Insect Enzyme Research Articles

Functional analysis of two polygalacturonase genes in Apolygus lucorum associated with eliciting plant injury using RNA interference

Salivary enzymes of many piercing-sucking insects lead to host plant injury. The salivary enzymes, polygalacturonase (PGs), act in insect feeding. PG family genes have been cloned from the mirid bug Apolygus lucorum, a pest of cotton and other host crops in China. We investigated the function of two PG genes that are highly expressed in A. lucorum nymphs (PG3-4) and adults (PG3-5), using siRNA injection-based RNA interference (RNAi). Accumulation of mRNA encoding both genes and their cognate proteins was significantly reduced (>60%) in experimental compared control green fluorescent protein (GFP) siRNA-treated mirids at 48 h post injection. Injury levels of cotton buds were also significantly reduced after injecting saliva isolated from PG3-4 and PG3-5 siRNA-treated A. lucorum. These results demonstrate that these two PG act in A. lucorum elicitation of plant injury.

Mode of Action of the Natural Insecticide, Decaleside Involves Sodium Pump Inhibition.

Decalesides are a new class of natural insecticides which are toxic to insects by contact via the tarsal gustatory chemosensilla. The symptoms of their toxicity to insects and the rapid knockdown effect suggest neurotoxic action, but the precise mode of action and the molecular targets for decaleside action are not known. We have presented experimental evidence for the involvement of sodium pump inhibition in the insecticidal action of decaleside in the cockroach and housefly. The knockdown effect of decaleside is concomitant with the in vivo inhibition of Na+, K+ -ATPase in the head and thorax. The lack of insecticidal action by experimental ablation of tarsi or blocking the tarsal sites with paraffin correlated with lack of inhibition of Na+- K+ ATPase in vivo. Maltotriose, a trisaccharide, partially rescued the toxic action of decaleside as well as inhibition of the enzyme, suggesting the possible involvement of gustatory sugar receptors. In vitro studies with crude insect enzyme preparation and purified porcine Na+, K+ -ATPase showed that decaleside competitively inhibited the enzyme involving the ATP binding site. Our study shows that the insecticidal action of decaleside via the tarsal gustatory sites is causally linked to the inhibition of sodium pump which represents a unique mode of action. The precise target(s) for decaleside in the tarsal chemosensilla and the pathway linked to inhibition of sodium pump and the insecticidal action remain to be understood.

An Efficient Antioxidant System in a Long-Lived Termite Queen.

The trade-off between reproduction and longevity is known in wide variety of animals. Social insect queens are rare organisms that can achieve a long lifespan without sacrificing fecundity. The extended longevity of social insect queens, which contradicts the trade-off, has attracted much attention because it implies the existence of an extraordinary anti-aging mechanism. Here, we show that queens of the termite Reticulitermes speratus incur significantly lower oxidative damage to DNA, protein and lipid and have higher activity of antioxidant enzymes than non-reproductive individuals (workers and soldiers). The levels of 8-hydroxy-2’-deoxyguanosine (oxidative damage marker of DNA) were lower in queens than in workers after UV irradiation. Queens also showed lower levels of protein carbonyls and malondialdehyde (oxidative damage markers of protein and lipid, respectively). The antioxidant enzymes of insects are generally composed of catalase (CAT) and peroxiredoxin (Prx). Queens showed more than two times higher CAT activity and more than seven times higher expression levels of the CAT gene RsCAT1 than workers. The CAT activity of termite queens was also markedly higher in comparison with other solitary insects and the queens of eusocial Hymenoptera. In addition, queens showed higher expression levels of the Prx gene RsPRX6. These results suggested that this efficient antioxidant system can partly explain why termite queens achieve long life. This study provides important insights into the evolutionary linkage of reproductive division of labor and the development of queens’ oxidative stress resistance in social insects.

Diverse subcellular localizations of the insect CMP-sialic acid synthetases.

The occurrence and biological importance of sialic acid (Sia) and its metabolic enzymes in insects have been studied using Drosophila melanogaster. The most prominent feature of D. melanogaster CMP-Sia synthetase (DmCSS) is its Golgi-localization, contrasted with nuclear localization of vertebrate CSSs. However, it remains unclear if the Golgi-localization is common to other insect CSSs and why it happens. To answer these questions, Aedes aegypti (mosquito) CSS (AaCSS) and Tribolium castaneum (beetle) CSS (TcCSS) were cloned and characterized for their activity and subcellular localization. Our new findings show: (1) AaCSS and TcCSS share a common overall structure with DmCSS in terms of evolutionarily conserved motifs and the absence of the C-terminal domain typical to vertebrate CSSs; (2) when expressed in mammalian and insect cells, AaCSS and TcCSS showed in vivo and in vitro CSS activities, similar to DmCSS. In contrast, when expressed in bacteria, they lacked CSS activity because the N-terminal hydrophobic region appeared to induce protein aggregation; (3) when expressed in Drosophila S2 cells, AaCSS and TcCSS were predominantly localized in the ER, but not in the Golgi. Surprisingly, DmCSS was mainly secreted into the culture medium, although partially detected in Golgi. Consistent with these results, the N-terminal hydrophobic regions of AaCSS and TcCSS functioned as a signal peptide to render them soluble in the ER, while the N-terminus of DmCSS functioned as a membrane-spanning region of type II transmembrane proteins whose cytosolic KLK sequence functioned as an ER export signal. Accordingly, the differential subcellular localization of insect CSSs are distinctively more diverse than previously recognized.

Insecticide ADME for support of early-phase discovery: combining classical and modern techniques.

The two factors that determine an insecticide's potency are its binding to a target site (intrinsic activity) and the ability of its active form to reach the target site (bioavailability). Bioavailability is dictated by the compound's stability and transport kinetics, which are determined by both physical and biochemical characteristics. At BASF Global Insecticide Research, we characterize bioavailability in early research with an ADME (Absorption, Distribution, Metabolism and Excretion) approach, combining classical and modern techniques. For biochemical assessment of metabolism, we purify native insect enzymes using classical techniques, and recombinantly express individual insect enzymes that are known to be relevant in insecticide metabolism and resistance. For analytical characterization of an experimental insecticide and its metabolites, we conduct classical radiotracer translocation studies when a radiolabel is available. In discovery, where typically no radiolabel has been synthesized, we utilize modern high-resolution mass spectrometry to probe complex systems for the test compounds and its metabolites. By using these combined approaches, we can rapidly compare the ADME properties of sets of new experimental insecticides and aid in the design of structures with an improved potential to advance in the research pipeline. © 2016 Society of Chemical Industry.

RNAi pest control and enhanced BT insecticidal efficiency achieved by dsRNA of chymotrypsin-like genes in Ostrinia furnacalis

Chymotrypsins (CTP) are a main type of proteolytic digestive enzymes in Lepidopteran insects. They can play a major role in degrading activated Bt toxin in some insect species, leading to decreased Bt toxicity and inducing insect resistance. Seven CTP-like sequences were obtained by screening transcriptome data of Asian corn borer, Ostrinia furnacalis. The expression analyses revealed that almost all of the CTP genes showed relatively higher transcript levels at the larval stage, and most showed relatively higher transcript levels in the saliva and midgut. We synthesized dsRNA from the seven CTP-like genes and fed it to larvae at the different instar stages. The dsRNAs from all seven CTPs significantly enhanced mortality of larvae from the first to the third instar larval stages, and only the dsRNA from CTP8 had higher lethality to fourth instar larvae. When larvae were fed on a diet containing 0.2 mg/ml dsRNA and Bt (5 μg/g diet), the 5-day mortalities were significantly enhanced, except for larvae treated with dsRNA from CTP5. The dsCTP16 plus Bt treatment resulted in a 5-day mortality of 100 %, and all of the dsCTPs accelerated insect death. These results indicated that members of the CTP-like gene family are effective RNAi targets for pest control. In particular, CTP8 represents an effective target for controlling the whole larval stage, and CTP16 is an effective target to enhance Bt insecticidal efficiency.

Xanthogalerucella luteola (Col.: Chrysomelidae) α-amylase affected by seed proteinaceous extract from datura, wild oat and amaranth seeds

The elm leaf beetle, Xanthogalerucella luteola (Muller) (Col.: Chrysomelidae) is a serious pest of elm trees and it has been distributed all over the world. The current study was undertaken to investigate the inhibitory effects of protein extracts of three weed seeds including datura Datura stramonium L., amaranth Amaranthus retroflexus L. and wild oat Avena fatua L. against X. luteola α-amylase using spectrophotometric assay as well as in gel assays. The effects of five concentrations of each seed proteinaceous extracts were tested on α-amylase activity of the larval gut. The results showed a dose dependent manner in inhibition of the insect enzyme. At the highest concentration of protein extracts (12 μg protein) of all three seed extracts including amaranth, wild oat and datura, the inhibition was 71, 79 and 31%, respectively. Whilst, at low concentration (0.75 μg protein), the inhibition observed was 15, 36 and 5%, respectively. Thus, the greatest inhibition percentage was obtained when proteinaceous extract of wild oat seed was used. These results were confirmed when in gel assays were performed. All three seed proteinaceous extracts had an optimum pH inhibition of 6.0. Thus, it is concluded that wild oat seed proteins are potentially good for detailed investigation in order to get a clear picture of its active compound/s and its structure-function relationship.

Production of FAME biodiesel in E. coli by direct methylation with an insect enzyme.

Most biodiesel currently in use consists of fatty acid methyl esters (FAMEs) produced by transesterification of plant oils with methanol. To reduce competition with food supplies, it would be desirable to directly produce biodiesel in microorganisms. To date, the most effective pathway for the production of biodiesel in bacteria yields fatty acid ethyl esters (FAEEs) at up to ~1.5 g/L. A much simpler route to biodiesel produces FAMEs by direct S-adenosyl-L-methionine (SAM) dependent methylation of free fatty acids, but FAME production by this route has been limited to only ~16 mg/L. Here we employ an alternative, broad spectrum methyltransferase, Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). By introducing DmJHAMT in E. coli engineered to produce medium chain fatty acids and overproduce SAM, we obtain medium chain FAMEs at titers of 0.56 g/L, a 35-fold increase over titers previously achieved. Although considerable improvements will be needed for viable bacterial production of FAMEs and FAEEs for biofuels, it may be easier to optimize and transport the FAME production pathway to other microorganisms because it involves fewer enzymes.

Proteomics comparison of aspartic protease enzyme in insects

Proteomics comparison of aspartic protease enzyme in insects

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems.

Expression profiles of lysozyme- and prophenoloxidase-encoding genes in Spodoptera species challenged with entomopathogenic fungus, Metarhizium anisopliae (Metchnikoff) Sorokin using qRT-PCR

Lysozyme and prophenoloxidase are two immunity-related enzymes in insect. In our attempt to understand their roles in defence, we profiled expressions of the encoding genes in Spodoptera exigua Hübner and Spodoptera litura Fabricius, during the infection process by an entomopathogenic fungus, Metarhizium anisopliae var. anisopliae. Gene expressions were analysed using quantitative real-time reverse transcription-PCR. Sampling was done at 0, 2, 12 and 24 h after infection, and when the infected larvae reached the moribund stage at 36 and 60 h after infection. The expression profiles of the two genes were similar in both larvae. At 24 h after infection, the lysozyme gene was expressed at a higher level in the infected larvae (fivefold) when compared to the healthy larvae, and reached the highest level (13-fold in S. exigua and 7-fold in S. litura) at the moribund stage. The expression levels of the prophenoloxidase gene in the infected larvae were similar to that of the control, except for a slight increase in expression at 2 and 24 h (~1.5-fold) after infection. These findings provide new information on the changes in transcriptional levels of LYZ and PPO, and indicate the importance of these genes in insect defence response against fungal infection.

English

Biochemical and feeding assays were performed in order to assess the bioactivity of trypsin inhibitors from sesame seeds to control Plodia interpunctella larvae. The biochemical assays were based on the ability of protease inhibitors (PI) present in sesame seeds to inhibit bovine pancreatic trypsin and digestive enzymes of insect larvae. The stability of the inhibitors was also assessed at different pH and temperature. The feeding bioassays were carried out in plates containing second instar larvae fed on diet with sesame protein extract. It was observed that all genotypes presented inhibition when tested with BPT, ranging from 51 to 90%, among them BRS Seda, CNPA G3 and CNPA G4 showed average of 69% of inhibitory activity for intestinal homogenate. The FII-fractions in these three genotypes showed thermal and pH stabilities at 40°C and 8.5, respectively. In feeding bioassay, P. interpunctella larvae were susceptible to SPCE of all three genotypes, revealing average mortality of 74% to SPCE and 45% to FII-fractions. Among all genotypes, BRS Seda was more effective to inhibition of digestive enzymes of insects and considered the more suitable genotype for further using in sesame breeding program to resistance to P. interpunctella. Key words: Sesamum indicum L., serine protease, stored grain pest, Lepidoptera.

Induction and activity of glutathione S-transferases extracted from Zonocerus variegatus (Orthoptera: Pyrgomorphidae) exposed to insecticides

Glutathione S-transferases (GSTs) have been recognized as important metabolic detoxifying enzymes in many phytophagous insects. However, the contribution of GST to insecticide resistance in Zonocerus variegatus (L.) has not been studied. Therefore, we carried out an initial study on the induction and kinetics of GST in Z. variegatus exposed to pyrethroids (PYRs) and Ocimum gratissimum leaf extract. Fifth-instar nymphs of Z. variegatus collected from cassava farms (with no history of insecticide exposure) on the campus of the University of Ibadan were reared to adult stage. Adult insects were divided into four groups of two replicates, each consisting of 30 insects. The groups were exposed to 20 ml of PYR insecticide and 25 mg/dl and 250 mg/dl of O. gratissimum leaf extract, respectively, while the last group served as the control. GST was extracted from the body tissues of the insects using the Bradford method and the kinetics of the enzyme was evaluated using the Lineweaver-Burk plots. No mortality was recorded in insects exposed to 25 mg/dl of O. gratissimum leaf extract, while mortality rates of 93.3 and 43.3% were recorded in insects exposed to PYR insecticide and 250 mg/dl of O. gratissimum leaf extract, respectively. The activity of GST was higher in insects exposed to the insecticides than in the control insects, while a higher enzyme activity was recorded in insects that died after exposure to the insecticides than in insects that were alive after exposure. The GST extracted from insects that were alive after exposure to PYR insecticide and O. gratissimum leaf extract exhibited a high affinity for the glutathione substrate compared with that extracted from insects that died after exposure. Higher GST activity in insects exposed to insecticides is indicative of the role of this enzyme in the metabolic detoxification of insecticides by Z. variegatus.

Phenobarbital induction and chemical synergism demonstrate the role of UDP-glucuronosyltransferases in detoxification of naphthalophos by Haemonchus contortus larvae.

We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC50) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field.

Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond.

Steroid hormones are responsible for the coordinated regulation of many aspects of biological processes in multicellular organisms. Since the last century, many studies have identified and characterized steroidogenic enzymes in vertebrates, including mammals. However, much less is known about invertebrate steroidogenic enzymes. In the last 15 years, a number of steroidogenic enzymes and their functions have been characterized in ecdysozoan animals, especially in the fruit fly Drosophila melanogaster. In this review, we summarize the latest knowledge of enzymes crucial for synthesizing ecdysteroids, the principal insect steroid hormones. We also discuss the functional conservation and diversity of ecdysteroidogenic enzymes in other insects and even non-insect species, such as nematodes, vertebrates, and lower eukaryotes.

Exoskeleton formation in Apis mellifera: Cuticular hydrocarbons profiles and expression of desaturase and elongase genes during pupal and adult development

Cuticular hydrocarbons (CHCs) are abundant in the superficial cuticular layer (envelope) of insects where they play roles as structural, anti-desiccation and semiochemical compounds. Many studies have investigated the CHC composition in the adult insects. However, studies on the profiles of these compounds during cuticle formation and differentiation are scarce and restrict to specific stages of a few insect species. We characterized the CHCs developmental profiles in the honeybee workers during an entire molting cycle (from pupal-to-adult ecdyses) and in mature adults (forager bees). Gas chromatography/mass spectrometry (GC/MS) analysis revealed remarkable differences in the relative quantities of CHCs, thus discriminating pupae, developing and newly-ecdysed adults, and foragers from each other. In parallel, the honeybee genome database was searched for predicted gene models using known amino acid sequences of insect enzymes catalyzing lipid desaturation (desaturases) or elongation (elongases) as queries in BLASTP analysis. The expression levels of six desaturase genes and ten elongase genes potentially involved in CHC biosynthesis were determined by reverse transcription and real time polymerase chain reaction (RT-qPCR) in the developing integument (cuticle and subjacent epidermis). Aiming to predict roles for these genes in CHC biosynthesis, the developmental profiles of CHCs and desaturase/elongase transcript levels were evaluated using Spearman correlation coefficient. This analysis pointed to differential roles for these gene products in the biosynthesis of certain CHC classes. Based on the assumption that homologous proteins may share a similar function, phylogenetic trees were reconstructed as an additional strategy to predict functions and evolutionary relationships of the honeybee desaturases and elongases. Together, these approaches highlighted the molecular complexity underlying the formation of the lesser known layer of the cuticular exoskeleton, the envelope.

Vertebrate Acyl CoA synthetase family member 4 (ACSF4‐U26) is a β‐alanine‐activating enzyme homologous to bacterial non‐ribosomal peptide synthetase

Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for β-alanine, and a KM of ~ 5 μm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that β-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.

Effect of Dietary Cadmium on the Activity of GlutathioneS-Transferase and Carboxylesterase in Different Developmental Stages of theOxya chinensis(Orthoptera: Acridoidea)

Glutathione S-transferases (GSTs) and carboxylesterases (CarEs) play important roles in the detoxification of endogenous and exogenous compounds. In this study, the biochemical effects of dietary cadmium (Cd) on the activities of GST and CarE in different developmental stages of the rice grasshopper Oxya chinensis Thunberg were studied. The results showed that the effects of the Cd concentration and developmental stage on GST activity were statistically significant. GST activity in O. chinensis increased at the highest Cd concentration in most nymphs, suggesting that GST is typically inducible by Cd. However, GST activity was inhibited in adults under Cd stress owing to life-stage-specific physiological characteristics. The results showed that the substrates, developmental stage, and Cd concentration had statistically significant effects on CarE activity. In most studies of CarE activity, the interaction between any two studied factors was statistically significant, although the interaction effects of the substrates, developmental stages, and Cd concentrations were not significant, which implied that the insect physiological condition and the external environmental may affect CarE activity. The results suggest that the insect's life stage and enzyme substrates should be considered when enzyme activity under Cd stress is studied.

Identification and abiotic stress response of a glutamine synthetase gene (AccGS) from the Asiatic honeybee, Apis cerana cerana (Hymenoptera: Apidae)

Glutamine synthetase (GS) is an essential detoxification enzyme that plays an important role in stress responses; however, little information regarding the function of this enzyme in hymenopteran insects is available. In the present study, we isolated and characterized the gene encoding GS in the Asiatic honeybee, Apis cerana cerana. Multiple alignments and a phylogenetic analysis of GS sequences showed that AccGS belongs to the GSII superfamily and clusters with invertebrate GSs. Real-time quantitative PCR data demonstrated that AccGS is expressed at all developmental stages and in all tissues, with the highest expression observed in the sixth larval instar and in the brain. Moreover, AccGS expression is highly regulated by environmental stress, including xenobiotic, temperature, and ultraviolet light stresses. A disc diffusion assay showed that the recombinant AccGS protein confers resistance to mercuric chloride (HgCl2) stress in E. coli. This suggests that AccGS may play multiple roles in early development and in environmental stress responses.

In vitro inhibition of glutathione S-transferases by several insecticides and allelochemicals in two moth species

Glutathione S-transferases (GSTs) are important detoxification enzymes in insects. This study examined the inhibitory effects of five insecticides (beta-cypermethrin, fenpropathrin, phoxim, abamectin, acetamiprid) and two plant-derived allelochemicals (quercetin, tannic acid) on GST activity in Micromelalopha troglodyta (Graeser) and Clostera anachoreta (Fabricius). GST activities were suppressed by all the insecticides and allelochemicals tested; the two allelochemicals were the most potent inhibitors, reducing GST activity in vitro in a dosage-dependent manner. Kinetic analyses of GST inhibition by quercetin and tannic acid in M. troglodyta and C. anachoreta suggested biochemical differences in their GSTs.