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Micron 65 (2014) 62–68

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uantitative and ultrastructural changes in the haemocytes ofpodoptera littoralis (Boisd.) treated individually or in combinationith Spodoptera littoralis multicapsid nucleopolyhedrovirus

SpliMNPV) and azadirachtin

l-Sayed H. Shauruba, Afaf Abd El-Meguida, Nahla M. Abd El-Aziza,b,∗

Department of Entomology, Faculty of Science, Cairo University, Giza, EgyptCollege of Science and Art, Dammam University, Saudi Arabia

r t i c l e i n f o

rticle history:eceived 22 December 2013eceived in revised form 16 April 2014ccepted 22 April 2014vailable online 30 April 2014

eywords:podoptera littoralisucleopolyhedroviruszadirachtinotal haemocyte countaemocyte ultrastructure

a b s t r a c t

The total haemocyte count (THC) and the possible ultrastructural alterations induced in the haemocytesof the fourth larval instars of the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera:Noctuidae), 96 h post-feeding on a semi-synthetic diet, treated with the LC50 of Spodoptera littoralis mul-ticapsid nucleopolyhedrovirus (SpliMNPV) and the LC50 of azadirachtin alone, and the LC25 of SpliMNPVcombined with the LC25 of azadirachtin were studied and compared to the control. Single treatmentwith the virus and azadirachtin or combined treatment significantly decreased the THC compared to thecontrol. There are five types of haemocytes in S. littoralis: prohaemocytes, plasmatocytes, granulocytes,spherulocytes and oenocytoids. The most common symptoms in granulocytes and plasmatocytes, themain affected cell types, due to viral infection were the presence of virogenic stroma, peripheral disper-sion of the chromatin and disappearance of the nucleoli. However, the most common symptoms in thesetwo types of haemocytes due to treatment with azadirachtin were the presence of rough endoplasmicreticulum filled with fibrous materials, due to probably apoptosis, in their cisternae and disorganiza-tion of mitochondria (looped, vacuolated and swollen). In addition, the cytoplasm of granulocytes wasvacuolated with the appearance of autophagic lysosomes, while plasmatocytes showed ruptured cellmembrane and folded nuclear envelope. Combined treatment with the NPV and azadirachtin induced the

same pathological changes which were recorded from individual treatment with the virus or azadirachtinto the same haemocytes. It can be concluded that the change in the THC and ultrastructure of granulocytesand plasmatocytes may affect the cellular-mediated immune response in S. littoralis. Moreover, it seemslikely that mitochondria were the target site of azadirachtin, as they were affected in both granulocytesand plasmatocytes treated with azadirachtin alone or in combination with SpliMNPV.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

The Egyptian cotton leafworm, Spodoptera littoralis (Boisd.)Lepidoptera: Noctuidae) is the major cotton pest in Egypt due tohe frequent foliar damage that it causes to this crop. Moreover, itlso attacks other economically important crops such as cucumber,otato, okra and egg plants.

Haemocytes are an essential component of the invertebratennate immune system (Strand, 2008). Their close similarities toertebrate blood cells enable their use as a tractable model for

∗ Corresponding author. Tel.: +20 1008011402; fax: +20 235728843.E-mail address: nahlasalem97@yahoo.com (N.M. Abd El-Aziz).

ttp://dx.doi.org/10.1016/j.micron.2014.04.010968-4328/© 2014 Elsevier Ltd. All rights reserved.

immunological research. The quantification of haemocytes in thehaemolymph (invertebrate blood) is one way of assessing the effi-cacy of the immune response because the absolute number ofhaemocytes changes during the course of infection (Rivers et al.,2002; De Andrade et al., 2010) and the haemocyte count in thehaemolymph is strongly correlated with the capacity to encapsu-late or phagocytose parasites (Pandey and Tiwari, 2012).

Baculoviruses (Baculoviridae) are natural insecticidal agentsactive in a wide range of insect pests (Yang et al., 2012). The major-ity of baculoviruses used as biological control agents are in the

genus Nucleopolyhedrovirus (NPV). They are ideal tools of inte-grated pest management programs (IPM), highly specific to theirhost insects, safe to the environment, humans, plants and naturalenemies (Ahmad et al., 2011).

E.-S.H. Shaurub et al. / Micron 65 (2014) 62–68 63

Fig. 1. Total haemocyte count (THC) (cells/mm3) of Spodoptera littoralis fourth larval instars, as a result of single treatment with the LC50 of the entomopathogenic virusSpliMNPV (8.90 × 108 PIB/ml) and the LC50 of azadirachtin (23.15 ppm), and combined treatment with the LC25 of SpliMNPV (10.40 × 106 PIB/ml) and the LC25 of azadirachtin( iation

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9.95 ppm). The graph bar on the top of each column represents the standard devignificantly different from each other (P < 0.05) using one-way analysis of variancepliMNPV.

On the other hand, azadirachtin, a tetranortriterpenoid iso-ated from the seeds of the neem tree Azadirachta indica A. JussMeliaceae) is a well known insect growth regulator of plantrigin (Rembold et al., 1982). The potent and specific effects ofzadirachtin illustrate its potential in insect control (Kubo andlocke, 1982) and can be mixed with NPV in IPM (Zamora-Avilést al., 2013).

Although the biology of S. littoralis has received a lot of attention,he alterations in the number and ultrastructure of the haemocytesn response to the entomopathogenic viruses and botanicals are soar not characterized. Therefore, the present study aims to inves-igate the effect of S. littoralis multicapsid nucleopolyhedrovirusSpliMNPV) and azadirachtin, either alone or in combination, onhe total haemocyte count (THC) and ultrastructure in S. littoralisourth instar larvae.

. Materials and methods

.1. Insects

A susceptible strain of S. littoralis was established fromgg masses obtained from Faculty of Agriculture, Cairo Uni-ersity. Newly hatched larvae were transferred into plasticoxes (18 cm × 12 cm × 10 cm) containing about 0.5 cm thick semi-ynthetic diet on which larvae were fed (Shorey and Hale,965). The pupae were sexed and placed in sterilized containers18 cm × 15 cm × 30 cm) for adult emergence. The containers wereined with tissue paper as an ovipositing stratum. Adult moths wereed on 10% sucrose solution. Insects were reared under laboratoryonditions of 25–27 ◦C, 65–70% RH and 12:12 h (L:D) photoperiod.

.2. SpliMNPV and azadirachtin

The viral isolate SpliMNPV (Baculoviridae) was provided byntomovirology Laboratory, Faculty of Agriculture, Cairo Universityn the form of a suspension in sterile distilled water, with a stockoncentration of 3.4 × 1010 polyhedral inclusion body (PIB)/ml. Thisuspension was stored at −20 ◦C till required. Azadirachtin (96%

urity) was obtained as a powder from Carl Roth GmbH + Co. Kg.

stock solution of azadirachtin (200 ppm) was prepared in sterileistilled water (Zamora-Avilés et al., 2013) and refrigerated at 4 ◦Cill use.

(S.D.) of the mean of three replicates. Columns followed by different letters wereVA). AZA = azadirachtin; NPV = SpliMNPV; AZA + NPV = azadirachtin combined with

2.3. Bioassay

A preliminary experiment was carried out to estimate the LC25and LC50 values of SpliMNPV and azadirachtin applied alone tonewly molted S. littoralis fourth larval instars, following the sur-face treatment bioassay described by Zamora-Avilés et al. (2013).Five viral concentrations (3.4 × 109, 3.4 × 108, 3.4 × 107, 3.4 × 106

and 3.4 × 105 PIB/ml) and six serial concentrations of azadirachtin(50, 25, 12.5, 6.25, 3.125 and 1.5625 ppm) were prepared. One ml ofeach concentration was poured onto the surface of the same semi-synthetic diet used for rearing, thoroughly swirled and air-dried.The treated diet was offered for 24 h to the fourth larval instars,pre-starved for 3 h. Then, the larvae were fed on fresh untreateddiet until pupation or death. The control experiment consisted oflarvae fed on the semi-synthetic diet treated with distilled wateronly. Each concentration was repeated three times of 50 larvae each(Total n = 150 larvae). All the experiments were incubated at 25 ◦C.The percentage of mortality was corrected using Abbott’s formula(Abbott, 1925). The LC25 and LC50 values were estimated using pro-bit analysis (Finney, 1971). They were 10.40 × 106 and 8.90 × 108

PIB/ml, respectively for the treatment with SpliMNPV; 9.95 and23.15 ppm, respectively for the treatment with azadirachtin. Thesame above procedures were also followed in case of combinedtreatment with SpliMNPV and azadirachtin, except for the use of amixture of NPV and azadirachtin (LC25:LC25).

2.4. THC and ultrastructure

Three groups of newly molted fourth larval instars of S. littoraliswere set up. They were respectively fed for 24 h on the semi-synthetic diet used for rearing, previously treated with the LC50of SpliMNPV, the LC50 of azadirachtin and the LC25 of SpliMNPVcombined with the LC25 of azadirachtin. Treated larvae were thenfed on untreated diet, as previously assayed in this study. A non-treated group was used as a control. After 96 h of treatment, theTHC and transmission electron microscopy (TEM) in non-treatedand treated haemocytes were undertaken. Each experiment wasrepeated three times.

For the THC, the haemolymph of treated and non-treated larvaewas collected on a glass slide by pricking a needle in the abdomen

and quickly drawn into Thoma White Blood Cells diluting pipetteup to 0.5 mark, and then diluted 20-times with Toissin’s solutionup to mark II. After shaking vigorously and discarding the firstthree drops, the haemolymph was taken over a Neubauer improved

64 E.-S.H. Shaurub et al. / Micron 65 (2014) 62–68

Fig. 2. (A–D) Transmission electron microscopic micrographs of normal plasmatocytes and granulocytes of Spodoptera littoralis fourth larval instars showing typical cellstructure. (A, 7000×): A portion of a plasmatocyte showing cell membrane (CM); cytoplasm (C); Golgi apparatus (G); lysosome (Ly); nucleus (N); nuclear envelope (NE);nucleolus (Nu); rough endoplasmic reticulum (RER). (B, 9880×): A portion of a plasmatocyte showing chromatin (Ch); a large number of mitochondria (M); free ribosomes( proce(

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arrows). (C, 9880×): A plasmatocyte showing cell membrane (CM) with cytoplasmicNE) with nuclear pores (NP); rough endoplasmic reticulum (RER).

aemocytometer (DHC-No1) and the cells were counted in the fourorner ruled squares in each of two chambers. The THC was calcu-ated by the formula suggested by Jones (1962).

For the TEM, the haemolymph of treated and non-treated lar-ae was collected directly, as previously described, in a fixative (4%lutaraldehyde in 0.2 M cacodylate buffer, pH 7.2). After 30 min fix-tion, the haemocytes were centrifuged for 10 min at 3000 rpm andhe pellets were embedded in 2.5% agarose. They were washed in 5%ucrose solution in 0.2 M cacodylate buffer, pH 7.2 and post-fixed in% osmium tetroxide in cacodylate buffer for 1 h. After dehydration

n a graded alcohol series, they were embedded in Epon-Aralditeesin. Ultrathin sections were examined under a Philips EM-420icroscope.

.5. Statistical analysis

The data obtained for the THC were statistically analyzed usingne-way analysis of variance (ANOVA) (SAS Institute, 2009). Wherehe difference was significant (at P < 0.05), the means were sepa-ated using Student’s t-test.

sses (CP); ribosomes (arrows). (D, 9880×): A granulocyte showing nuclear envelope

3. Results and discussion

The results shown (Fig. 1) indicate that treatment of S. lit-toralis fourth larval instars with the LC50 of SpliMNPV, the LC50 ofazadirachtin and the LC25 of SpliMNPV combined with the LC25of azadirachtin significantly decreased (P < 0.05) the THC com-pared to the control, with t-values of 0.0059, 0.0026 and 0.0027,respectively. This decrease was about 68.25, 73.25 and 73.70%,respectively. Similarly, Gitanjali et al. (1999) reported that therewas a general decline in the THC of Spodoptera litura (Fabr.) larvaein response to NPV, particularly in younger larvae. On the otherhand, De Andrade et al. (2010) found that infection of Anticarsiagemmatalis Hübner larvae with Anticarsia gemmatalis multicapsidnucleopolyhedrovirus (AgMNPV) did not affect the THC.

According to Rosenberger and Jones (1960), the presence ofpathogens in the haemocoel of the insects can activate their defensesystem, causing alterations in the THC. However, there is no consen-

sus in associating the defense response to the decrease or increasein the THC. Some authors (Ratcliffe et al., 1985; Morton et al.,1987; Rivers et al., 2002) elicited that the presence of pathogenswould cause a decrease in the number of circulating haemocytes

E.-S.H. Shaurub et al. / Micron 65 (2014) 62–68 65

Fig. 3. (A–C) Transmission electron microscopic micrographs showing ultrastructural changes in the plasmatocytes and granulocytes of fourth larval instars of Spodopteralittoralis 96 h post-infection with the LC50 of the entomopathogenic virus SpliMNPV (8.90 × 108 PIB/ml). (A, 27,000×): A portion of a plasmatocyte nucleus (N) showing migratedchromatin (Ch); virus rod (arrow) in the process of being occluded by elongate profile (EP); nuclear envelope (NE); virus rods (V); virogenic stroma (VS); polyhedron (P). (B,2 lyhedn ar env

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8,000×): A portion of a plasmatocyte nucleus showing enveloped virions (EV); poucleus showing migrated chromatin (Ch); enveloped virions (arrow heads); nucle

ue to nodulation and encapsulation around the invaders as wells degranulation of some cell types. In contrast, Richards anddwards (1999) and Russo et al. (2001) showed that the presence ofathogens in the haemocoel stimulated the haemopoiesis resulting

n increase in the number of the cells in the haemolymph.In agreement with the results obtained, treatment with

zadirachtin decreased the THC in S. litura (Ayyangar and Rao, 1990;harma et al., 2003) and Danaus chrysippus (L.) larvae (Pandey et al.,

008). The decrease in the number of azadirachtin-treated haemo-ytes may be due to the formation of nodules comprised of groupsf haemocytes or the inhibition of the brain hormone secretionTiwari et al., 2006; Pandey and Tiwari, 2011).

ron (P); multi-embedded polyhedra (Ph). (C, 27,500×): A portion of a granulocyteelope (NE); virus rods (arrows); virogenic stroma (VS).

Several authors (Kumar, 1998; Bajwa and Ahmad, 2012;Zamora-Avilés et al., 2013) suggested that combining azadirachtinwith NPV causes severe damage to the peritrophic membraneand midgut secretory cells, with concomitant decrease in theactivities of digestive enzymes, thereby facilitating the easy pen-etration of the active viral bodies and proliferation for subsequentpathogenic effects. This suggestion may account for the relativedecrease (17.16%) in the THC in case of combined treatment with

azadirachtin and SpliMNPV compared to the treatment with SpliM-NPV alone in the present study (Fig. 1).

There are five types of haemocytes in S. littoralis fourth instar lar-vae: prohaemocytes, plasmatocytes, granulocytes, spherulocytes

66 E.-S.H. Shaurub et al. / Micron 65 (2014) 62–68

Fig. 4. (A–D) Transmission electron microscopic micrographs showing ultrastructural changes in the plasmatocytes and granulocytes of fourth larval instars of Spodopteralittoralis 96 h post-treatment with the LC50 of azadirachtin (23.15 ppm). (A, 4900×): A granulocyte showing cytoplasm (C) with large vacuoles (Vac); normal nucleus (N)with nucleolus (Nu); double-layered nuclear envelope (NE); abnormal mitochondria (arrows); pseudopodia (PS). (B, 9880×): A granulocyte showing residual bodies (B);autophagic lysosome (Ly); polymorphic mitochondria (looped, LM; swollen, SM; vacuolated, VM); normal nucleus (N); rough endoplasmic reticulum (RER) containing fibrousmaterials in their cisternae (arrows). (C, 7000×): A plasmatocyte showing ruptured cell membrane (CM); mitochondria (M); normal nucleus (N) with nuclear envelope (NE)a e cistf ia (SM

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nd nucleolus (Nu); rough endoplasmic reticulum (RER) with fibrous materials in tholded nuclear envelope (NE); vacuolated mitochondria (VM); swollen mitochondr

nd oenocytoids. At TEM level, plasmatocytes and granulocytesere the main cell types affected by the present treatment, whereas

n case of prohaemocytes, spherulocytes and oenocytoids the effectf treatment was negligible. The normal (control) plasmatocytesnd granulocytes show well developed cell membrane with cyto-lasmic processes, rough endoplasmic reticulum, mitochondria,ucleus, ribosomes, Golgi apparatus and primary lysosomes. Theucleoli have dense chromatin dispersed in clumps with onlyccasional batches near the nuclear membrane (Fig. 2(A–D)). Theouble-layered nuclear envelope has an outer layer with ribonu-leoprotein granules attached to it and an inner layer which appearso be uncoated (Fig. 2(A and D)). Free ribosomes are scattered in

he cytoplasm (Fig. 2B). The nuclear pores are located at intervalsround the envelope (Fig. 2D).

Fig. 3 shows SpliMNPV-infected plasmatocytes and granulo-ytes of S. littoralis fourth larval instars. A virogenic stroma and

ernae (arrows). (D, 14,000×): A portion of a plasmatocyte showing lysosomes (Ly);).

naked viral rods were observed in the nuclei of infected plasma-tocytes. Elongate profiles, which had the same diameter as thenucleocapsids, were found, which were considered to be capsids(Fig. 3A). Moreover, infected plasmatocytes showed dense bodies ofvarious sizes, which appeared to be the initial sites for polyhedronformation (Fig. 3B). On the other hand, SpliMNPV-infected gra-nulocytes contained unenveloped virions, but only few envelopedvirions were observed (Fig. 3C). Prior to the appearance of virus par-ticles in the nuclei of both infected plasmatocytes and granulocytes,the chromatin had dispersed into the peripheral area and the nucle-oli disappeared, with concomitant appearance of the virogenicstroma (Fig. 3(A and C)). Similarly, Zhang et al. (2002) found that

when the haemocytes of S. litura larvae were infected with Auto-grapha californica multicapsid nucleopolyhedrovirus (AcMNPV),virogenic stroma and viral nucleocapsids were observed in theinfected nuclei and the chromatin was migrated to the periphery.

E.-S.H. Shaurub et al. / Micron 65 (2014) 62–68 67

Fig. 5. (A–C) Transmission electron microscopic micrographs showing ultrastructural changes in the plasmatocytes and granulocytes of fourth larval instars of Spodopteralittoralis 96 h post-treatment with the LC25 of the entomopathogenic virus SpliMNPV (10.40 × 106 PIB/ml) combined with the LC25 of azadirachtin (9.95 ppm). (A, 9880×):A granulocyte showing cytoplasm (C) filled with vacuoles (Vac); chromatin (Ch) begins to migrate toward the nuclear envelope (NE); autophagic lysosome (Ly); abnormalmitochondria (M); rough endoplasmic reticulum (RER). (B, 7000×): A plasmatocyte showing cytoplasm (C) with large vacuoles (Vac); marginal chromatin (Ch); nucleus (N)filled with unenveloped virions (arrows); virogenic stroma (VS); curved mitochondria (arrow head). (C, 14,000×): A portion of a plasmatocyte nucleus showing marginalc clear e(

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hromatin (Ch); elongate profile (EP); enveloped virions (EV) aligned near the nuarrows).

oughton et al. (1999) recorded also similar observations in theixth larval instars of Agrotis ipsilon (Hufn.) infected with Agrotispsilon multicapsid nucleopolyhedrovirus (AgipMNPV).

Kislev et al. (1969) reported that NPV formation was found toake place mainly in the plasmatocytes, and to a much lesser extentn the granulocytes and oenocytoids. Kawarabata (1974) estimatedhat more than 80% of the infectivity in NPV-infected haemolymphas due to the unenveloped virions. This indicates that latter were

he highly infectious agents. Several authors suggested that virusarticles pass through the basement membrane of the midgut cellsf insects to infect the haemocoel (Summers, 1969; Tanada andeutenegger, 1970). On the other hand, Tanada and Hess (1976)uggested that the haemocoel can be infected with NPV by a differ-nt route, where unenveloped nucleocapsids may emerge throughhe nuclear pore into the cytoplasm, bud through the basal cell

embrane and then enter the haemocoel.Fig. 4 shows azadirachtin-treated granulocytes and plasmato-

ytes of S. littoralis fourth larval instars. The cytoplasm of treatedranulocytes was extremely vacuolated. These vacuoles contained

nvelope; multi-embedded polyhedra (Ph); virogenic stroma (VS); virus particles

degenerated organelles, which appeared as cell debris. Cytoplasmicextrusions similar to pseudopodia appeared protruding from thesehaemocytes (Fig. 4A). Dark residual bodies, autophagic lysosomesand polymorphic mitochondria (looped, vacuolated and swollen)were found (Fig. 4B). As to treated plasmatocytes, the cell mem-brane was ruptured (Fig. 4C) and the nuclear envelope was folded(Fig. 4D), thus the surface area of the nucleus increased. Vacuo-lated and swollen mitochondria were also observed (Fig. 4D). Bothtreated granulocytes and plasmatocytes showed rough endoplas-mic reticulum filled with fibrous materials in their cisternae (Fig. 4Band C), which may be apoptotic response (Hossain and Richardson,2011). Nuclei were not affected due to treatment with azadirachtin.Similarly, Sharma et al. (2003) observed vacuolization of the cyto-plasm and degeneration of the organelles in the plasmatocytes andgranulocytes of S. litura larvae treated with Neem gold. Conse-

quently, the cellular immune system had collapsed (Sharma et al.,2008).

The same pathological changes induced in the granulocytes andplasmatocytes of S. littoralis larvae due to individual treatment with

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8 E.-S.H. Shaurub et al.

pliMNPV and azadirachtin were also observed in case of combinedreatment. The cytoplasm of both cell types was vacuolated and the

itochondria were disorganized (Fig. 5A and B). Chromatin clumpsigrated to the periphery of the nucleus (Fig. 5A–C). The viral rods

nvaded the nuclei of plasmatocytes, and virogenic stroma wasell developed (Fig. 5B and C). Also, treated plasmatocytes showeducleocapsids aligned near the nuclear envelope and the elongaterofiles were formed to encapsulate viral rods (Fig. 5C).

In conclusion, it appears that the ingested SpliMNPV andzadirachtin penetrate into the haemocoel and have direct effectsn the haemocytes of S. littoralis fourth instar larvae. The sup-ression of the THC and the pathological changes induced inhe plasmatocytes and granulocytes indicate that SpliMNPV andzadirachtin might act as immunosuppressants. This finding mighte taken into consideration in evaluating the success of microbial

nsecticides and botanicals as control agents for S. littoralis. More-ver, mitochondria were the probable site of action of azadirachtin,s they were affected in both granulocytes and plasmatocytesreated with azadirachtin alone or in combination with SpliMNPV.

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