph of parasitized and unparasitized larvae of H. armigera (Fig. 3). In case of PR, the cells were either round, oval, or elliptical in unparasitized larvae while in parasitized larvae, the PR was abnormal, and the cytoplasm occupied most of cell forming a very thin layer around the nucleus. PLs are highly polymorphic cells with their shapes varying from being spindle shaped to one with a very pointed end. In unparasitized larvae, these cells were oval with a large centrally located nucleus; however, this type of shape was not observed in parasitized larvae. GRs are also variable in shape and size and are easily distinguished by the presence of vacuoles in the cytoplasm, but this was not observed in the parasitized larvae of H. armigera. SPs, ADs, and OEs show radical differences in parasitized larval hemolymph. There was a drastic difference in DHC of parasitized and control larvae of H. armigera (Table 1). The larvae of H. armigera parasitized by C. blackburni reveal a significant increase in percentages of PRs and GRs, while in unparasitized larvae, the percentage of PLs and SPs significantly increased (p b 0.001; F = 4 and t = 44.72). However, there were no significant differences in percentage of ADs and OEs between parasitized and unparasitized larvae. In parasitized larvae of H. armigera, the amount of PLs was
A
1. Prohemocyte A
B
2. Plasmatocyte A
B
5. Adipohaemocyte
B
4. Sperulocyte
3. Granulocyte A
B
A
B
6. Oenocytoid
Fig. 3. Morphology of haemocytes viz. prohemocyte, plasmatocyte, granulocyte, sperulocyte, adipohemocyte, and oenocytoid of unparasitized (A) and parasitized (B) Helicoverpa armigera after Giemsa staining.
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Table 1 The percentage of differential hemocyte count (DHC) in the parasitized and unparasitized larvae of Helicoverpa armigera. Cellular type (%) H. armigera
PR
PL
GR
SP
AD
OE
Unparasitized Parasitized
46⁎ 55.39
15.36⁎ 12.85
14.60⁎ 21.42
26.44⁎ 8.57
1.91⁎ 1.18
0.33⁎ 0.23
⁎ Values are significantly different from each other at p b 0.001; F = 4 and t = 44.721 with 4 degrees of freedom.
lower but cell aggregation and spreading of cells were observed. Cell aggregation was observed in hemolymph of parasitized larvae and morphology of PL differed in hemolymph of unparasitized and parasitized larvae of H. armigera (Fig. 4). Morphometric analysis of the different hemocytes types found in the hemolymph of unparasitized and parasitized H. armigera larvae is shown in the Tables 2A and B, respectively. Histological study of larval midgut of H. armigera midgut parasitized by C. blackburni In the present investigation, the midgut of fourth instar unparasitized larvae of H. armigera had many histological differences compared to parasitized larvae. In unparasitized larvae, the diameter of midgut was larger than that observed in parasitized insects and is lined with a prominent peritrophic membrane (Fig. 5A). The columnar epithelial cells with a granular cytoplasm are seen with the nuclei located centrally or distally. The basal layer of the epithelial cells rested on the basement membrane while the tip of the cell was toward the lumen (Fig. 5A). Large spongy flask-shaped cells, also referred to as goblet cells, are seen interspersed between the columnar cells and open into the lumen of the midgut (Fig. 5A). Regenerative cells are observed in small groups at the base of the epithelial cells. In contrast, the peritrophic membrane is not observed in parasitized larvae and, in general, shows the destruction of epithelial cells (Fig. 5B). There is a complete dissolution of midgut layers with the columnar epithelial cells detached completely (Fig. 5B). The nuclei of the columnar epithelial cells migrate toward the apex and eventually disintegrate. In addition, no demarcation of goblet and regenerative cells were seen (Fig. 5B). The columnar epithelial cells become loose, damaged, shrunk, and are distorted completely with the cytoplasmic residues scattered with no distinct cell boundaries. Disruption of microvilli of the columnar cells and formation of vacuoles in the lumen were also observed. Discussion In recent years, crop protection based on appropriate use of ecofriendly biological control agents has been recognized as a valuable
A
tool in sustainable and stable pest management programs. In light of this understanding, extensive work has been done on various species of parasitoids implicated as effective biocontrol agents (Mustafizur, 1970; Thompson, 1982; Powell, 1989; Ohnuma and Kainoh, 1992; Bell et al., 2000; Shi et al., 2002). The present study was initiated to understand the effects of parasitization by the endoparasitic wasp, C. blackburni on larvae of H. armigera with focus on food consumption, growth, and development. Being an egg-larval parasitoid, C. blackburni oviposit on the eggs of H. armigera and then develop a complex and unique association with the growing host larvae deriving nutrition from the host hemolymph. As a result, parasitized larvae consume less food affecting its growth and development. This may be attributed partly to the space occupied by the developing parasitoid larvae within the host hemocoel restricting the possibility of increase in size of the host. Consequently, this affects growth as observed in the present study with the unparasitized larva increasing in size and weight during the last 3 instars while the parasitized larvae had much lower weight gain and food consumption rates. Jackson et al. (1978) reported that the first 2 instars of C. blackburni feed on the hemolymph of the pink bollworm, Pectinophora gossypiella, while the 3rd instar damages the larval gut leading to less uptake of food. Parasitization affecting growth and development of the host insect has been reported in several host-parasite interactions, viz., Apantales rubecula (Mustafizur, 1970), Hyposoter exiguae (Thompson, 1982), Microplitis demolitor and M.croceipes (Powell, 1989), Ascogaster retinulates (Ohnuma and Kainoh, 1992), and Hyposoter didymator and Chelonus iniatus (Morales et al., 2007). It has been suggested that parasitization results in developmental arrest of the host to divert nutrients to support parasite development. Hemocytes play a vital role in defense mechanisms and are important indicators for growth and metamorphosis of insects. Physiological mechanisms of phagocytosis, encapsulation, and other related defense mechanisms primarily depend upon the availability of circulating immune cells (Sanjayan et al., 1996). The total number of circulating hemocytes in parasitized larvae of H. armigera was significantly reduced due to directly induced immune suppression. This is reflected in the reduction in numbers of PLs and SPs, which plays a major role in cellular immune responses. In parasitized larvae, the PLs tend to aggregate forming multiple layers as a result of which cell morphology changes. Six types of hemocytes were observed in hemolymph of 3rd instar of H. armigera, which were severely affected due to parasitization. The decrease in THC in parasitized insects indicates a severe and drastic decrease in the proportion of available metabolically active cells and reduces the ability of insect's defense system. Physiological mechanisms of phagocytosis, encapsulation, and other related defense mechanisms primarily depend upon the availability of circulatory immune cells (Sanjayan et al., 1996) and declined number of THC in insects showed an adverse effect on its growth and development (Ahmad, 1995).
B
Fig. 4. Cell aggregation and morphology of plasmatocytes (PL) in hemolymph of unparasitized (A) and parasitized Helicoverpa armigera (B).
Y. Sanap et al. / Journal of Asia-Pacific Entomology 19 (2016) 65–70
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Table 2 Morphometric analysis of the different hemocytes types found in the hemolymph of unparasitized (A) and parasitized (B) H. armigera larvae.
A C.D. (μm) N.D. (μm)
PR
PL (spherical)
PL (oval)
GR (large)
GR (small)
SP
AD
OE
Max Min Max Min
3.42 ± 0.88 1.84 ± 0.47 2.64 ± 0.68 2.35 ± 0.60 5.43 ± 0.l3 4.90 ± 0.32 0.52 ± 0.04
3.83 ± 0.98 1.66 ± 0.43 2.77 ± 0.71 1.85 ± 0.47 5.93 ± 3.95 4.20 ± 0.28 1.73 ± 3.67
2.57 ± 0.81 1.51 ± 0.48 1.64 ± 0.51 2.35 ± 0.74 3.26 ± 0.32 3.12 ± 0.31 0.13 ± 0.01
4.35 ± 1.30 3.68 ± 1.10 2.31 ± 0.73 2.66 ± 0.84 12.68 ± 1.81 4.85 ± 0.48 7.82 ± 1.33
0.70 ± 0.24 1.78 ± 0.63 0.82 ± 0.29 1.26 ± 0.44 1.21 ± 0.15 0.85 ± 0.10 0.35 ± 0.05
4.32 ± 1.1 4.20 ± 1.0 1.93 ± 0.4 1.86 ± 0.48 14.25 ± 0.95 2.83 ± 0.18 11.42 ± 0.77
5.31 ± 1.30 3.11 ± 0.80 1.12 ± 0.29 1.11 ± 0.28 13.93 ± 0.92 0.98 ± 0.006 12.94 ± 0.91
17.10 ± 7.64 14.27 ± 6.38 4.45 ± 1.99 4.30 ± 1.92 193.11 ± 38.35 15.02 ± 3.00 178.09 ± 35.35
Max Min Max Min
1.77 ± 0.45 1.90 ± 0.49 1.88 ± 0.48 1.49 ± 0.38 2.65 ± 0.20 2.24 ± 0.14 0.40 ± 0.06
1.78 ± 0.46 3.99 ± 1.03 1.80 ± 0.46 2.36 ± 0.61 6.56 ± 0.32 3.40 ± 0.22 3.16 ± 0.1
1.60 ± 0.50 2.12 ± 0.67 1.77 ± 0.56 1.59 ± 0.50 2.71 ± 0.27 2.23 ± 0.22 0.48 ± 0.005
4.52 ± 1.40 2.99 ± 0.94 1.50 ± 0.47 1.35 ± 0.42 12.72 ± 0.23 4.85 ± 0.16 7.87 ± 0.04
1.18 ± 0.41 0.89 ± 0.31 1.30 ± 0.46 1.13 ± 0.40 1.21 ± 0.14 0.85 ± 0.10 0.36 ± 0.04
2.86 ± 0.73 4.13 ± 0.37 1.45 ± 0.37 1.24 ± 0.32 9.60 ± 0.95 1.43 ± 0.1 8.17 ± 0.85
2.01 ± 0.52 1.89 ± 0.49 1.07 ± 0.27 1.04 ± 0.26 3.00 ± 0.20 0.88 ± 0.05 2.12 ± 0.15
5.83 ± 2.61 3.30 ± 1.47 3.76 ± 1.68 3.76 ± 1.68 16.34 ± 3.26 11.09 ± 2.21 5.25 ± 1.05
C.A. (μm2) N.A. (μm2) Cy.A. (μm2) B C.D. (μm) N.D. (μm) C.A. (μm2) N.A. (μm2) Cy.A. (μm2)
Values are presented as mean of experiments ± SEM. C.D. = cellular diameter, N.D. = nuclear diameter, C.A. = cellular area, N.A. = nuclear area, Cy.A. = cytoplasmic area.
Hence, hemocyte can be used as an indicator for change and hemocyte based information may be used to taking preventative measures to save the economically important insects and control the insect pests. Histological studies on the midgut of parasitized larvae of H. armigera show the destruction of the peritrophic membrane besides extensive damage to the midgut epithelial cells. The hypertrophied columnar cells release granular material into the lumen as well as disruption of microvilli prevent the active transport of K+ ions from the hemolymph to the gut lumen (Anderson and Harvey, 1966; Moffett, 1979; Harvey et al., 1983; Zeiske et al., 2000), thereby resulting in poor uptake of nutrients by the parasitized larvae (Barbeta et al., 2008). As a result of parasitization, similar damage to columnar cells have been observed in Anticarsia gemmatalis (Matos et al., 1999), S. litura (Im et al., 1988), Autographa
californica (Keddie et al., 1989), Lymantria dispar (Adams et al., 1994), S. exigua (Flipsen et al., 1995; Knebel-Morsdorf et al., 1996), Agrotis segetum (Oballe et al., 1996), Zethenia rufescentaria (Lin et al., 1999), and H. armigera (Kumari and Singh, 2013). Circulating hemocytes (blood cells) play a vital role in defense mechanisms against microbes and parasitoids in the hemocoel. Hemocytes are vital components of the insect immune system and are biochemically very sensitive having multiple functions such as nodule formation, phagocytosis, encapsulation, synthesis, and transport of nutrients and hormones for proper growth and wound healing (Figueiredo et al., 2006). In the last 250 years, several studies on hemocytes and their role in insect immunity have been described (Swammerdam, 1758; Cuenot, 1897; Wigglesworth, 1939; Jones, 1959; Gupta, 1985; Pandey et al., 2003;
A
RC
ML
ME
BM
PM S C
L
GC
BM BB
ME
CC PM
L BM
B
L
RC L
BM
RC
Fig. 5. Light microscopy of unparasitized (A) and parasitized (B) midgut section of Helicoverpa armigera. Various parts viz. Midgut epithelium (ME), lumen (L), peritrophic membrane (PM), basement membrane (BM) columnar cells (CC), goblet cells (GC). *Cavity of Globet cell, brush border (BB) of the columnar cells, are as labeled. Muscular layer (ML) stem cells (SC) located along the basement membrane (BM) between columnar and goblet cells (GC) have been shown.
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Pandey and Tiwari, 2011, 2012). In the present study, we observe that C. blackburni have a unique and complex association with their host which develops in the hemocoel and feed on hemolymph of host larvae thereby deriving nutrients from the host. This results in damage to the midgut cells and consequent damage to the digestive system of H. armigera eventually leading to death. Conclusions In the present study, we undertook a hematological and histological approach to investigate the effect of C. blackburni on growth and development in the larvae of H. armigera. The results suggest that C. blackburni retards growth and development of H. armigera with drastic reduction in food consumption besides affecting the midgut cells that eventually leads to death. This study provides an insight where important changes take place due to parasitism in H. armigera larvae and suggest that C. blackburni could be useful as an effective biocontrol agent with other known pest controlling methods. Acknowledgments YS thanks to UGC for Research Fellowship and VVD is thankful to the Science and Engineering Research Board, Department of Science and Technology, Government of India, New Delhi, for funding under Young Scientist Scheme (SB/YS/LS-260/2013). Technical assistance of Miss Jyoti Chintalchere and Mr. Sachin Lakare is also acknowledged. References Adams, J.R., Sheppard, C.A., Shapiro, M., Tompkins, G.J., 1994. Light and electron microscopic investigations on the histopathology of the midgut of gypsy moth larvae infected with LdMNPV plus a fluorescent brightener. J. Invertebr. Pathol. 64, 156–159. Ahmad, S., 1995. Oxidative stress from environmental pollutants. Arch. Insect Biochem. 29, 135–157. Anderson, N.E., Harvey, W.R., 1966. Active transport in the Cecropia midgut II. Fine structure of the midgut epithelium. J. Cell Biol. 31, 107–134. Andrejko, M., Mizerska-Dudka, M., 2012. Effect of Pseudomonas aeruginosa elastase B on level and activity of immune proteins/peptides of Galleria mellonella hemolymph. J. Insect Sci. 12, 88. Barbeta, B., Marshal, A.T., Gillon, A., Craik, D., Anderson, M., 2008. Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proc. Natl. Acad. Sci. U. S. A. 105, 1221–1225. Beckage, N.E., Riddiford, L.M., 1978. Developmental interactions between the tobacco hornworm Manduca sexta and its braconid parasite Apanteles congregatus. Entomol. Exp. Appl. 23, 139–151. Bell, H.A., Marris, G.C., Bell, J., Edwards, J.P., 2000. The biology of Meteorus gyrator (Hymenoptera: Braconidae), a solitary endoparasitoid of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae). Bull. Entomol. Res. 90, 299–308. Couchman, J.R., King, P.E., 1979. Effect of the parasitoid Diaeretiella rapae on the feeding rate of its host Brevicoryne brassicae. Entomol. Exp. Appl. 25, 9–15. Cuenot, L., 1897. Les globules sanguins et les organes lymphoides des invertebres (Revue critique et nouvelles recherches). Arch. Ann. Microbiol. 1, 153–192. Danyk, T., Mackauer, M., Johnson, D., 2005. Reduced food consumption in the grasshopper Melanoplus sanguinipes (Orthoptera: Acrididae) parasitized by Blaesoxipha atlanis (Diptera: Sarcophagidae). Can. Entomol. 137, 356–366. Duodu, Y.A., Antoh, F.F., 1984. Effects of parasitism by Apanteles sagax (Hym.: Braconidae) on growth, food consumption and food utilization in Sylepta degogata larvae (Lep.: Pyralidae). Entomophaga 29, 63–71. Figueiredo, M.B., Castro, D.P., Nogueira, N.F.S., Garcia, E.S., Azambuja, P., 2006. Cellular immune response in Rhodnius prolixus: Role of ecdysone in haemocyte phagocytosis. J. Insect Physiol. 52, 711–716. Flipsen, J.T.M., Martens, J.W.M., Van Oers, M.M., Vlak, J.M., Vanlet, W.M., 1995. Passage of the Autographa californica nucleopolyhedrosis virus through the midgut epithelium of Spodoptera exigua larvae. Virology 208, 328–335. Grossniklaus-Burgin, C., Wyler, T., Pfister, R., Lanzrein, B., 1994. Biology and morphology of the parasitoid Chelonus inanitus (Braconidae, Hymenoptera) and effects on the development of its host Spodoptera littoralis (Noctuidae, Lepidoptera). Invertebr. Reprod. Dev. 25, 143–158. Gupta, A.P., 1985. Cellular Elements in the Hemolymph. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology Biochemistry Pharmacology. Pergamon Press, New York, pp. 400–451. Harvey, W.R., Cioffi, M., Dow, J., Wolferberger, M.G., 1983. Potassium ions transport ATPase in insect epithelia. J. Exp. Biol. 106, 91–117. Im, D.J., Shephard, B.M., Aguda, R.M., 1988. Pathogenicity and histopathology of a nuclear polyhedrosis virus of Spodoptera litura (Fab.). Insect Sci. Appl. 9, 539–542.
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