Generation of recombinant baculovirus carrying Gal-1 gene
To investigate whether the overexpression of galectin-1 could improve the biopesticide potential of baculovirus, a recombinant baculovirus, vABpRpGal1, carrying the Gal-1 gene under the control of the very-late polyhedrin promoter and dsRed gene as a reporter gene was generated. A control virus carrying only the dsRed gene was also generated (Fig. 1A). IPLB-Sf-21 cells infected with vABpRpGal1 exhibited red fluorescence 48 h post infection (Fig. 1B), and the expression of Gal-1 in infected cells was confirmed by western blot analysis (Fig. 1C).
Recombinant baculovirus carrying gal-1 gene significantly induced higher mortality in S. litura larvae
To evaluate the toxicity of recombinant vABpRpGal1 in S. litura larvae, the virus was added once to the artificial feed on day one of the experiment, larvae were observed for 7 days, and the mortality was recorded daily. There was no difference between the vABpRpGal1 and control virus groups in the survival rate (over 95% for both) (Fig. 2A). Larvae fed with vABpRpGal1 were smaller (Fig. 2B), and therefore, the surviving larvae were placed on sweet potato leaves (one leaf per larva) to assess their appetite. The larvae from the control virus group ingested more than 50% of the leaves, whereas the larvae from the vABpRpGal1 group ingested less than 50% of the leaves (Fig. 2C and 2D), indicating that vABpRpGal1 reduced larval appetite.
To evaluate the effect of continuous vABpRpGal1 exposure in larvae, viruses were added to the artificial feed daily for 7 days. The larvae fed with the control virus had a survival rate of 80% on day 7, whereas larvae fed with vABpRpGal1 had a survival rate of less than 40% on day 5 and less than 20% on day 7 (Fig. 3A). The analysis of body length showed that, starting on day 3 of the experiment, larvae in virus groups exhibited slower growth rates compared to those in the control group, and larvae in the vABpRpGal1 group started to show signs of growth arrest on day 5 and had the shortest body length on day 6 (Fig. 3B and 3C). These results suggested that galectin-1 could negatively influence the appetite and growth of S. litura larvae.
Galectin-1 increases the vulnerability of S. litura larvae to baculovirus by suppressing the immune system
Galectin-1 was shown to bind to receptors on the T cell surface in mammals and subsequently induce apoptosis, resulting in a suppressed immune system. This is also a tactic used by cancer cells to avoid immune system attacks. To evaluate the effect of vABpRpGal1 infection on the immune response of S. litura, the expression of genes involved in the Toll pathway was measured as pathogen-induced humoral immunity in insects mediated mainly via the Toll pathway. Upstream toll-like receptors and downstream anti-bacterial peptide cecropin expression levels increased in larvae infected with the control virus, suggesting that pathogen infection elicited an immune response. In contrast, the expression levels of upstream toll-like receptors (Toll), Myd88, Pelle, and downstream anti-bacterial peptide cecropin were lower in the vABpRpGal-1 group (Fig. 4A), suggesting that Gal-1 had an inhibitory effect on the Toll pathway. Similar results were obtained when the expression of genes involved in the IMD pathway, including pgrp, fadd, and relish, was analyzed (Fig. 4B), indicating that Gal-1 suppressed the expression of genes involved in the immune response of host larvae.
Phagocytosis is the primary cell-mediated immune response in insects. To evaluate the effect of Gal-1 on cell-mediated immunity, the phagocytic activity of hemocytes collected from S. litura larvae 48 h after infection with baculovirus was measured using FITC-labeled E. coli. Phagocytosed E. coli emitted green fluorescence when observed under a fluorescence microscope, while the fluorescence from un-phagocytosed E. coli was masked by trypan blue dye. Fluorescence microscopy images showed green fluorescence in the control and control virus groups, whereas significantly less green fluorescence signals were detected in the vABpRpGal1 group (Fig. 4C, left). Phagocytic activity (phagocytosis) was determined based on the ratio of the number of fluorescent cells to the total number of cells. The phagocytic activity was inhibited following viral infection, and the activity was further inhibited by the expression of Gal-1 (Fig. 4C, right). Combining these results, we concluded that Gal-1 exerted an inhibitory effect on both humoral and cell-mediated immune responses.
Recombinant baculovirus expressing Gal-1 has potential as a bio-pesticide in the field
To evaluate the potential of recombinant baculoviruses expressing Gal-1 as bio-pesticides, a field trial was conducted. Eight to ten lettuce plants were planted in a box (40 cm × 60 cm, Fig. 5A), and the leaves were sprayed with a solution containing control virus, vABpRpGal1, or sterile water for the control group. In total, 40 or 80 S. litura larvae were evenly distributed on the leaves. To evaluate the toxicity of recombinant vABpRpGal1 in S. litura larvae, leaves were sprayed with virus once on day 1 of the experiment. Larvae were observed for 1 to 7 days, and the mortality was recorded daily. The survival rate was measured and photographs were taken of the leaves ingested by the larvae after 2 days (cabbage) and 3 days (lettuce). Ingested lettuce photographs showed that larvae without viral infection ingested almost all the leaves, whereas larvae infected with the control virus ingested approximately half of the leaves, and larvae infected with vABpRpGal1 ingested the fewest leaves (Fig. 5B). The survival rate for the control and control virus groups was 95% and 85%, respectively, whereas it was only 52.5% for the vABpRpGal1 group (Fig. 5C). The other two lettuce replicates showed the same trend (Fig. 5D and 5E).
Similar results were obtained when cabbage was used as the food source, with a survival rate of 97.5% for the control, 82.5% for the control virus, and 70% for the vABpRpGal1 groups (data not shown). These results indicated that overexpression of Gal-1 increased the lethality of baculovirus in S. litura larvae. To evaluate the bio-pesticide potential of recombinant vABpRpGal1 in other pests, a field experiment was conducted using fall armyworm (Spodoptera frugiperda) and diamondback moth larvae, and similar results were obtained as those with S. litura larvae (Fig. 6A and 6B). When the experiment was conducted using insects with economic value, such as honeybees, recombinant vABpRpGal1 did not exert any toxicity (Fig. 6C), suggesting its safety in the field for non-host insects. Generally, using viruses to control pests results in worse outcomes compared to using bacteria. The insecticidal efficiency of recombinant baculovirus expressing Gal-1 was compared with another bio-insecticide, Bacillus thuringiensis, and the results showed that the insecticidal effects (growth and survival rate) of the Gal-1 virus did not differ significantly from those of B. thuringiensis (Fig. 6D). Thus, the constructed recombinant virus expressing Gal-1 exhibits potential for practical application as a bio-pesticide.