Investigations into the cellular and humoral immune mechanisms of Spodoptera litura (Fabricius) against potential entomopathogens

doi:10.21203/rs.3.rs-4660383/v1

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Investigations into the cellular and humoral immune mechanisms of Spodoptera litura (Fabricius) against potential entomopathogens

https://doi.org/10.21203/rs.3.rs-4660383/v1

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Entomopathogens are foreign agents which elicits immune responses in insects. The insect innate immune system can identify and overcome these foreign-agents through cellular and humoral processes. The current study reports the cellular and humoral immune responses in Spodoptera litura to different entomopathogens including Beauveria bassiana, Metarhizium anisopliae, Bacillus thuringiensis and Nuclear Polyhedrosis virus (Sl NPV). In case of cellular immunity haemocytes are responsible for a number of defensive mechanisms in insects such as phagocytosis, nodulation, encapsulation, etc. Humoral immune responses include the recognition of invading pathogen either as bacteria, fungi or viruses followed by the immediate de novo synthesis of antimicrobial peptides (AMPs) and their secretion into the hemolymph. These peptides are mainly synthesized by the fat body and in a lesser degree by the haemocytes, integument, gut, salivary glands and reproductive structures. Third instar larvae were treated with the test entomopathogens and haemolymph was collected at different time intervals i.e., 1, 3, 6, 12, 24, 48 and 72 hours after infection (HAI) to study the cellular [Total Haemocyte Count (THC), Differential Haemocyte Count (DHC), Phagocytosis and Nodulation] and humoral (PO and Lysozyme activity) immune responses. Plasmatocytes (PLs) and Granulocytes (GRs) were observed as the main circulating haemocytes which are involved in the immune mechanism in the S. litura larvae. The immediate response of larvae to entomopathogens infection was a significant increase of total circulating hemocytes. This increase included both granulocytes and plasmatocytes. However, after 3 days from the immune challenge, the number of circulating hemocytes was significantly lower. Consequently, the larvae showed Phagocytosis, nodulation, increased PO and Lysozyme activity in the immediacy of the challenge, but progressively the phenomenon decreased.

Spodoptera litura

Entomopathogens

Cellular immune responses

Humoral immune responses

Insects frequently interact with a diverse group of microorganisms in nature. Numerous microorganisms have been known to show adverse effect on insects by invading, colonising and killing them. The term "Entomopathogens" refers to the diverse collection of insect pathogens, which includes viruses, bacteria, fungi, protozoa, nematodes, etc.

Entomopathogens are usually recognized as harmful foreign agents that cause immunological reactions which are managed by different immune cells and tissues in insects. The insect innate immune system can identify and get rid of specific diseases and parasites through cellular and humoral processes (Lemaitre and Hoffmann, 2007). Haemocytes are considered as the main immune cells in insects (Strand, 2008 and Hillyer and Strand, 2014). Insects use haemocytes, which are present in haemocoel, for numerous cellular and humoral defence systems to protect themselves against harmful microorganisms. Granulocytes (GRs) and Plasmatocytes (PLs) are the important haemocytes that are involved in immune mechanism of lepidopteran insects (Li et al., 2022).

Nodulation, encapsulation and phagocytosis are examples of cellular immune responses. Humoral immune responses include the production of various antimicrobial peptides, proteins, reactive oxygen and nitrogen species, lysozyme activating system and prophenoloxidase activating system, which results in the coagulation and melanization of haemolymph (Bogdan et al., 2000 and Nappi et al., 2000). Pattern Recognition Receptors (PRRs), found on the surface of insect cells, interact with Pathogen Associated Molecular Patterns (PAMPs), which are found on the surface of pathogens and include Peptidoglycans, Lipopolysaccharides (LPSs) and β-1,3 glucans, to cause the humoral response (Zhong et al., 2017).

When compared to other responses, the immunological pathways used by insects to defend themselves against viruses are quite diverse. Although there are several families of viruses that are harmful to insects, baculoviruses are the most extensively researched and well understood. The majority of baculoviruses are extremely host specific and can only replicate in a small number of closely related hosts (Black et al., 2022). As soon as the infection spreads from the trachea to the haemocoel, it becomes systemic. However, the main antiviral defence appears to be RNA interference (RNAi) (Bronkhorst and Van Rij, 2014 and Hillyer, 2016). Cellular and humoral responses have also been recognized as potential antiviral defence mechanisms.

Similar to viruses, bacterial entomopathogens must enter the host through an orifice, such as a wound or spiracle, or through oral consumption. Nodulation and phagocytosis are the two common cellular immune reactions for bacterial invasion. Depending on the kind of LPSs in the bacterial cell wall, humoral responses involve the Phenoloxidase (PO) cascade and Antimicrobial Peptides (AMP) synthesis by activation of either the Toll or Immune Deficiency (IMD) pathway (Myllymäki et al., 2014 and Satyavathi et al., 2014).

In case of entomofungal pathogens, the establishment of appressorium or invasion through orifices like spiracles, wounds, or oral ingestion allow fungal entomopathogens to directly penetrate the host cuticle (Talbot, 2019). They enter through the cuticle and make their way to the haemolymph. The immune system of the host can be suppressed by a range of compounds produced by fungal diseases (Boucias et al., 1995) and they can also release toxins that kill insects (Kershaw et al., 1999). Along with humoral and cellular immunological responses, a fungal infection will cause the initiation of complex proteolytic cascades, such as the Prophenoloxidase (PPO) pathway, which sets off the melanization response (Cerenius et al., 2010).

Tobacco caterpillar, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae, is a destructive polyphagous pest that infests a variety of crops, including cotton, tomato, capsicum, potato, soybean, okra, clover and onion (Saleem et al., 2016) and is known to cause extensive damage to crops. In addition to its economic importance as a pest, S. litura is an ideal insect for demonstrating the cellular and humoral mechanisms of defence to insect pathogens. To further understand the mechanisms of disease development and insect defence system, S. litura larvae were exposed to test entomopathogens viz., Beauveria bassiana, Metarhizium anisoplae,

Bacillus thuringiensis var. kurstaki and S. litura Nuclear Polyhedrosis virus (Sl NPV) and cellular and humoral immune responses were monitored.

Insect Rearing

The Spodoptera litura larvae were collected from the college farm of College of Agriculture, Professor Jayashankar Telangana State Agricultural Univesrity (PJTSAU) and farmer fields in Rajendranagar, Hyderabad. The collected larvae were maintained on the artificial diet (Gupta et al., 2005) with Relative Humidity 70–75% and temperature 20 ± 2˚c under laboratory conditions. The larvae which were in the stage to pupate were transferred into the tray containing soil. After pupation, the pupae were collected in a tray and covered with a muslin cloth. The emerged adults with a male and female ratio of 2:3, were transferred into the cages containing 15% sucrose solution (Santharam, 1985). The egg masses were collected and maintained in the Insect Pathology Laboratory, Department of Entomology, College of Agriculture, PJTSAU, Rajendranagar, Hyderabad.

Entomopathogens and Inoculation procedure

Commercial formulations of different test entomopathogens i.e., Beauveria bassiana, Metarhizium anisopliae, Bacillus thuringiensis and Nuclear Polyhedrosis virus (Sl NPV) (Table 1) used in the present study were sourced from a noted agri biotech company, AgriLife (India) Private Limited, Hyderabad. Leaf dip method for Bt and Sl NPV and topical application for entomopathogenic fungi were used for inoculating the larvae. Once S. litura larvae moulted to 3rd instar, they were starved for 24 hours before inoculating with the leaf dip method. For inoculating the larvae with Bt and Sl NPV, the castor leaves were dipped in suspensions of Bt for treatment three and in viral suspensions for treatment four as described in Table 1. The treated castor leaves were then dried for 30 seconds and the dried leaves were placed on the filter paper within a petri dish (one leaf per petri dish). Two larvae per petri dish were released and four such replications for each treatment were maintained. Then the larvae were allowed to feed on the infested leaves. Since oral infection is not the primary infection route for entomopathogenic fungi, for treatment with B. bassiana and M. anisopliae the third instar larvae were topically exposed by adding 10µl of the solution with a sampler device, following which they were placed on filter paper for 30 seconds and transferred two larvae per petri dish and four such replications were maintained for the treatment. For control, castor leaves were dipped in 10µl of Tween 80 (0.05%) solution, then dried for 30 seconds and placed in the petri dish. Two larvae per petri dish were released.

Table 1

Entomopathogens used for experimentation
Treatment	Dosage
T1 - Beauveria bassiana 1%WP	5 g / l
T2 - Metarhizium anisopliae 1%WP	5 g / l
T3 - Bacillus thuringiensis var. kurstaki 20SL	2 ml/l
T4 - Nuclear Polyhedrosis virus (Sl NPV)	250 LE / ha
T5 - Control (Untreated check)

Collection of haemolymph from infected larvae

Haemolymph was collected by cutting the tip of a proleg of the infected larvae from each treatment, previously anesthetized on ice using fine scissors and needle (Rosenberger and Jones, 1960). Gentle pressure was applied on the insect abdomen for getting more quantity of haemolymph (Barkat et al., 2002). The collected haemolymph from the infected larvae was used to prepare blood smears, by spreading a drop (20µl) of haemolymph on a glass slide and smeared by drawing a second slide across the first at an angle of 45° and then dried at room temperature. The air-dried haemolymph smear slides were then dipped into methanol two times and air dried. Slides were then stained with Giemsa stain (diluted five times with phosphate-buffer saline (PBS) and filtered before use) for 20 min and then rinsed in distilled water. The smear was washed in 0.02% acetic acid followed by rinsing in distilled water. After drying, permanent microscopy slides were prepared using Permount or Canada balsam.

Characterization of the haemocytes

In order to identify haemocyte types, the shape, size and the cytoplasm constituents of cells were observed. About 10 slides (4 replications) were prepared from 8 untreated individuals for identifying different haemocyte types based on the identification keys set by Gupta (1979). The haemolymph drawn from the untreated individual was diluted with 0.9% NaCl solution, about 20µl of diluted haemolymph was placed on a slide using micropipette, stained with Giemsa stain and observed under the phase contrast microscope to identify different types of haemocytes.

Identification of cellular immune responses of S. litura to different test entomopathogens

Haemocytes count

Total haemocyte counts were calculated for S. litura larvae after infection with different test entomopathogens and also from the untreated larvae in the control.

Determination of Total Haemocyte Count (THC)

The total haemocyte count of S. litura larvae were calculated after the infection with entomopathogens by using Neubauer haemocytometer. The sampling of the haemolymph was done by using Thoma white cell pipette. Haemolymph was collected on a glass slide and quickly sucked up into Thoma white cell pipette up to the mark of 0.5. The end of the pipette was wiped and diluting fluid (glacial acetic acid, 1 ml; distilled water, 100 ml; gentian violet, 0.3%) was drawn up to the mark of 11, for producing a dilution of 1/20, (1 part of blood: 20 parts of WBC fluid). The contents of the pipette were properly mixed and first three drops were discarded (Jones, 1962) and Neubauer’s chamber was charged with the 200µl of the dilutant. Leave the haemocytometer for 2 minutes to settle the cells and reduce their movement. The haemocytes were counted from the four corner squares (white cell squares) in each of the two chambers according to the following formula (Gupta and Sutherland, 1968)

Haemocytes in five 1 mm squares x dilution x Depth of chamber	x	1000
Number of 1 mm squares counted

Determination of Differential Haemocyte Count (DHC)

Differential Haemocyte Count (DHC) from the stained slides was carried out with the help of a cell counter by following the battlement method (Perveen and Ahmad, 2017). The film was examined systematically by observing traversed three fields along the edge and two fields down starting at the thin end of the smear. This sequence was continued until minimum of 200 cells were enumerated and various classes of haemocytes was also observed (Mahmood and Yousaf, 1985).

Phagocytosis

Phagocytic activity was determined by counting the cells containing spores in a Neubauer haemocytometer. Observations were made on phase contrast microscope (Zibaee et al., 2011).

Nodulation

Injected larvae were chilled on ice, haemolymph was collected in a capillary tube and 200µl was placed onto a haemocytometer for nodule counting (Zibaee and Malagoli, 2014).

Identification of humoral immune responses of S. litura to different test entomopathogens

Collection of haemolymph

Approximately 40 µl of haemolymph was obtained from each individual larva. Two larvae for each replication and four replications for each treatment were used for the study. The collected haemolymph was mixed with anticoagulant solution in the ratio of 4:5, immediately (400 µl haemolymph: 500 µl of anticoagulant solution) (0.01 M EDTA, 0.1 M glucose, 0.062 M NaCl, and 0.026 M citric acid, pH 4.6) (Azambuja et al., 1991).

PO preparation

The diluted haemolymph was centrifuged at 12,000 rpm for 5 min, and then the supernatant was removed and the pellet washed using phosphate buffer (K₂HPO₄ 65 mM, KH₂PO₄ 2.6 mM, NaCl 400 mM and NaN₃ 3 mM; pH = 6.5) (two times) (Leonard et al., 1985). The pellet was homogenized after adding 500 µl of cold phosphate buffer, and then centrifuged at 12,000 rpm for 15 min. The Haemocyte Lysate Supernatant (HLS) was transferred into new microcentrifuge tubes. Samples (10 µl) were preincubated with phosphate buffer at 30°C for 30 min, then 50 µl of 10 mM L-dihydroxyphenylalanine (L-DOPA) as substrate was added and incubated for 5 min at 30°C. PO activity was measured using a spectrophotometer (S 2100 Diode Array spectrophotometer) at 490 nm wavelength. One unit was described as 0.01 absorbance increase at 490 nm/min (Zibaee et al., 2011). Activity in treated assays were compared with that of control.

Lysozyme Activity

After collecting the haemolymph, the activity of lysozyme was evaluated according to Garriga et al. (2020) with slight modifications. Briefly, N-Phenylthiourea crystals were added to the samples to prevent activation of the PO system. Samples were centrifuged twice at 250 x g for 5 minutes at 4°C, and then at 1600 x g for 10 minutes at 4°C, collecting the supernatant. Cell-free hemolymph was diluted 1:10 with sterile PBS. Thereafter, 100 µl of the samples were added to 150 µl of Micrococcus lysodeikticus (0.45 mg/ml in 30mM phosphate buffer, pH 7.2; Sigma-Aldrich), with an optical density of 0.6–0.7, which was used as substrate to measure lysozyme relative activity. M. lysodeikticus alone and cell-free haemolymph without addition of the bacterium was used as control. Absorbance at 450 nm was measured every 30 s for 10 min. in 96-well plates using a Bio-Rad iMark™ Microplate Absorbance reader (Bruno et al., 2022).

Data Analysis

Each treatment was replicated four times and the data was analyzed by using Completely Randomized Design (CRD). The data was subjected to the standard statistical analysis using techniques of analysis of variance. Differences between samplings were considered statistically significant at a probability more than 5% (p ≤ 0.05).

Cellular immune responses of S. litura to the selected entomopathogens

The cellular immune responses (Fig. 1) of S. litura in terms of important parameters were studied and outcome of the studies are discussed.

Determination of Total Haemocyte Count (THC) through light microscopy

Giemsa staining of larval haemolymph and light microscopy observations showed that the THC (Fig. 1a) count was increased up to 24 Hours After Infection (HAI) for all entomopathogens other than Bacillus thurangiensis var. kurstaki. In case of Bt treated larvae the count was increased up to 48 HAI (Table 2). Bt treated larvae showed highest number of haemocytes at 48 HAI (458 ± 3.21^a). However, later the count started decreasing. Entomopathogenic fungi treated larvae showed lowest number of haemocytes in comparison to other test entomopathogens. The order of THC observed in different test entomopathogens were Bt > Spodoptera litura Nuclear Polyhedrosis Virus > Beauveria bassiana ≥ Metarhizium anisopliae. Untreated control showed least THC in comparison with any of the test entomopathogen (Fig. 2).

Table 2

Changes in Total Haemocyte Count (THC) in *S. litura* larvae infected with selected entomopathogens
Treatment	1 HAI	3 HAI	6 HAI	12 HAI	24 HAI	48 HAI	72 HAI
B. bassiana	143 ± 2.08^bc	145 ± 2.65^c	149 ± 1.53^c	148 ± 2.08^c	148 ± 2^c	139 ± 2.08^c	121 ± 2.65^d
M. anisopliae	140 ± 2.52^cd	140 ± 2.52^cd	141 ± 1.25^d	144 ± 3^c	142 ± 1.53^d	138 ± 2.08^c	109 ± 1.73^e
Bt	151 ± 2.65^a	158 ± 2.65^b	280 ± 2.08^a	349 ± 1.73^a	438 ± 1.53^a	458 ± 3.21^a	204 ± 2.08^a
Sl NPV	148 ± 1.53^ab	180 ± 1^a	183 ± 2.31^b	201 ± 2.65^b	234 ± 2.52^b	184 ± 1.53^b	164 ± 1.53^b
Control	135 ± 2.65^d	134 ± 2^d	137 ± 0.88^d	142 ± 1.73^c	141 ± 1.15^d	139 ± 0.58^c	136 ± 1.53^c
*Different alphabets denote significant differences among treatments (P ≤ 0.05).

The overall THC overtime in S. litura larvae infected with different entomopathogens is summarized in Table 1. The number of total haemocytes after the infection of B. bassiana and Sl NPV were found to be increased up to 24 HAI. Bt showed highest number of haemocytes among all the entomopathogens and the number increased up to 48 HAI (458 ± 3.21^a). On the other hand, M. anisopliae showed increased number of total haemocytes up to 12 HAI (144 ± 3^c) towards which the larvae showed lowest THC among all the test entomopathogens.

Determination of Differential Haemocyte Count (DHC) through light microscopy

Giemsa staining of larval haemolymph and light microscopy observations revealed that PLs (Table 3) (Fig. 1c) and GRs (Table 4) (Fig. 1b) were the important haemocytes which are mainly involved in immune responses. The count of PLs and GRs increased up to 24 HAI for all entomopathogens other than Bt. In case of Bt treated larvae the PLs (156 ± 3.21^a) and GRs (194 ± 2.08^a) count was found to be increased up to 48 HAI. Bt treated larvae showed highest number of PLs and GRs up to 48 HAI. However, later the count started decreasing. Entomopathogenic fungi treated larvae showed lowest number of PLs and GRs in comparison to other test entomopathogens. The order of PLs count observed in different test entomopathogens were Bt > Sl. NPV > B. bassiana > Control > M. anisopliae (Fig. 3) and that of GRs was Bt > Sl NPV > B. bassiana > M. anisopliae (Fig. 4). Untreated control showed least GRs in comparison with any of the test entomopathogen.

Table 3

Changes in total Plasmatocytes (PLs) number in *S. litura* larvae infected with selected entomopathogens
Treatment	1 HAI	3 HAI	6 HAI	12 HAI	24 HAI	48 HAI	72 HAI
B. bassiana	61 ± 1.73^bc	64 ± 2.65^b	84 ± 2.08^b	85 ± 1.53^c	89 ± 2.52^c	77 ± 2.31^c	58 ± 2.52^c
M. anisopliae	43 ± 2.52^d	52 ± 2.31^c	68 ± 1.53^c	43 ± 1.53^e	39 ± 2.08^e	26 ± 2.65^e	25 ± 1.73^e
Bt	72 ± 2.08^a	89 ± 2.65^a	96 ± 1.53^a	109 ± 2.52^a	136 ± 2.52^a	156 ± 3.21^a	92 ± 2.08^a
Sl NPV	2 ± 1.53^b	68 ± 1.53^b	89 ± 1.53^a	96 ± 2.05^b	108 ± 2.31^b	98 ± 2.89^b	76 ± 2.52^b
Control	55 ± 2^c	54 ± 1.73^c	54 ± 2.08^d	56 ± 1.53^d	57 ± 2.52^d	56 ± 2.08^d	36 ± 2.65^d
*Different alphabets denote significant differences among treatments (P ≤ 0.05).

Table 4

Changes in total Granulocytes (GRs) number in *S. litura* larvae infected with selected entomopathogens
Treatment	1 HAI	3 HAI	6 HAI	12 HAI	24 HAI	48 HAI	72 HAI
B. bassiana	58 ± 2^c	91 ± 2.08^c	92 ± 2.08^c	95 ± 2.31^c	72 ± 2.52^b	53 ± 1.53^c	32 ± 2.52^c
M. anisopliae	52 ± 2^c	46 ± 2.65^d	53 ± 2.08^d	61 ± 2.52^d	54 ± 2.08^c	32 ± 2.08^e	28 ± 2.08^c
Bt	97 ± 2.08^a	147 ± 2.31^a	158 ± 2.65^a	189 ± 3^a	194 ± 2.08^a	92 ± 1.73^a	58 ± 2.52^a
Sl NPV	67 ± 2.65^b	118 ± 2.65^b	120 ± 1.53^b	121 ± 3^b	76 ± 2^b	60 ± 1.53^b	48 ± 2.08^b
Control	35 ± 2.08^d	42 ± 2.89^d	39 ± 2.08^e	45 ± 2.08^e	48 ± 2.89^c	46 ± 2.52^d	42 ± 2.52^b
*Different alphabets denote significant differences among treatments (P ≤ 0.05).

The overall DHC overtime in S. litura larvae infected with different entomopathogens is summarized in Tables 3 and 4. The number of PLs and GRs after the infection of

B. bassiana was increased up to 24 HAI. In case of Sl NPV the number of PLs was increased up to 24 HAI and GRs up to 12 HAI. Whereas Bt showed highest number among all the entomopathogens and the number of PLs and GRs increased up to 48 HAI and 24 HAI, respectively. On the other hand, M. anisopliae showed increased number of PLs and GRs up to 6 HAI and 12 HAI respectively towards which the larvae showed lowest DHC among all the entomopathogens.

Effect of test entomopathogens on phagocytosis

Infection of S.litura larvae with different test entomopathogens showed that maximum phagocytosed cells were observed in Bt and minimum was observed in case of

M. anisopliae. The order of phagocytosis observed in different test entomopathogens were Bt > Sl NPV > B. bassiana = M. anisopliae. Untreated control showed least phagocytic activity in comparison with any of the test entomopathogen (Fig. 5).

The number of phagocytosed cells increased up to 24 HAI when treated with

B. bassiana (36 ± 2.52^c), M. anisopliae (32 ± 1.53^c) and Sl NPV (49 ± 2.08^b). On the other hand, Bt showed highest phagocytic activity 48 HAI (126 ± 1.53^a) (Table 5).

Table 5

Number of phagocytosed cells observed in *S. litura* larval haemolymph after infection with selected entomopathogens
Treatment	1 HAI	3 HAI	6 HAI	12 HAI	24 HAI	48 HAI	72 HAI
B. bassiana	3 ± 0.58^c	16 ± 2.52^c	22 ± 2.65^c	29 ± 2.52^c	36 ± 2.52^c	13 ± 1.15^c	8 ± 0.58^bc
M. anisopliae	1 ± 0.58^c	14 ± 1.73^c	19 ± 1.53^c	24 ± 1.53^c	32 ± 1.53^c	10 ± 0.58^c	6 ± 1.15^c
Bt	26 ± 2.52^a	47 ± 2.08^b	65 ± 1.15^a	89 ± 1.53^a	96 ± 1.53^c	126 ± 1.53^a	37 ± 1^a
Sl NPV	19 ± 1.53^b	58 ± 1.53^a	59 ± 2.31^b	61 ± 2.85^b	49 ± 2.08^b	24 ± 1.15^b	10 ± 1.53^b
Control	-	2 ± 0.58^d	3 ± 0.58^d	2 ± 0.58^d	1 ± 0.58^d	-	-
*Different alphabets denote statistical differences among treatments (P ≤ 0.05).

Effect of test entomopathogens on nodulation

Infection of S. litura larvae with different test entomopathogens showed that maximum nodule formation (Fig. 1d) was observed in Bt (72 ± 2.31^a) and minimum was observed in case of M. anisopliae (32 ± 2.52^c). The order of nodulation observed in different test entomopathogens was Bt > Sl NPV > B. bassiana ≥ M. anisopliae. Untreated control showed least number of nodules in comparison with any of the test entomopathogen (Fig. 6).

The number of nodules formed were increased up to 24 HAI for all entomopathogens. However, the number started decreasing later (Table 6).

Table 6

Number of nodules formed in *S. litura* larval haemolymph after infection with selected entomopathogens
Treatment	1 HAI	3 HAI	6 HAI	12 HAI	24 HAI	48 HAI	72 HAI
B. bassiana	10 ± 0.58^c	12 ± 1^c	24 ± 1^c	33 ± 1.53^c	38 ± 1.53^c	12 ± 1.53^c	8 ± 0.58^b
M. anisopliae	-	5 ± 1.15^d	28 ± 1.73^c	28 ± 1.53^c	32 ± 2.52^c	17 ± 2.65^c	2 ± 0.58^c
Bt	24 ± 0.58^a	41 ± 1.73^a	56 ± 2.52^a	68 ± 1.53^a	72 ± 2.31^a	34 ± 2^a	32 ± 2^a
Sl NPV	15 ± 0.58^b	35 ± 2^b	48 ± 2.08^b	59 ± 3.21^b	45 ± 2.08^b	25 ± 2.08^b	29 ± 2.65^a
Control	-	1 ± 0.58^d	3 ± 0.58^d	2 ± 0.58^d	1 ± 0.58^d	-	-
*Different alphabets denote statistical differences among treatments (P ≤ 0.05).

Humoral immune responses of S. litura to the selected entomopathogens

The humoral immune responses of S. litura in terms of important parameters were studied and outcome of the studies are discussed.

Effect of test entomopathogens on Phenoloxidase (PO) activity

The highest activity of PO was observed after infection with Sl NPV at 3 HAI then followed by the Bt (6.91 ± 0.16^a) and minimum was observed when infected with

M. anisopliae. The order of PO activity observed in different test entomopathogens were Bt > Sl NPV > B. bassiana > M. anisopliae. Untreated control showed least PO activity in comparison with any of the test entomopathogen (Fig. 7).

The PO activity was increased up to 24 HAI in all entomopathogens. On the other hand, Sl NPV showed highest PO activity at 3 HAI (9.45 ± 0.30^a) (Table 7).

Table 7

Phenoloxidase (PO) specific activity (µmol/min) measured in *S.litura* larval haemolymph after infection with selected entomopathogens
Treatment	1HAI	3HAI	6HAI	12HAI	24HAI	48HAI	72HAI
B. bassiana	0.98 ± 0.15^a	2.97 ± 0.14^c	3.94 ± 0.09^c	3.94 ± 0.08^b	4.31 ± 0.0.20^b	1.03 ± 0.20^b	0.92 ± 0.19^a
M. anisopliae	0.84 ± 0.23^a	0.63 ± 0.18^d	2.87 ± 0.10^d	2.91 ± 0.15^c	3.02 ± 0.07^c	0.73 ± 0.10^b	0.64 ± 0.08^a
Bt	1.34 ± 0.56^a	6.12 ± 0.27^b	6.58 ± 0.59^a	6.82 ± 0.14^a	6.91 ± 0.16^a	5.32 ± 0.39^a	2.86 ± 1.14^a
Sl NPV	1.21 ± 0.31^a	9.45 ± 0.30^a	5.01 ± 0.37^b	3.01 ± 0.09^d	2.38 ± 0.66^c	1.38 ± 0.44^b	1.21 ± 0.27^a
Control	0.83 ± 0.23^a	0.67 ± 0.10^d	0.64 ± 0.09^e	0.73 ± 0.19^e	0.68 ± 0.15^d	0.71 ± 0.08^b	0.62 ± 0.10^a
*Different alphabets denote statistical differences among treatments (P ≤ 0.05).

Effect of test entomopathogens on Lysozyme activity

The highest activity of lysozyme was observed after infection with Bt (5.91 ± 0.56^a) and minimum activity was observed when infected with M. anisopliae. The activity was increased up to 24 HAI in all entomopathogens (Fig. 7). The order of Lysozyme activity observed in different test entomopathogens were Bt ≥ Sl NPV > B. bassiana > M. anisopliae (Fig. 8). Untreated control showed least lysozyme activity in comparison with any of the test entomopathogens (Table 8).

Table 8

Lysozyme specific activity (µmol/min) measured in *S. litura* larval haemolymph after infection with selected entomopathogens
Treatment	1 HAI	3 HAI	6 HAI	12 HAI	24 HAI	48 HAI	72 HAI
B. bassiana	1.35 ± 0.04^bc	1.42 ± 0.32^b	2.45 ± 0.34^b	2.48 ± 0.44^bc	3.96 ± 0.16^bc	1.92 ± 0.43^c	0.82 ± 0.02^cd
M. anisopliae	0.98 ± 0.09^c	1.32 ± 0.18^bc	1.49 ± 0.27^c	1.81 ± 0.55^bc	3.15 ± 0.13^c	0.92 ± 0.06^d	0.74 ± 0.08^d
Bt	2.43 ± 0.28^a	2.91 ± 0.08^a	3.89 ± 0.21^a	5.78 ± 0.52^a	5.91 ± 0.56^a	4.2 ± 0.08^a	1.21 ± 0.07^a
Sl NPV	1.54 ± 0.07^b	3.12 ± 0.14^a	3.62 ± 0.17^a	3.45 ± 0.68^b	4.61 ± 0.61^ab	2.56 ± 0.05^b	1.09 ± 0.02^ab
Control	0.95 ± 0.04^c	1.08 ± 0.41^c	1.17 ± 0.35^c	1.15 ± 0.47^c	1.13 ± 0.47^d	0.86 ± 0.06^d	0.94 ± 0.04^bc
*Different alphabets denote statistical differences among treatments (P ≤ 0.05).

The environmental concerns raised by the continued use of chemical pesticides, the rising of resistant strains of insect pests and the suitable conditions for the utilization of entomopathogens in crop fields, represent promising premises for their use as biological control agent of S. litura in India. The identification of haemocyte types of an insect represents the first step of a comprehensive immune characterization and it is fundamental for understanding how the immune system may react against a specific pathogen. The Total Haemocyte Count was observed to be increasing sharply initially after the inoculation of the entomopathogens however later decreased with time. These was well documented in Chilo suppressalis walker larvae after the infection by the several natural entomopathogenic fungi (Shahriari et al., 2021). Cellular immunological response of the greater wax moth, Galleria mellonella to Aspergillus niger-1,3-glucan changes the THC which pointed to the activation of G. mellonella cellular processes in response to the administration of A. niger α-1,3-glucan. The DHC increased sharply in the initial few hours, but they gradually decreased later. Observation in alterations of phagocytosis as a cellular response in the greater wax moth G. mellonella exposed to A. niger-1,3-glucan. The creation of haemocyte aggregates and alterations in phagocytosis have been reported. (Stączek et al., 2020).

Through light microscopy observations, two basic types of haemocytes viz., Plasmatocytes (PLs) and Granulocytes (GRs) were identified in larvae of S. litura. These types of haemocytes were well characterized in other lepidopteran larvae (Lavine and Strand, 2002; Nakahara et al., 2009). Due to the similarity in cell shape between S. litura and Bombyx mori, we utilised the results of the study by Nakahara et al. (2009). It is essential to note that the specific response of PLs and GRs can vary depending on the type of entomopathogen, the dose of the pathogen injected and the insect species being studied. Additionally, the time frame in which these changes occur can vary as well. In S. litura larvae, entomopathogen isolates altered both total and differential haemocyte numbers. Although different entomopathogens had impacts with varying intensities, the period between 24 and 48 HAI had the greatest increase in the number of haemocytes. After the first 24 to 48 HAI spike, there was a decline at 72 HAI.Several previous studies also described this type of variation in haemocytes after pathogenic stresses such as in Schistocerca gregaria L. (Orthoptera: Acrididae) (Gunnarsson and Lackie (1985), Melanoplus sanguinipes Fabricius (Orthoptera: Acrididae) Bidochka and Khachatourians, 1987), Periplaneta americana L. (Blattaria: Blattidae) Spodoptera exigua Hubner (Lepidoptera: Noctuidae) (Hung and Boucias, 1992), Galleria mellonella L. (Lepidoptera, Pyralidae) (Sewify and Hashem, 2001), Reticulitermes flavipes Kollar (Isoptera: Rhinotermitidae) (Chouvenc et al., 2009), Oxya japonica Thunberg (Orthoptera: Acrididae) and Eurygaster integriceps Puton (Hemiptera: Scutelleridae) (Zibaee et al., 2011).

One of the primary cellular reactions of insects to pathogens is nodule formation, especially when the invader is too large to be absorbed by phagocytosis (Chouvenc et al., 2009). Entomopathogenic extracts stimulated phagocytosis and nodulation in S. litura larvae. The highest number of nodules, encapsulated cells and phagocytic cells were observed at 12 to 48 HAI concurrently with the rise in the number of circulating haemocytes. Solter et al. (1997) and Solter and Maddox (1998) suggested that encapsulation and nodule development were associated with infections in lepidopteran larvae. Haemocyte adherence to the surface of non-self-targets causes cellular encapsulation or nodulation (Ling and Yu, 2006). Comparative studies on Eurygaster integriceps, Ephestia kuehniella and Zophobas morio when exposed to five different B. bassiana isolates to examine several defensive responses. They found that at 24 hours, the highest nodulation rate was observed (Seyedtalebi et al., 2017).

PO cascade and melanization are intimately connected with encapsulation. POs are stocked pre-enzymes or zymogens in insect hemolymph and they become activated upon wounding or infection as part of the innate immune response (Kanost & Gorman, 2008). The enzymes have two biochemical functions in hydroxylation of tyrosine to form L-dihydroxyphenylalanine, and oxidizing o-diphenols to form quinones (Gorman et al., 2007). After forthcoming reactions, quinones form melanin, which is deposited on the surface of encapsulated parasites, hemocyte nodules and wound sites (Kanost & Gorman, 2008). In our experiments, the highest activity of PO was found after 6 and 12 h post-injection which it corresponds to the hemocyte increase and nodule formation. Results may be explained by melanin deposition as a complement of the nodule formation process. The melanin deposition (melanization) of nodules and capsules around non-self-target is one of the important defensive responses in insects (Gillespie et al., 1997; Christensen et al., 2005; Michel & Kafatos, 2005; Nappi & Christensen, 2005; Chouvenc et al., 2009; Zibaee et al., 2011; Black et al., 2022). Melanin deposition blocks absorption of nutrients by parasites leading to their killing due to starvation (Chen & Chen, 1995). Also, formation of cytotoxic reactive oxygen and nitrogen intermediates during melanin synthesis causes to kill invading organisms (Nappi & Christensen, 2005).At the very least, a preliminary nonspecific component of the lysozyme activity to the entomopathogens was found. There is an increase in lysozyme activity at 24 HAI with B. bassiana blastospores in infected G. mellonella larvae (Wojda et al.,2009). Lysozymes are the proteins which are widely distributed and are found in insects, vertebrates, plants and microorganisms. They are muramidases that cause the lysis of the peptidoglycan layer of bacterial cell by hydrolyzing the 1-4-β-linkage between the N-acetylmuramic acid and N-acetylglucosamine residues. The enhanced activity of lysozyme in infected insects suggests that, unlike its constitutively produced vertebrate equivalent, this protein is inducible in lepidopterans (Daffre et al., 1994).

In conclusion, our study demonstrated that S. litura larvae present at least two different types of circulating hemocytes, in agreement with previous observations performed in other lepidopteran models. Our experiments evidenced a different susceptibility of S. litura larvae to different test entomopathogens. In all cases the effects of the entomopathogens involved cell-mediated immunity that is initially triggered and then strongly reduced. The immediate response of larvae to entomopathogens infection was a significant increase of total circulating hemocytes. This increase included both granulocytes and plasmatocytes. However, after 3 days from the immune challenge, the number of circulating hemocytes was significantly lower. Consequently, the larvae showed Phagocytosis, nodulation, increased PO and Lysozyme activity in the immediacy of the challenge, but progressively the phenomenon decreased. Further research is required in order to completely understand the effects of entomopathogens on immune system of S. litura. These results combined with field mortality caused by entomopathogens will lead to the arsenal of bio-compatible controller for the S. litura.

Funding

This study was funded by Professor Jayashankar Telangana State Agricultural University (PJTSAU), Hyderabad, Telangana, India.

Author Contribution

Adama Thanuja, conducted research work and wrote the main manuscript.SJ Rahman, Helped in the research work and reviewed the manuscriptP. Rajanikanth and Bharati N Bhat reviewed the manuscript

Acknowledgements

The authors are highly thankful to Central Instrumentation Cell (CIC), teaching & non-teaching staff of Department of Entomology, PJTSAU, Hyderabad, India for providing all the facilities and guidance while carrying out the research at insect pathology lab of Department of Entomology, College of Agriculture, PJTSAU, Rajendranagar.

Data availability

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

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Investigations into the cellular and humoral immune mechanisms of Spodoptera litura (Fabricius) against potential entomopathogens

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Abstract

Figures

Introduction

Materials and methods

Insect Rearing

Entomopathogens and Inoculation procedure

Collection of haemolymph from infected larvae

Characterization of the haemocytes

Haemocytes count

Determination of Total Haemocyte Count (THC)

Determination of Differential Haemocyte Count (DHC)

Phagocytosis

Nodulation

Collection of haemolymph

PO preparation

Lysozyme Activity

Data Analysis

Results

Determination of Total Haemocyte Count (THC) through light microscopy

Determination of Differential Haemocyte Count (DHC) through light microscopy

Effect of test entomopathogens on phagocytosis

Effect of test entomopathogens on nodulation

Effect of test entomopathogens on Phenoloxidase (PO) activity

Effect of test entomopathogens on Lysozyme activity

Discussion

Conclusion

Declarations

Funding

Author Contribution

Acknowledgements

Data availability

References

Additional Declarations

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