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Use of Proteinase Inhibitors as Insecticides

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The Plagues of Egypt are a well-known to many, to send a message to the Pharaoh, the eighth of which saw Moses have God send was a plague of locusts to destroy crops. Luckily those were mere allegorical stories. What is true is that crop yields for ancient societies have always been plagued pests destroying crops. Even in modern times pests are a major issue for farmers. Effective modern delivery methods of insecticides to fight pests such as chemical sprays like the infamous roundup by Monsanto have proven to be effective, but face issues such as potential poisons lingering in the environment, insects acquiring resistance to them over time, not being very specific in their targets often killing everything they are sprayed on, and tend to be quite toxic. A more effective method has been the use of biological pesticides, where plants in question generate toxins required to fight insects specifically without being toxic. One methods of delivery that has shown promise has been to use proteinase inhibitors.

Proteases are found in almost all organisms playing important roles in biological processes by breaking down proteins via hydrolytic reactions (Lin et al., 2017). If proteases are overexpressed in a cell it can lead to many proteins being degraded needlessly affecting cell function (Lin et al., 2017). As a result, proteases need to be properly controlled and one of the most efficient ways to do so is with protease inhibitors. When proteinase inhibitors (PI's) bind to digestive enzymes it causes them to become unreactive (Broadway and Duffey, 1986). The pancreas would then have to secrete additional proteinases itself to fight the inhibition from inhibitors so that normal digestion can occur and prevent re-absorption of protease-inhibitor enzyme complex (Broadway and Duffey, 1986). If this process is left unhindered it would lead to loss of essential amino acids which could have be used to build more proteases (Broadway and Duffey, 1986). This loss of amino acids results in an amino acid deficiency that leads to growth, development and survival issues of the organism (Fatemeh and Bandani, 2011). What makes proteinase inhibitors attractive for controlling pests is that they are encoded by single genes and are effective over wide ranges of pests, making them more practical than methods that rely on inhibiting complex pathways with similar targets (Fatemeh and Bandani, 2011). A substantial amount of development has gone towards finding suitable proteinase inhibitors for plants to express. Insects have also been found to be adapting against the use of protease inhibitors, with some being able to overexpress gut proteases or create new enzymes that are harder to inhibit (Nath et al., 2015). This only shows that the study of proteinase inhibitors still has a long ways to go and will require much more study. Some of the techniques studied in recent years to deter pests include use of trypsin inhibitor against lepidopteran larvae and serine proteinase inhibitors against mosquitos.

It was believed that a trypsin inhibitor that was expressed in the germinating seed of Dolichos biflorus would be effective the gut proteins of P. brassicae and S. littoralis. To test this theory researchers extracted the PI from germinating various seed samples then sterilizing seeds in ethanol and water and growing them, collecting the plant seeds from flowering plants at different time intervals and then grinded them up into fine powder and processed it with acetone to get a crude extract (Nath et al., 2015). Researchers were able to measure and extract the amount of trypsin inhibitor in each seed from the crude extract via centrifugation and found that the more days after germination when they extracted the trypsin inhibitor the less the seed had (Nath et al., 2015). This decline coincided with noted decreases in soluble protein activity in the seeds after germination which could be attributed to degradation of proteins including inhibitors and proteases during germination (Nath et al., 2015). To measure trypsin inhibitor effectiveness against gut proteases of organisms, researchers also dissected the midgut of P. brassicae and S. littoralis and centrifuged it to isolate for a source of trypsin, which they found (Nath et al., 2015). To measure activity of inhibitor against gut proteases, researchers measured optical density after mixing inhibitor and gut proteases together (Nath et al., 2015). It was found in this study that all trypsin inhibitors extracted from seeds exhibited inhibitory response against P. brassicae, researchers then took the sample (HPK4) that showed highest inhibitory action and tested it against proteases of S. littoralis, and found that it also exhibited inhibitory activity making it ideal an ideal candidate for an insecticide (Nath et al., 2015). To test inhibitor ability as an insecticide researchers used cabbage leaf discs coated in trypsin inhibitor extracted from HPK4 seeds as it had the highest activity, and fed it to newly hatched larvae for 5 days (Nath et al., 2015). The experiment measured the amount of the leaf disc eaten and fecal matter produced from larvae over the 5 day period using different concentrations of trypsin inhibitor ranging from 0.025mg to 2.5mg (Nath et al., 2015).  It was found that P. brassicae larvae were sensitive to inhibitor as they showed mortality rate ranging from 10% to 80% (at 0.025mg and 2.5mg concentration respectively) after 5 days of feeding (Nath et al., 2015). Larval fecal matter was also noted to having 38% less gut trypsin and other soluble proteins compared to fecal matter in control samples, and with less of cabbage leaf being eaten in all samples compared to control due to massive dying off of larvae (Nath et al., 2015).  Larval death could be attributed to lack of essential amino acids in insects body due to trypsin inhibitor (Nath et al., 2015). Reduced soluble protein in fecal matter was attributed to less intestinal aberration and the fact that much less of the leaf was eaten overall (Nath et al., 2015).  Similar results were found in complementary study regarding the effects on S. littoralis, with low survival rates, reduced larval mass, and decreased soluble protein concentration in fecal matter compared to control (Nath et al., 2015). Overall researchers were able to conclude that the PI's (trypsin inhibitor in this case) from various germinating seed strains were able to inhibit gut proteinases of many lepidopteran larvae such as P. brassicae and S. littoralis (Nath et al., 2015).  It is worth noting that while this experiment used PI's as a spray over plants, researchers indicate that this holds promising future for crops to express higher levels of proteinase inhibitors naturally to fight pest populations in a way without spraying toxic pesticides (Nath et al., 2015).

Another tested method to fight pests was with serine proteinase inhibitors. Mosquitoes are a major vector of disease carrying with them malaria, west nile virus, and etc, mosquitoes are found around the world and it has been reaffirmed many times that the best way to control mosquitos is through controlling populations (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013).  This topic has been studied extensively and is known that the majority of digestive enzymes for mosquitoes are serine proteases (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). For this experiment researchers looked into Ae. aegypti a mosquito strain which is found commonly around the world (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). It was determined that mosquito larvae would be the ideal target for this protease inhibitors because they are aquatic and feed constantly which would give inhibitors the greatest chance to succeed in killing them as they are in early development (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). To determine which serine proteases were present in mosquito larvae researchers performed a kinetic assay of larval midgut proteins and determined that the gut extract was a host to trypsin-like, elastase-like, and chymotrypsin-like enzymatic activities (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). Researchers then used a phage display system to select an inhibitor for all of these digestive proteases, finding that HiTI a trypsin inhibitor from the horn fly would suffice because of successful detection for HiTI on an active M13 phage as it was fused to coat one of the coat proteins (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013).  Researchers then created a  combinatorial HiTI inhibitor library and tested for their ability to inhibit the serine proteases in mosquito larvae and their ability to inhibit the trypsin-like, chymotrypsin-like and elastase-like proteases (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). Amino acid analysis of phagemid DNA indicated that for HiTI, the largest area for variation occurred in the P1 position of the enzyme (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). This was important because it appeared this position determined the specificity of the HiTI inhibitor, as it was found that if a basic amino acid was present in P1 it would inhibit trypsin, while chymotrypsin would be inhibited if hydrophobic amino acids was present, and if small aliphatic amino acids are present then elastase would be inhibited (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). Among the library created mutants that would inhibit one of the three noted above were found with majority of clones not being proteases (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). Researchers then proceeded to purify and clone the HiTI variants into a plamids which experessed in P.pastoris (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013).It was found that the wild type HiTI was able to inhibit the trypsin-like enzymes but not the remaining two (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). Researchers were able to find 3 mutants that inhibited trypsin-like enzymes but did not inhibit elastase-like and chymotrypsin-like enzymatic reactions, two variants were found to inhibit chymotrypsin with high selectivity for bovine chymotrypsin, one of which (T23) was also found to be an inhibitor for elastase-like proteases as well reaffirming the researchers belief in specific selection by inhibitors for different proteases (Soares, Soares Torquato, Alves Lemos, & Tanaka, 2013). The results of this research are promising because this study provides a better understanding of mosquito gut enzymes and provides a framework for insecticide development based on protease inhibitors for mosquitos.

In conclusion, it was found that proteinase inhibitors are a viable methods for controlling pests whether it is controlling mosquito populations by feeding larvae serine proteinase inhibitor or coating crops with trypsin inhibitors and feeding it to pests. Much of the work currently has been formed around determining which proteinase inhibitors would match well with their intended target. In order to expand the scope and make proteinases a functional insecticide it would seem more work needs to be done around delivery systems, whether it be as a chemical insecticide spray or genetically altering plants to overexpress proteinase inhibitors. Over time as this field becomes more developed we can be able to determine the effects of these inhibitors on humans. The concept has been proven and more work is needed to make them viable, proteinase inhibitors have a strong future and once the kinks have been worked out then there is no doubt they will be massively successful in increasing crop yields and fighting off pests.

References

Dantzger, M., Vasconcelos, I. M., Scorsato, V., Aparicio, R., Marangoni, S., & Macedo, M. L. R. (2013). Bowman-Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential. Phytochemistry, 118(August), 224-235. http://doi.org/10.1016/j.phytochem.2015.08.013

Lin, H., Lin, X., Zhu, J., Yu, X.-Q., Xia, X., Yao, F., … You, M. (2017). Characterization and expression profiling of serine protease inhibitors in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). BMC Genomics, 18(1), 162. http://doi.org/10.1186/s12864-017-3583-z

Nath, A. K., Kumari, R., Sharma, S., & Sharma, H. (2015). Biological activity of Dolichos biflorus L. trypsin inhibitor against lepidopteran insect pests. Indian Journal of Experimental Biology, 53(September), 594-599.

Soares, T. S., Soares Torquato, R. J., Alves Lemos, F. J., & Tanaka, A. S. (2013). Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display. Insect Biochemistry and Molecular Biology, 43(1), 9-16. http://doi.org/10.1016/j.ibmb.2012.10.007

Broadway, R. M., & Duffey, S. S. (1986). Plant proteinase inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. Journal of Insect Physiology, 32(10), 827-833. http://doi.org/10.1016/0022-1910(86)90097-1

Saadati, F., & Bandani, A. R. (2011). Effects of Serine Protease Inhibitors on Growth and Development and Digestive Serine Proteinases of the Sunn Pest, Eurygaster integriceps. Journal of Insect Science (Online), 11(72), 72. http://doi.org/10.1673/031.011.7201


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