Midgut Of Spodoptera Exigua Research Articles

Characterization of CRY2AB-toxin-binding proteins in Spodoptera exigua midgut

Characterization of CRY2AB-toxin-binding proteins in Spodoptera exigua midgut

Three GPI-anchored alkaline phosphatases are involved in the intoxication of Cry1Ca toxin to Spodoptera exigua larvae.

Glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (ALP) isoforms are crucial for the intoxication of crystal proteins (Cry) to several insect species. We cloned five SeALPs from the larval midgut of Spodoptera exigua, a major pest of many crops. All five SeALPs contain a signal peptide at the N-terminus, a phosphatase domain, and a GPI-anchor site at the C-terminus. Additionally, the sequences encode two or three potential N-glycosylation sites. The five SeALPs were highly expressed at the larval stage, especially in the larval gut or Malpighian tubules. Ingestion over four consecutive days of double-stranded RNAs (dsRNAs) targeting SeALP1, SeALP2, SeALP3, SeALP4, and SeALP5 significantly reduced the corresponding mRNA levels by 60.0%, 40.0%, 65.6%, 48.1%, and 69.1% respectively, compared with the levels in control larvae that fed on non-specific dsRNA (dsEGFP). When larvae that previously ingested phosphate buffered saline (PBS)-, dsEGFP-, or five dsSeALPs-overlaid diets were then exposed to a diet containing Cry1Ca, the larval mortalities after six days were 70.0%, 71.8%, 49.1%, 54.9%, 65.3%, 52.5%, and 77.4%, respectively. ANOVA analysis revealed that the larvae that previously fed on the dsSeALP1-, dsSeALP2-, and dsSeALP4-overlaid diets had significantly lower mortalities than those that previously ingested the PBS-, dsEGFP-, dsSeALP3- and dsSeALP5-overlaid diets. The results suggest that SeALP1, SeALP2 and SeALP4 are involved in the intoxication of Cry1Ca to S. exigua larvae.

Proteolytic activation of Bacillus thuringiensis Vip3Aa protein by Spodoptera exigua midgut protease

Proteolysis of Vip3Aa by insect midgut proteases is essential for their toxicity against target insects. In the present study, proteolysis of Vip3Aa was evaluated by Spodoptera exigua midgut proteases (MJ). Trypsin was verified involved in the activation of Vip3Aa and three potential cleavage sites (Lys195, Lys197 and Lys198) were identified. Four Vip3Aa mutants (KKK195197198AAA, KK197198AA, KK195198AA and KK195197AA) were designed and constructed by replacing residues Lys195,197,198, Lys197,198, Lys195,198 and Lys195,197 with Ala, respectively. Proteolytic processing assays revealed that mutants KK197198AA, KK195198AA and KK195197AA could be processed into 66kDa activated toxins by trypsin or MJ while mutant KKK195197198AAA was not cleaved by trypsin and less susceptible to MJ. Bioassays demonstrated that mutants KK197198AA, KK195198AA and KK195197AA were toxic against S. exigua resembled that of wild-type Vip3Aa, however, the LC50 of mutant KKK195197198AAA against S. exigua was higher than wild-type. Those results suggested that proteolysis by MJ was associated with the insecticidal toxicity of Vip3Aa against S. exigua. It also revealed that trypsin played an important role in the formation of Vip3Aa activated toxin. Our studies characterized the proteolytic processing of Vip3Aa and provided new insight into the activation of this novel Bt toxin.

Enhancement of Bacillus thuringiensis Cry1Ac and Cry1Ca toxicity against Spodoptera exigua (Hübner) by suppression of a chitin synthase B gene in midgut

The chitin synthase B (CHS-B) gene was cloned from midgut of Spodoptera exigua (SeCHS-B). SeCHS-B contains an open reading frame of 4572 nucleotides, encoding a protein of 1523 amino acids with a predicted molecular mass of approximately 174.6kDa. The SeCHS-B mRNA was expressed in all developmental stages and specifically in the midgut tissue by qRT-PCR analysis. Expression of SeCHS-B gene was suppressed by feeding double-stranded RNA (dsCHS-B, 400ng/larva) in the third and fifth instar larvae of S. exigua. Suppression of the SeCHS-B gene expression significantly increased 20% and 25% of mortality on pupation and adult emergence of S. exigua. Also, feeding of dsCHS-B significantly enhanced Cry toxin toxicity to third instar larvae as 34% in 10μg Cry1Ac and 29% in 0.8μg Cry1Ca at 6days after treatment. These results suggest that the SeCHS-B gene plays an important role in development of S. exigua and RNA interference may apply to effective pest control with B. thuringiensis.

The Spodoptera exigua (Lepidoptera: Noctuidae) ABCC2 Mediates Cry1Ac Cytotoxicity and, in Conjunction with Cadherin, Contributes to Enhance Cry1Ca Toxicity in Sf9 Cells.

In insects, the mode of Cry1A toxins action has been studied in detail and many receptors that participate in the process are known. Recent evidence has revealed that an ABC transporter (ABCC2) is involved in conferring resistance to Cry1A toxins and that ABCC2 could be a receptor of Cry1A. However, it is not known whether Cry1Ca interacts with the same receptor proteins as Cry1A. In this study, we report the cloning of an ABC transporter gene, SeABCC2b, from the midgut of Spodoptera exigua (Hübner) larvae, and its expression in Sf9 cells for a functional analysis. The addition of Cry1Ca and Cry1Ac to Sf9 cell culture caused swelling in 28.5% and 93.9% of the SeABCC2-expressing cells, respectively. In contrast, only 7.4% and 1.3% of the controls cells swelled in the presence of Cry1Ca and Cry1Ac. Thus, SeABCC2b-expressing Sf9 cells had increased susceptibility to Cry1Ca and Cry1Ac. Similarly, S. exigua cadherin (SeCad1b) expressed in Sf9 cells caused 47.1% and 1.8% of the SeCad1b-expressing cells to swell to Cry1Ca and Cry1Ac exposure. Therefore, Sf9 cells expressing SeCad1b were more sensitive to Cry1Ca than Cry1Ac. Together, our data suggest that SeABCC2b from S. exigua mediates Cry1Ac cytotoxicity and, in conjunction with SeCad1b, contributes to enhance Cry1Ca toxicity in Sf9 cells.

Dissimilar Regulation of Antimicrobial Proteins in the Midgut of Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Toxins or Baculovirus.

Antimicrobial peptides (AMPs) and lysozymes are the main effectors of the insect immune system, and they are involved in both local and systemic responses. Among local responses, midgut immune reaction plays an important role in fighting pathogens that reach the insect body through the oral route, as do many microorganisms used in pest control. Under this point of view, understanding how insects defend themselves locally during the first phases of infections caused by food-borne pathogens is important to further improve microbial control strategies. In the present study, we analyzed the transcriptional response of AMPs and lysozymes in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae), a polyphagous pest that is commonly controlled by products based on Bacillus thuringiensis (Bt) or baculovirus. First, we comprehensively characterized the transcripts encoding AMPs and lysozymes expressed in S. exigua larval midgut, identifying 35 transcripts that represent the S. exigua arsenal against microbial infection. Secondly, we analyzed their expression in the midgut after ingestion of sub-lethal doses of two different pore-forming B. thuringiensis toxins, Cry1Ca and Vip3Aa, and the S. exigua nucleopolyhedrovirus (SeMNPV). We observed that both Bt toxins triggered a similar, wide and in some cases high transcriptional activation of genes encoding AMPs and lysozymes, which was not reflected in the activation of the classical systemic immune-marker phenoloxidase in hemolymph. Baculovirus ingestion resulted in the opposed reaction: Almost all transcripts coding for AMPs and lysozymes were down-regulated or not induced 96 hours post infection. Our results shed light on midgut response to different virulence factors or pathogens used nowadays as microbial control agents and point out the importance of the midgut immune response contribution to the larval immunity.

RNA interference-mediated knockdown of three putative aminopeptidases N affects susceptibility of Spodoptera exigua larvae to Bacillus thuringiensis Cry1Ca

Aminopeptidase N (APN) isoforms in insects have been documented to be involved in the mode of action of insecticidal crystal proteins (Cry) from Bacillus thuringiensis. Here we cloned two novel Seapns from the larval midgut of Spodoptera exigua, a major pest of many crops of economic importance in China. According to a phylogenetic analysis, these two novel SeAPNs, along with the four SeAPN isoforms already described, belong to six different clades. All the six SeAPNs share similar structural features. From N- to C-terminus a signal peptide, a gluzincin aminopeptidase motif, a zinc binding/gluzincin motif, and a glycosylphosphatidylinositol-anchor sequence are located. The six Seapn genes were highly expressed at the larval stage, especially in the larval gut. Ingestion during four consecutive days of double-stranded RNAs (dsRNAs) targeting Seapn1, Seapn2, Seapn3, Seapn4, Seapn5 and Seapn6 significantly reduced corresponding mRNA levels by 55.6%, 45.5%, 43.2%, 56.8%, 45.4%, and 46.0% respectively, compared with those recorded in control larvae fed on non-specific dsRNA (dsegfp). When the larvae that previously ingested phosphate buffered saline (PBS)-, dsegfp-, or six dsSeapns-overlaid diets were then exposed to a diet containing Cry1Ca, the larval mortalities were 71.2%, 69.3%, 52.0%, 77.2%, 43.3%, 62.0%, 65.4% and 53.8% respectively recorded after 6days. ANOVA analysis revealed that the larvae previously fed on dsSeapn1-, dsSeapn3-, and dsSeapn6-overlaid diets had significantly lower mortalities than those previously ingested PBS-, dsegfp-, dsSeapn2-, dsSeapn4- and dsSeapn5-overlaid diets. Thus, these results suggest that SeAPN1, SeAPN3 and SeAPN6 may be candidate receptors for Cry1Ca in S. exigua.

RNA interference of cadherin gene expression in Spodoptera exigua reveals its significance as a specific Bt target

Cadherin is a calcium-dependent cell surface molecule and has been proposed to be a target molecule of Bacillus thuringiensis Cry proteins. Based on a partial cadherin gene obtained from 454 pyrosequencing transcripts of the fifth instar Spodoptera exigua midgut, a full open reading frame of cadherin gene (SeCad1) was cloned. Its predicted amino acid sequence encodes extracellular 9 cadherin repeats, a transmembrane, and intracellular domain. SeCad1 gene was expressed in all developmental stages specifically in gut tissue by RT-PCR analysis. Expression of SeCad1 gene was suppressed by both injection and feeding of its specific double-stranded RNA (dsRNASeCad1) in the fifth instar. The suppression of SeCad1 expression did not significantly influence on pupal and adult developments of S. exigua. However, the larvae treated with dsRNASeCad1 (100ng/larva) significantly reduced susceptibility to B. thuringiensis ssp. aizawai. In contrast, the dsRNASeCad1-treated larvae did not show any change in susceptibility to B. thuringiensis ssp. kurstaki. These results suggest that SeCad1 is a specific target molecule to Cry1C toxin from B. thuringiensis in S. exigua, but not to Cry1A toxin.

A new gene superfamily of pathogen-response (repat) genes in Lepidoptera: Classification and expression analysis

Repat (REsponse to PAThogens) genes were first identified in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae) in response to Bacillus thuringiensis and baculovirus exposure. Since then, additional repat gene homologs have been identified in different studies. In this study the comprehensive larval transcriptome from S. exigua was analyzed for the presence of novel repat-homolog sequences. These analyses revealed the presence of at least 46 repat genes in S. exigua, establishing a new gene superfamily in this species. Phylogenetic analysis and studies of conserved motifs in these hypothetical proteins have allowed their classification in two main classes, αREPAT and βREPAT. Studies on the transcriptional response of repat genes have shown that αREPAT and βREPAT differ in their sequence but also in the pattern of regulation. The αREPAT were mainly regulated in response to the Cry1Ca toxin from B. thuringiensis but not to the increase in the midgut microbiota load. In contrast, βREPAT were neither responding to Cry1Ca toxin nor to midgut microbiota. Differential expression between midgut stem cells and the whole midgut tissue was studied for the different repat genes revealing changes in the gene expression distribution between midgut stem cells and midgut tissue in response to midgut microbiota. This high diversity found in their sequence and in their expression profile suggests that REPAT proteins may be involved in multiple processes that could be of relevance for the understanding of the insect gut physiology.

Functional interactions between members of the REPAT family of insect pathogen‐induced proteins

Studies on the transcriptional response to pathogens in the insect larval gut have shown the regulation of several genes after the infection. Repat (REsponse to PAThogens) genes were first identified in Spodoptera exigua midgut as being up-regulated in response to the exposure to Bacillus thuringiensis toxins and baculovirus. Recently, new members of the REPAT family showed a constitutive up-regulation in a B. thuringiensis-resistant population. Based on a yeast two-hybrid screening, we have detected the interaction of REPAT1 with other members of the REPAT family, leading to the discovery of a new member: REPAT8. The functional role of this interaction was shown by following the changes of the subcellular localization of REPAT1 in the presence of REPAT8. REPAT1 alone was localized exclusively in the cytoplasm, while the presence of REPAT8 led to the migration of REPAT1 to the nucleus. Finally, analysis of the expression pattern of eight REPAT members has shown that B. thuringiensis-related treatments (Cry1Ca toxin, Xentari™ product and an acrystalliferous strain) induced a general up-regulation of repat genes, especially of repat2. In contrast, no significant effect was detected after treatment with Escherichia coli or Enterococcus sp., or by the presence of microbiota in the midgut. The results suggest that the different repat genes play different roles in response to pathogens.

REPAT, a new family of proteins induced by bacterial toxins and baculovirus infection in Spodoptera exigua

Insect larvae spend most of their time eating and the digestive tract is the most crucial barrier for the entrance of many pathogens. In our study, suppression subtractive hybridization (SSH) was used to compare Spodoptera exigua midgut gene expression between larvae exposed to the Bacillus thuringiensis Cry1Ca toxin and non-exposed insects. Based on the SSH results, full cDNA sequences coding for four homologous proteins were obtained. Quantitative and semi-quantitative RT-PCR showed the increased expression of the genes coding for these proteins after exposure to different B. thuringiensis toxins as well as after infection with baculovirus. The proteins were named REPAT after their increased expression in Response to Pathogen. REPAT1, a member of this family, was recombinantly expressed using the baculovirus expression system, revealing the glycosylated nature of the protein. Recombinant baculoviruses expressing REPAT1 were used to infect larvae from S. exigua, showing that expression of REPAT1 was reducing the virulence of baculovirus to the infected larvae. Together, these results suggest a role for REPAT1 in mitigating pathological effects.

Effects of Gamma Radiation on the Ultrastructure and Acid Phosphatase Activity of the Mid-Gut of the Adult Beet Armyworm, Spodoptera exigua1,2

The effects of gamma radiation on the mid-gut of adult beet armyworms, Spodoptera exigua (Hubner), were investigated with the light and electron microscope. Control moths (0 krad) and moths irradiated with a 137Cs source (20, 40, and 120 krad) were sacrificed 24 and 132 h post-emergence. In control moths, microvilli, a single nucleus, rough endoplasmic reticulum, mitochondria, lysosomes, and a Golgi complex comprise the normal cytoarchitecture of individual columnar epithelial cells. These epithelial cells are supported by a continuous basal lamina, and inner circular and outer longitudinal muscle bands. Tissues of irradiated animals show no disruptions in the nucleus, various organelles, membranes or muscle layers. It appears that sub-sterilizing dosages (20 krad) and sterilizing dosages (40 krad) do not seriously effect the ultrastructure of the mid-gut initially (24 h postemergence) or with an increase in time (132 h postemergence). Males irradiated with 120 krad show no radiation effect to the mid-gut, although this dosage produces 50% mortality in 132 h (5-6 days). Resistance to the effect of gamma radiation is correlated with the absence of mitotic cell division. Cytochemical studies reveal no increase in acid phosphatase activity when mid-gut tissue from 120-krad-irradiated animals is compared to controls (0 krad). These results further confirm the extreme radioresistance of the mid-gut of Spodoptera exigua .