1 Introduction

The cotton leaf worm, Spodoptera littorals (Bosid.) causes severe damage for cotton plants and subsequently leads to economic losses in cotton industry in Egypt. This pest develops resistance to the majority of chemical insecticides due to uncontrolled intensive use of these insecticides. Most chemical insecticides kill pest insects swiftly, but are also toxic to beneficial insects and other species in the agro ecosystem [1].To avoid unfavorable side effects of these insecticides on non-target organisms and environment, alternative safe and effective microbial insecticides have been initiated [1]. Insect viruses and entomopathogenic bacteria, fungi, and nematodes have been investigated as biological control agents of S. littoralis [2].Entomopathogenic bacteria, particularly Bacillus thuringiensis Berliner (Bt), is the most commonly used microbial insecticides. Cry toxins enjoy the advantages of highselectivity and the possibility of the application by sprays or transgenic plants [1]. According to Sanahuja et al. [3], Bt’s insecticidal activity is attributed to its proteinaceous crystals and vegetative insecticidal protein contents, which are produced during sporulation and vegetative growth phases, respectively Kumar et al. [4].

Baculovirus, the second most popular microbial insecticide, has been employed as a safe bio- insecticide because of its effectiveness and specificity as well as the presence of infectious protein crystals in it Zhang et al. [5]. The Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) is a baculovirus that has been evaluated, registered, and applied for control of S. littoralis, as well as the fall armyworm, Spodoptera frugiperda, and the tobacco cutworm Spodoptera.

litura in Africa, America and Japan Abdel-Khalik et al. [6], El-Sheikh; [7],Takatsuka et al. [8].

Similar to liver of vertebrates and hepatopancreas of crustaceans, the insect fat body (FB) has multiple metabolic functions and takes parts in the metabolisms of lipids, carbohydrates and proteins [9] and [10]. According to Wigglesworth [11] and Kritsky [12], it produces trehalose, desaminates and transamination of amino acids. Also, it removes calcium salts, urates, and other nitrogenated products from the hemolymph. The FB cells in Lepidoptera have the ability to phagocytose and take part in tissue remodelling during metamorphosis [11].

They have the ability to maintain a balance between the food resource and energy demands during the insect development. During the periods of insect actively feeding, FB accumulates molecules to be used in the periods of food leakage for developmental demands. Also, FB cells may change their activity in response to the nutritional and hormonal signals to supply the insect growth needs, metamorphosis and reproduction [13, 14]. However, these changes may also occur under various pathological and environmental conditions.

2 Materials and methods

2.1 Tested insects

The used insects are a laboratory strain of the cotton leaf worm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) which had been reared in the laboratory without any exposure to chemicals and was obtained as egg masses from the Research Division of the cotton leaf worm, Plant Protection Research Institute, Dokki—Giza, Egypt.

2.2 Compounds

Three commercial bio insecticides were used in this investigation. All tested bio-agents were obtained as wettable powder produced by the Plant Protection Research Institute, Bio pesticide Production Unit, Dokki- Giza, Egypt. They were as follows:

2.2.1 a. Protecto® WP 9.4% (Bacillus thuringiensis var. kurstaki)

Bacillus thuringiensis (Kingdom: Eubacteria; Order: Bacillales; Family: Bacillaceae) is a gram-positive spore-forming bacterium that produces crystalline proteins called deltaendotoxins during its stationary phase of growth. This product is used at rate of 300 gm/feddan with LC50 value is 3.2 × 102 Iu/ml. Feddan is equal to 4200 square meter.

2.2.2 b. Viruset® WP 4% (Spodoptera littoralis ucleopolyhedrosis virus [SpliNPV])

Nucleopolyhedrosis virus (NPV) is a double stranded DNA virus that belongs to baculoviruses subgroup (Family: Baculoviridae). The virus was isolated from diseased larvae and formulated to be used at rate of 300 gm/feddan and LC50 value of 1 × 102 PIBs/ml.

2.2.3 c. Profect® WP 5% + 2% (Btk + SpliNPV)

This product is a mixture of 5% of Bacillus thuringiensis var. kurstaki and 2% of SpliNPV. The product is used at rate of 400 gm/feddan and LC50 value is 5 × 103 Iu/ml and 1.6 × 102 PIBs/ml.

2.3 Preparing of insects for dissection

A newly ecdysed 2nd instar larva were fed on castor oil leaves treated with the determined LC50 of each compound for 24 h, then reared on untreated leaves until the symptoms of infection appeared. Infected larva were anesthetized and fixed in wax plate. Insects were dissected by cutting along their backs with an entomological scissors. The gut and other organ were examined then photographed under a stereo-microscope. Uninfected larva was used as control.

2.4 Specimen preparation for electron microscopy

After dissection of larva, mid gut was separated and preserved in a fixative solution. The resin embedded specimens were mounted in special holders which at the same time fit the microtome. Ultrathin sections were obtained using an ultra-cut E microtome equipped with a diamond knife (Diatome, Switzerland). Sections for TEM analysis were collected on carbon coated for more supports then stained with uranyl acetate and lead citrate [15]. After staining, sections were examined in a SEM electron microscope equipped with a ProScan slow scan CCD camera. Preparations and ultrathin sections were observed at 80 kV using a JEOL JEM -2100 at EM Unit, Mansoura University, Egypt.

3 Results and discussion

3.1 Morphological studies

3.1.1 Internal organs of untreated larvae

The Fat tissue (Fig. 1A–C) of spopdoptera littoralis larva was a whitish, spongy mass, suspended in the hemocoel and supported by the tracheal system and involved the organs of body cavity.

Fig. 1
figure 1

Structural analysis of the internal tissues of spodoptera littoralis un treated larvae. A shows foregut (arrow) and midgut (arrow head). B shows hind gut (arrow). C shows fat tissue

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3.1.2 Internal organs of larvae treated with viruset compound

The treated larvae with viruset (Fig. 2A–C) appeared with reduced layer of fat tissue and tracheal system moderately attached to the gut.

Fig. 2
figure 2

Structural analysis of the internal tissues of spodoptera littoralis larvae infected with viroset compound. A shows foregu t(arrow) and hind gut (arrow head). B shows mid gut (arrow). C shows fat tissue

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3.1.3 Internal organs of larvae treated with Protecto compound

Larvae showed yellowish, reduced layer of fat tissue and less attached tracheal system to the gut (Fig. 3).

Fig. 3
figure 3

Structural analysis of the internal tissues of spodoptera littoralis larvae infected with protecto compound. A shows foregu t(arrow) and mid gut (arrow head). B shows mid gut (arrow), Ac refers to alementary canal. C shows fat tissue of the insect

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3.1.4 Internal organs of larvae treated with profect compound.

The treated larvae showed a very thin layer of fat tissue and damaged tracheal system (Fig. 4).

Fig. 4
figure 4

Structural analysis of the internal tissues of spodoptera littoralis larvae infected with profect compound. A foregut (arrow) and mid gut (arrow head). B shows alementary canal (AC). C shows fat tissue of the insect

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3.2 Cytological studies

3.2.1 Ultra structure of mid gut of untreated larvae

See Fig. 5.

Fig. 5
figure 5

Transmission electron micrograph of gut wall of spodoptera littoralis larvae. A Gc- goblet cell, Mv- microvilli, arrow-Glycogen granules, ch-chromatin, N-nucleus. The glycogen droplets increased in size and quantities, distributed all over the epithelial cells. Normal goblet cell appeared with normal microvilli. B-Arrow-trachea, Bl-basal lamina, Cm-circular muscle. The gut cell contained many glycogen granules. A thin basal lamina and a lot of tracheoles underneath the plasma membrane

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3.2.2 Ultra structure of mid gut of larvae treated with viruset compound:

See Fig. 6.

Fig. 6
figure 6

Transmission electron micrograph of gut wall of spodoptera littoralis larvae treated with viruset compound. A Gc-highly infected goblet cell with fully degenerated microvilli, arrow- degenerated nucleus. B Red arrow—fully formed polyhedral of the virus, white arrow- processed polyhedral, N—nucleus of epithelial cell. C Arrow—polyhedra stuff the gut wall of epithelial cell. Late stage of virus infection resulting in complete hydrolysis of the epithelial gut wall and formation of liquefied gut content

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3.2.3 Ultra structure of mid gut of larvae treated with protecto compound

The cytoplasm of the epithelial cells become highly vacuolated as the vacuoles containing disintegrated glycogen granules (Fig. 7A–C). A part of epithelial cell in its way to be engulfed in the gut lumen (Fig. 7B). Bt invading microvilli of epithelial cell (Fig. 7B).

Fig. 7
figure 7

Transmission electron micrograph of sector of gut wall of spodoptera littoralis larvae treated with protecto compound. A Arrows-Glycogen (lipid) vacuoles. Stars—glycogen vacuole in different stages of degeneration. B Red arrows-sections of bacterial cells. Ec- part of epithelial cell in its way to be engulfed in the gut lumen. Star- disintegrated glycogen vacuole. C Gc-goblet cell. Arrow- sections of Bt accumulated in the gut lumen. Arrow head- disintegrated glycogen vacuole.Mv- microvilli of epithelial cells invaded with Bt. N- hypertrophied nucleus of epithelial cell having dispersal chromatin granules all over the nucleoplasm

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3.2.4 Ultra structure of mid gut of larvae treated with profect compound

See Fig. 8.

Fig. 8
figure 8

Transmission electron micrograph of sector of gut wall of spodoptera littoralis larvae treated with profect compound. A Arrow-glycogen granules. B arrow-BT in the cytoplasm, thick arrow- glycogen granules. N-hypertrophied nucleus with chromatin granules scattered all over the nucleoplasm. C-arrows-trachea, cy-cytoplasm, thick arrow-basal lamina

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4 Discussion

Fat body, is a vital multi-functional tissue found in the visceral cavity of insect life stages [16, 17]. It carries out a variety of tasks, such as maintaining bacterial endosymbionts [18], storage of urate during development [19], synthesizing, releasing, and storing a variety of macromolecules [18]. It acts as a source of humoral factors, and role in immune functions [20].

The primary metabolites of insects include three major categories: lipids, proteins and carbohydrates. Our research showed that bacterial and viral infections may change these major categories in the host larvae. According to Arrese and Soulages [16], this effect may be associated with the insect fat body involved in synthesizing, storing and secreting lipids, proteins and carbohydrates. According to our study of the pathological morphology of fat-body tissues, viral and bacterial infection results in the destruction of the fat bodies, which may be one of the causes of the obvious changes in the host larva’s major primary metabolite content. Schmid-Hempel [21], agreed with our conclusions and pointed out that virus infection results in the host’s body physically losing nutrients, which are then given to the invading organisms to consume for their own growth and reproduction.

Our findings showed that larva treated with the profect compound, which is a mixture of bacteria and viruses, had a fatter body than larva treated with the bacteria and viruses separately. Dantzer, [22] agreed with us and provided an explanation for the observed rise in the lipid and protein contents of the infected larvae. They pointed out that the host’s body causes stress and immunological responses to fight the invasive viruses. The host’s metabolic rate rises as a result of these significant energy and resource demands.

The midgut of the control larvae is composed of a single layer of columnar and goblet cells, as well as other epithelial cells, which are adhered to the muscular connective tissue by a basement membrane. Each epithelial cell has numerous microvilli. The cytoplasm contains a large number of organelles, such as mitochondria, lysosomes, free ribosomes, and rough endoplasmic reticulum. It has also, a spherical nucleus with a normal chromatin distribution and a typical nuclear envelope. A significant chamber known as the goblet cavity and an apical entry into the midgut lumen serve as the goblet cell’s distinguishing features. Mitochondria is present in the numerous cytoplasmic extensions that line and expand into this cavity. The flattened nucleus of goblet cell is located in the basal region, just below the goblet cavity.

When the insect consumes a diet containing Bt formulation, the typical structure of the midgut of the larva changed. The Bt d-endotoxin crystals are dissolved in the midgut to produce one or more protoxin proteins. Specific receptors, which may be glycoproteins, are present in the brush border membrane of the midgut epithelium [23]. The Cry toxins may create pores that allow the passage of ions and small molecules like sucrose by inserting into the cell membrane. Osmotic inflow and cell lysis come from the disruption of the cell membrane’s selective permeability. Thus the integrity of the epithelium is destroyed and the insect dies from starvation and/or septicemia [24].

In this study, the histopathological changes in the third instar S. littoralis larvae’s midgut caused by B. thuringensis treatment were examined. The Bt action may be responsible for the histopathological alterations in the gut. These alterations include the loosening of the midgut columnar cells from the basement membrane and from one another. Cell vacuolization, microvilli destruction, and the passing of epithelial cell contents into the midgut lumen with nuclear migration to the apical membrane occur. Similar histopathological effects in midgut of E. kuehniella larvae treated with Cry toxins were found by [25].

In some cells, the microvilli have entirely separated from the apical surface. The contents of the epithelial cells are engulfed into the intestinal lumen once the microvilli detach. Dead cells detached from the epithelium have been seen in the mid gut lumen of numerous mosquitoes infected with several Plasmodium species, according to reports f Paskewitz et al. [26], Han et al. [27] Zieler and Dvorak [28]. They suggested that this might be a common response to various stressful situations.

Uncontrolled cell division, the development of rounded cells with little cytoplasm and a relatively big nucleus taking up the majority of the cell’s surface area are one of the main effects of epithelial cells. Convoluted plasma membrane is noticed. The lysosomes separated from the cells leaving white vacuoles in its place. Nuclear sheath destruction, chromatin clumping and nuclear envelope shrinkage were also noted. These conclusions are in agreement with those of Almeida et al. [29], Suryani et al. [30]. According to research by Barbeta et al. [31], epithelial cell displacement and hypertrophy from the basal lamina decrease the insect’s ability to digest food. (Nasiruddin and Mordue [32] reported that, endoplasmic reticulum fragmentation, cytoplasm vacuolation, and microvilli disruption are the main reason of this hypertrophy.

Additionally, our findings demonstrated that columnar and goblet cells deteriorated and microvilli were damaged when the larvae consumed Bt endotoxin. Also, the goblet cells and epithelial cells dissolved, with its center engulfed into the gut lumen. The disruption of the midgut epithelial cell microvilli by Bt has been observed in other insect hosts by [33] and [34]. This result suggests that Bt toxins initially modify the microvillar membranes.

Similar reports of vacuolation of the Bt-affected midgut epithelial cells in lepidopterous hosts were made by Mathavan et al. [33] and Lane et al. [35]. They noted the extrusion of cytoplasmic globules from midgut cells that affected with Bt. According to Percy and Fast [36], Sutter and Raun [37], and Endo and Nishitsutsuji-Uwo [38], other lepidopteran species also exhibit similar extrusions. The nucleoplasm of the severely hypertrophied nucleus is scattered with folded and dispersed chromatin.

Some cells have microvilli that have entirely separated from the apical surface. The contents of the epithelial cells are absorbed into the gut lumen following the detachment of the microvilli. It is possible that dying epithelial cells shed into the lumen or phagocytosed by viable neighbour epithelial cells are part of a remodelling process of the midgut, as suggested for some mammalian organs [39].However, dead cells detached from the epithelium have also been discovered in the midgut lumen of multiple mosquitoes carrying different Plasmodium species [26] and [28]. They suggested that this may be a typical reaction to various stress circumstances.

Baculoviruses (Bv) enter the haemocoel tissues through the basal lamina after occluded derived virions (ODVs) infect midgut cells. It’s probable that the virus is telling columnar epithelial cells to release proteases at the basal lamina because the basal lamina is made up of protein. The midgut epithelium is not a primary site of viral replication for the majority of baculoviruses, but infected cells create the initial BV supply required to infect other organs in larvae. Infected midgut cells shed off as a virus defense mechanism, and new cells are produced to help in the recovery of gut. Allowing recovery of midgut epithelium enables the host to consume food, develop, and this allows the replication of virus.

According to Engelhard et al. [40], Kirkpatrick et al. [41], and Washburn et al. [42], the tracheal system is the main route by which viruses spread from the midgut. The tracheal system, or external insect respiratory system starts as apertures along sides of the insect called spiracles. Tracheal tubes extensively branch when they pass through spiracles and enter the haemocoel. These branches transition abruptly into a network of very small tubes called tracheoles. Tracheoles impregnate nearly every tissue including the midgut epithelium. When a tracheolar cell infected by a midgut cell-derived BV, the BV progeny will gain access to the hemocoel’s deep tissues.

The fat body which serves as the liver of the insect is an important component of baculovirus replication [43]. I is responsible for sugar storage and lipid metabolism in insect. In the lepidopteran larva, the fat body is an amorphous and protuberant organ running throughout the insect. The tissue is highly accessible to BV and the energy-rich cells of the fat body are ideal for producing abundant virus progeny. The fat body becomes full up with OBs, giving an opalescent, puffy appearance to the larvae just before it dies. Exploiting the inner tissues of the insect host gives the virus the advantage of being able to produce enormous numbers of progeny. Most baculoviruses release OBs from the host by tissue liquefaction and the rupture of cuticle after death. As the liquefied remains ooze out from the dead host, OBs are broadly dispersed along food surfaces that are eaten by a new host.

Within 30 min of infection, viral early genes are expressed, and its protein products, together with virion protein and begin to enable DNA replication [44].The structural alteration of the nucleus, known as nuclear hypertrophy, results in an enlarged nucleus. This is in line with our observations, which demonstrated that the nucleus hypertrophy and chromatin was dispersed throughout the nucleoplasm.

When Anticarsia gemmatalis is exposed to squamocin, midgut cells undergo autophagy, which results in damage to microvilli and severe cytoplasmic vacuolization. Our findings matched what Costa et al. [45] discovered in the midgut digestive cells of A. aegypti larvae exposed to squamocin. According to Gaban et al. [46], the mid gut is severely affected when A. aegypti larvae treated with novel chemical insecticide demonstrate high secretory activity and droplets discharged to the gut lumen from the apical region of the cell.

When combining the virus and bacteria together a significant effect on the mid gut epithelium and visceral muscle occur. Columnar and goblet cells are totally broken down. After the microvilli have been completely separated from the cell, lysis takes place. The same results observed by Knaak and Fiuza [47], who noted the destruction of microvilli and lyses of Anticarsia gemmatalis cells treated with Bacillus thuringensis and nuclear polyhedrosis virus. They demonstrated that there were no alterations in the intestinal cells of A. gemmatalis larvae in the individual treatments with Bt.k. and AgNPV up to 6 h after application. After 6 h, Btk and AgNPV together already showed significant cytoplasmic vacuolization, leading to cellular disorganization. Salama et al. [48] and Mahmoud et al. [49] supported our findings. They clarified that following B.t and NPV treatment, vegetative cells and spores of B.t penetrated the midgut epithelial cells, followed by penetration of the virus. The potential entry of B. t and the ensuring release of toxin led to a partial breakdown of the cell organelles, which made it easier for the virus to enter from nearby fat tissues. Magholli et al. [50] explained that the synergistic effects of the two pathogens, B.t and NPV, on Heliothis armigera may be due to the delay in pre-pupal stage.