Suyog S. Kuwar

Suyog S. Kuwar

Research Scientist | Molecular biologist | Protein Engineering| | Entomologist | Mentor - looking for opportunity!

PhD Project

PhD project

Summary:

Insects have the most diverse habitats and hence the food sources. They are good in adaptation to food related challenges. One such challenge is protease inhibitors, a defense metabolite from plants produced in response to herbivory.

Insect midgut is the least defended site in order to control insects. The PIs can inhibit the proteases of insects, especially digestive proteases and retard the growth and development. However, millions of years of co-evolution between the host plants and insects, insects have acquired specific physiological adaptation of digestive and detoxificative enzymes to plant defenses. Insects have managed to feed on PI containing plants and known to produce PI-insensitive proteases when fed on PI-supplemented artificial diet. Our work reduce the knowledge gap to better understand the insect adaptation to PI. For this we tried to answer following questions:

1. How many digestive serine protease genes are present in the Helicoverap armigera and in sister species S. frugiperda and M. sexta?

  • There are at least 114, 86 and 77 serine protease sequences in H. armigera , S. frugiperda and M. sexta genomes, respectively.
  • Serine proteases are highly conserved at their catalytic residues, H57 D102, S195, and N-terminal signature sequence.
  • There are five major groups in the gene family; one group is azurocidine like, with mutated catalytic residues His57 to Ser and/ or Ser195 to Leu/Ile, might not be active and two groups of chymotrypsin differing in the pro-peptide length.
  • Most genes were found in large gene clusters and good correlation between intron conservation and phylogenetic relationships of serine proteases.

Ready for submission: BMC Genomics

2. Does H. armigera larvae has feeding choice when given PI-supplemented and PI-free diet, and how the PIs effect on life history traits?

  • Neonate and 4th instar H. armigera larvae prefers PI-free diet compare to PI-supplemented artificial diet.
  • Larvae on PI-supplemented diet weighed less compared to PI-free diet, trypsin-specific PIs had a stronger effect on mean larval weight than other PIs.
  • Pupal weights were also reduced for larvae fed on PI-supplemented diet.
  • Reduction of trypsin activity, but not of chymotrypsin activity was observed in larvae fed on PI-supplemented diets compared to PI-free diet.

Under review

3. How are digestive proteases regulated in the midgut of H. armigera larvae in response to a plant protease inhibitor, SKTI (soybean Kunitz trypsin inhibitor)?

  • We performed a microarray experiment in the midgut of H. armigera larvae fed on SKTI-free and SKTI-supplemented artificial diet.
  • Although initially the expression of several trypsins and chymotrypsins increased, eventually the expression of some trypsins decreased, while the number of chymotrypsins and their expression increased in response to SKTI. Diverged serine proteases were also differentially expressed.
  • We validated the gene expression of candidate genes using qRT-PCR at different time points (12, 24, 48, 72 and 96 h).
  • Carbohydrate metabolism and immune defense genes were affected in response to SKTI ingestion.
  • Enzyme assays revealed reduced trypsin-specific activity and increased chymotrypsin-specific activity in response to SKTI.
  • We observed poor larval growth on the SKTI-supplemented diet until 24 h, however, after 48 h larvae attained comparable weight to that of SKTI-free diet.

Published

4. How are the digestive and detoxificative genes regulated in the midgut of H. armigera in response to two hosts (cotton and soybean) leaves diet, containing different defense metabolites?

  • We performed comprehensive microarray experiments in the midgut of H. armigera larvae fed on cotton leaves, soybean leaves.
  • We observed diet specific expression of serine protease, cytochrome P450 monooxygenase, UDP-glycosyl transferase, glutathione S-transferase and esterase gene families.
  • Enzyme assays reveal reduced trypsin, chymotrypsin and amylase activity in the lumen of soybean leaf and cotton leaf fed larvae compared to artificial diet fed larvae.
  • We measured the enzyme activity in response to cottons most abundant secondary toxic metabolite, gossypol, in the larvae fed on gossypol-supplemented artificial diet.
  • We found reduced trypsin, chymotrypsin and amylase activity in response to gossypol feeding.
  • The activity was reduced in the larvae fed cotton leaves, followed by gossypol-supplemented artificial diet and soybean leaves.
  • We found excess loss of enzymes in the frass of larvae fed on gossypol-supplemented artificial diet compared to artificial diet without it.
  • We observed reduced larval growth on the leaf diets compared to artificial diet; in addition, larvae grow slower on soybean leaves compared to cotton leaves.
  • We observed a hormesis effect in larval performance when larvae were fed on gossypol-supplemented artificial diet.

Manuscript ready for submission: Insect Molecular Biology

5. Identification of PI-sensitive and PI-insensitive proteases at translational level?

  • We used affinity purification of midgut lumen content against an SKTI-immobilized column to separate SKTI-sensitive proteases from SKTI-insensitive proteases.
  • We analyzed SKTI-column-bound and unbound fractions from the affinity purification by comparing the protein spots on 2D gel electrophoresis and mass spectrometry.
  • We managed to conclusively identify the PI-insensitive proteases in the larvae fed on SKTI-supplemented diet compared to SKTI-free artificial diet, however, due to high sequence similarity the presence of other proteases is also likely.
  • Feeding on SKTI-supplemented diet not only affect on proteases but also amylases and carboxypeptidases.

Manuscript in preparation

6. How the digestion is regulated in H. armigera , and when exactly the effect of PI occurs to increase expression of proteases?

  • We measured the enzyme activity in the midgut lumen and frass of H. armigera larvae fed on SKTI-supplemented or SKTI-free artificial diet for up to 96 hours at 3 h intervals.
  • We observed no diurnal regulation of trypsin, chymotrypsin or total protease activity in the midgut lumen and frass of the larvae fed on either artificial diet.
  • We found induced gene expression of trypsins and chymotrypsins in the midgut of larvae fed on SKTI-supplemented diet compared to SKTI-free artificial diet, after 06 and 09 h post feeding. In addition, there were some protease genes induced as early as 03 h of SKTI ingestion.

In conclusion, our study provides a comprehensive resource of all the digestive proteases from H. armigera , S. frugiperda and M. sexta. Findings about life history traits and the differential feeding behavior of H. armigera larvae on PI-free and PI-supplemented diets. The differential regulation of trypsins and chymotrypsins at the transcript and protein levels, in addition a rebound in growth rate by using SKTI-insensitive proteases in H. armigera. We identified SKTI-sensitive and SKTI-insensitive proteases in the midgut of in H. armigera at the transcript and translational level in response to feeding on SKTI. H. armigera regulates its digestive and detoxificative genes in plastic, but intricate manner on two host leaf diets. Finally the onset of induced protease gene expression in response to feeding on SKTI-supplemented diet and no diurnal regulation of proteases in H. armigera. The study of this multigene family in the light of adaptation against plant secondary metabolites and protease inhibitors will help in understanding the mechanisms of adaptation in general and perhaps in design of possible pest control applications.