Stages of aestivation as a physiological state and the related biochemical composition in the grain chinch bug (Macchiademus diplopterus)

The grain chinch bug (Macchiademus diplopterus Distant) is a phytosanitary pest, endemic to the Western Cape in South Africa. At the start of the aestivation phase of their lifecycle, grain chinch bugs seek sheltering sites, which potentially include fruit and fruit trees if orchards are near host plants. Aestivating grain chinch bug on export fruit is considered contaminant or hitchhiker phytosanitary pests. Previous studies have indicated that the grain chinch bug has the ability to become more tolerant of thermal stresses as they progress through their aestivation cycle. To examine the potential physiological changes that occur during aestivation, molecular (soluble protein identification) and biochemical (macromolecule) analyses were performed on the insects before entering aestivation, as well as early, mid, mid‐late and late aestivation periods. Analyses provided useful information on the abundance and identity of individual soluble proteins and concentration of macromolecules, indicating whether compounds are up‐ or down‐regulated throughout the aestivation cycle. The focus of this investigation was to examine the influence of heat shock proteins and proteins involved in energy production and metabolism throughout the aestivation period. Results provide insight into the thermo‐tolerance capabilities or mechanisms of the grain chinch bug. The significant decrease in the number of individual proteins identified in samples before aestivation compared to early aestivation indicated the insects' progression into a hypometabolic state. During the early, mid and mid‐late aestivation periods (from December to May), large volumes of fruit are exported from South Africa. An increase in abundance of proteins, such as smHsp20, Hsp10, 70, 80 and 90, occurred during the mid/mid‐late aestivation period compared with the early period. This indicated the potential role of heat shock proteins in the insect's ability to increase its thermo‐tolerance at a later stage within the aestivation cycle.


INTRODUCTION
The evolutionary success of insects is evident by their widespread distribution over many ecological niches.The ability of insects to survive in extreme environments, such as, at high latitudes or altitudes, indicates the evolution of biochemical and physiological strategies that reduce the negative influence of stressors, like high and low temperatures (Doucet et al., 2009).Adaption strategies, which include behavioural changes, as well as physiological responses, play a crucial role for survival during unfavourable conditions.Some insects, such as certain species of moths (Lepidoptera) undertake long migratory flights to escape low winter temperatures (Riley et al., 1995;Zhang et al., 1981).In contrast, other insects seek shelter within local areas, such as tree bark crevices, which buffer them from extreme environmental temperature changes (Dingle, 1996;Doucet et al., 2009).
Often this shelter-seeking strategy is coupled with physiological changes to further mitigate against stresses such as low or high temperature, radiation, and host availability.Thermal physiological changes are referred to as cold or heat hardening (Scott, 2020).Cold and heat hardening are conceptually similar, and include strategies for suppression of metabolic rate, retention of body water/fluids, conservation and reprioritization of metabolic reserves, and mechanisms to preserve and stabilize organs and cells for weeks or months to increase the insect's survivability (Hochachka & Guppy, 1987;Sejerkilde et al., 2003;Storey & Storey, 2012).
Macchiademus diplopterus (Distant) (Hemiptera: Lygaeidae) is an indigenous pest of cultivated grain crops and wild grasses in South Africa and a phytosanitary pest on export fruit due to its shelter-seeking behaviour as it moves into aestivation during the summer months (Johnson & Addison, 2008;Myburgh & Kriegler, 1967;Slater & Wilcox, 1973).Aestivation is defined as a prolonged period of dormancy, which is used a survival strategy by the grain chinch bug, M. diplopterus from December through June.When host plants desiccate, adult M. diplopterus move to surrounding shelter sites.These could include fruit orchards, in which case aestivating bugs may seek shelter in the stalk or the calyx ends of fruit and consequently pose a phytosanitary risk to trading partners (Annecke & Moran, 1982;Myburgh & Kriegler, 1967;Slater & Wilcox, 1973).
The high thermal tolerance levels of M. diplopterus, in particular chilling tolerance, was first identified when export fruit that was under cold storage during transit, was intercepted with live insects, and thus the consignments were rejected (Malumphy, 2011;Malumphy et al., 2012;Myburgh & Kriegler, 1967).The heat tolerance capabilities of M. diplopterus were observed during research conducted by Johnson and Neven (2011).Their research indicated that M. diplopterus was the most tolerant species of three phytosanitary pest species tested in that study, namely M. diplopterus, Phlyctinus callosus [Boherman] and Thaumatotibia leucotreta [Meyrick], when exposed to combination heat and controlled atmosphere treatments (CATTS).Research conducted by Smit (2019), also illustrated the ability of M. diplopterus to withstand hightemperature controlled atmosphere (CATTS) treatments.Thermotolerance research conducted by Okosun (2012) indicated that aestivating M. diplopterus could tolerate the high and low temperatures that could be used as phytosanitary treatments, making these less viable options for control.Pre-exposure of M. diplopterus to low temperatures through rapid and gradual pre-cooling, also enabled the insects to tolerate prolonged cold storage treatments.Okosun (2012) noted that the physiological state of the aestivating adult M. diplopterus could be the contributing factor, enabling the insect to be more tolerant of thermal stresses than actively metabolizing insects.Additionally, Okosun (2012) proposed that M. diplopterus becomes more tolerant of thermal stresses as it progresses further into the aestivation phase of its life cycle.
The ability of an insect to control production, metabolism, storage and transport of energy is a key element in its ability to maintain functions at a survival level during aestivation for prolonged periods.Energy reserves are stored in animal cells as glycogen, sugars or lipids and used on demand as a fuel source and in response to changing temperatures (Steele, 1985;Storey & Storey, 1986).Lipids are a key reserve class of molecules used by insects during diapause or aestivation, as lipids contain four times the energy available per unit mass than sugars.Another crucial key to surviving aestivation through molecular compositional changes enhances stress responses such as antioxidant defences necessary to survive (Storey & Storey, 2012).The main functions of these compositional changes are to protect the cells during dormancy, and as a defence mechanism when transitioning from a low metabolic state to normal metabolism, defending against the rapid generation of reactive oxygen species (Hermes-Lima & Zenteno-Savín, 2002;Storey & Storey, 2012).Mechanisms to protect cells and cellular function against thermal and desiccation stresses include the production of heat shock proteins (Clark & Worland, 2008;Goto et al., 1998;Goto & Kimura, 1998).Heat shock proteins have also been associated with aiding in the recovery from cold shock and are upregulated during diapause (Denlinger et al., 2001;Rinehart et al., 2006).Many heat shock proteins are water soluble proteins located in the cytoplasm and in organelles such as the nucleus and mitochondria.Heat shock proteins such as Hsp70 are localized in mitochondria, nucleus and nucleolus and are essential in the cytoplasm (Ali & Banu, 1991).Cytoplasmic Hsp70 can be classified into two categories, heat shock inducible protein 70 (Hsp70) and heat shock cognate or constitutive protein 70 (Hsc70) (Zhang et al., 2015).Low levels of Hsp70 are expressed during non-stress conditions but are quickly up regulated or induced by environmental stresses.In contrast, Hsc70 is expressed during normal conditions and can remain unchanged or slightly up regulated during stress (Daugaard et al., 2007;Mahroof et al., 2005;Zhang et al., 2015).In this study, the physiology of aestivating M. diplopterus was examined in order to identify molecular and biochemical compositional changes that may occur during aestivation.Identifying these changes could provide insight into how and why M. diplopterus becomes more thermo-tolerant and is, therefore, able to withstand thermal phytosanitary treatments.The role of heat shock proteins, proteins involved in energy regulation and other defence mechanisms are discussed to determine and better understand their potential influence on the thermo-tolerant strategies of M. diplopterus.

Determination of macromolecules-lipids, sugars, glycogen and proteins
Extraction and analysis methods for the determination of total protein, lipid, sugar and glycogen content of freeze-dried samples of M. diplopterus were modified from Yuval et al. (1998), Olson et al. (2000), Lee et al. (2004) and Yi and Jean (2011), and the detailed modified protocol is described below.
Extraction procedure A 2% Na 2 SO 4 solution was added to a microtube containing the ten insects per replicate, an electric grinding pestle (Cordless motor pellet pestle, Kimble, USA) was then used to grind samples for 30 s to 1 min.
Ground samples were centrifuged (Eppendorf, 5415R, Germany) for 1 min at 15000 rpm and the supernatant was used in further analyses.

Quantification
Total protein measurement A Bradford protein assay protocol (Bio-Rad Protein Assay Kit I #5000001, Bio-Rad) was followed to determine the total protein content of an aliquot taken from the supernatant produced in the extraction described above (BioRad, 2015).Absorbance was measured at 595 nm using a microplate reader (Varioskan, Thermo Electron Corporation, USA).For the determination of lipid, sugar and glycogen content, a solution of chloroform and methanol (2:1) was added to the remaining $200 μL supernatant.Samples were vortexed and centrifuged at 15000 rpm for 4 min.The supernatant was separated into three layers: sugars in the top layer ($300 μL); glycogen as a viscous layer in the middle; and lipids in the bottom layer ($700 μL).
Each layer per sample was transferred to a separate marked microtube.
The lipids and sugars were analysed immediately, and the glycogen layer was washed with methanol and stored at À20 C for later analysis.

Total lipids measurement
The lipid layer was evaporated using nitrogen gas (95% N 2 ) to create a pellet.Sulfuric acid (95%-98%) in a ratio of 2: 1 was added to sample, and samples were then heated at 90 C using a heating block (Thermomixer compact, Eppendorf, Germany), for 10 min.After cooling to room temperature, phosphoric acid with vanillin was added, and the sample was placed on a shaker for 20 min at room temperature.
Phosphoric acid and vanillin reagent was prepared by heating 25 mL of ddH 2 0 then adding 150 mg vanillin, mix well.After which add 100 mL of phosphoric acid (85-86%), mix well before use.Absorbance was measured at 530 nm using the microplate reader.

Sugar measurement
The anthrone reagent (mixture of 25 mL sulfuric acid and 50 mg anthrone) was added to the supernatant containing the sugar layer and inverted several times, after which it was boiled for 7.5 min.After heating, the samples were placed on ice and absorbance readings were taken at 620 nm on the microplate reader (Varioskan, Thermo Electron Corporation, USA).

Glycogen measurement
Water (200 μL ddH 2 0) was added to the glycogen pellet and heated at 70 C for 20 min.Anthrone was then added to the sample and boiled for 7.5 min.After the samples had cooled to room temperature, absorbance readings were taken at 630 nm on the microplate reader.

Extraction procedure
The method of Unruh et al. (2008) was modified as follows for the extraction of soluble proteins.For each of the 3 replicates, 250 μL of buffer solution was added to 10 mg insects in a microtube.The buffer solution consisted of TBS, 1 M EDTA (pH 8), 0.1% Triton, 0.05% ß-mercaptoethanol and 1X protease inhibitor (10:1:100:1, v/v).
Samples were ground using a motorized pestle (Cordless motor pellet pestle, Kimble, USA) for three sessions (roughly 1 min per session) and then vortexed.Samples were centrifuged for 4 min at 14000 rpm.
The supernatant was transferred to a clean tube, and 250 μL of buffer was added to the pellet.The pellet with buffer was then ground for another three rounds (roughly 1 min per round).Supernatants were combined and centrifuged for 4 min at 14000 rpm.

Quantification
Soluble protein content quantification and analysis were carried out at the Centre for Proteomic and Genomic Research (CPGR) in Rondebosch, Cape Town, South Africa.Samples were transferred to protein Lobind tubes (Sigma 666,505), and cold acetone (Sigma 34,850) was used to precipitate proteins through overnight incubation at À20 C.
Samples were then centrifuged for 15 min at 4 C at 21000 Â g.After the supernatant was removed, the pellet was washed three times with cold acetone after which the pellets were air-dried.Protein pellets were solubilized by resuspending them in 50 μL of 50 mM triethylammonium bicarbonate (TEAB) and 2% Sodium dodecyl sulphate (SDS) and placed at 95 C for 5 min.After which samples were centrifuged for five minutes at 10000 Â g.Quantification was performed using the QuantiPro BCA assay kit (Sigma QPBCA) (SpectraMax 190, USA).
A total of 50 μg of protein from each sample was transferred to a protein LoBind plate (Merck, 0030504.100).The protein was then reduced using tris (2-carboxyethyl) phosphine (TCEP) which was added to a final concentration of 10 mM TCEP and incubated at 60 C for 1 h.Samples were cooled to room temperature and then alkylated with methylmethanethiosulphonate (MMTS, Sigma 208,795) which was added to a final concentration of 10 mM MMTS and incubated at room temperature for 15 min.HILIC (hydrophilic interaction liquid chromatography) magnetic beads were added at an equal volume to that of the sample and a ratio of 5:1 total protein.The plate was then incubated at room temperature on a shaker at 900 rpm for 30 min, allowing for binding of the proteins to the beads.After binding, the beads were washed twice with 500 μL of 95% ACN for 1 min.Trypsin, made up in 50 mM TEAB was added at a ratio of 1:10 total protein and the plate was incubated at 37 C on the shaker for 4 h for digestion.

Electrophoresis proteins
Soluble protein samples that were extracted were separated using SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) based on their molecular weight.A broad range protein standard (BioRad 1,610,317) was used as marker for molecular weights.A volume of 10 μL sample was loaded onto gel for electrophoresis.A Mini Protean tetra system (Biorad, USA) was used at 200 V for roughly 30 min.After separation was complete, the gel was washed with distilled water before staining of protein bands.

Staining of gel
A BioRad Coomassie stain (50 mL) was used to stain the gel.The stain was added to the gel and shaken for 60 min.Gel was rinsed with distilled water until no stain was present in washing liquid for visualization.Larger proteins move slower through the matrix than smaller proteins.Gel bands of $80-85 kDA cute for further analysis.

Gel section digestions
Protein content analysis of gel sections was also carried out at CPGR.Gel bands were destained twice with 100 mM ammonium bicarbonate (AmBic), 50% acetonitrile (ACN) for 45 min with agitation at room temperature.Excess liquid was removed, and the gel pieces were dehydrated with ACN and subjected to vacuum centrifugation for 5 min.Protein was then reduced by rehydrating the gel pieces in 2 mM tris-carboxyethyl phosphine (TCEP), made up in 25 mM AmBic, followed by agitation at room temperature for 15 min.Excess liquid was removed, and protein alkylated by covering the gel pieces in 20 mM iodoacetamide (IAA), made up in 25 mM AmBic, and incubating in the dark at room temperature for 30 min.After alkylation, the gel pieces were washed three times with 25 mM AmBic at room temperature for 15 min with agitation.Excess liquid was removed, and gel pieces were dehydrated as before.Protein was digested by rehydrating the gel pieces in 0.02 mg/mL trypsin made up in 50 mM Ambic.
Gel pieces were incubated on ice for 1 h, and excess liquid was removed.Subsequently, the gel pieces were then covered with 50 mM AmBic and digested overnight at 37 C.After digestion, the excess liquid was transferred to a new tube, and the gel pieces were soaked in 0.1% TFA for 1 h at 37 C. Excess liquid was removed and added to the first extract.Samples were then dried by vacuum centrifugation and the buffer replaced by analytical grade water.Samples were dried down once more and resuspended in 0.1% formic acid (I, 2% ACN made up in analytical grade water for LCMS analysis. LCMS analysis was conducted with a Q-Exactive quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) coupled with a Dionex Ultimate 3000 nano-HPLC system.Peptides were loaded on a C18 trap column (300 μm Â 5 mm Â 5 μm) (Thermo Fisher Scientific, USA).The trap is then switched in-line with the analytical column for loading of peptides for 26 min.Thereafter, the trap is switched offline so as to prevent loading of hydrophobic contaminants.Chromatographic separation was performed with a PepAcclaim C18 column (75 μm Â 25 cm Â 2 μm) (Thermo Fisher Scientific, USA).
The solvent system employed was solvent A: LC water (Burdick and In-gel samples were processed by in-gel digestion.In-solution samples were digested using an automated HILIC magnetic beadbased workflow.In preparation for the HILIC magnetic bead workflow, beads were aliquoted into a new tube and the shipping solution removed.Beads were then washed with 250 μL wash buffer (15% ACN, 100 mM Ammonium acetate (Sigma 14,267) pH 4.5) for one minute.This was repeated once.The beads were then resuspended in loading buffer (30% ACN, 200 mM Ammonium acetate pH 4.5).The rest of the process described hereafter was performed using a Hamilton Mass-STAR robotics liquid handler (Hamilton, Switzerland).A total of 50 μg of protein from each sample was transferred to a protein LoBind plate (Merck, 0030504.100).Protein was reduced with tris (2-carboxyethyl) phosphine (TCEP; Sigma 646,547) which was added to a final concentration of 10 mM TCEP and incubated at 60 C for one hour.Samples were cooled to room temperature and then alkylated with methylmethanethiosulphonate (MMTS; Sigma 208,795) which was added to a final concentration of 10 mM MMTS and incubated at room temperature for 15 min.HILIC magnetic beads were added at an equal volume to that of the sample and a ratio of 5:1 total protein.The plate was then incubated at room temperature on the shaker at 900 RPM for 30 min for binding of protein to beads.After binding, the beads were washed twice with 500 μL of 95% ACN for one minute.For digestion Trypsin (Promega PRV5111), made up in 50 mM TEAB was added at a ratio of 1:10 total protein and the plate was incubated at 37 C on the shaker for four hours.After digestion, the supernatant containing peptides was removed and dried down.
Samples were resuspended in 0.1% TFA (TFA, Sigma T6508) prior to clean up by Zip-Tip (Sigma Z720070).Thereafter, samples were dried down once more and then resuspended in LC loading buffer: 0.1% FA, 2.5% ACN.Peptides were then analysed by LCMS, and relative quantification results were obtained.

Statistical analysis of data
Statistical analysis of total proteins, lipids, glycogen, and sugars was analysed using a one-way analysis of variance with STATISTICA version 13 (Statsoft Inc., 2017).ANOVA-generated P-values and the significant differences between means w're determined using Fisher's least significant differences (LSD) test with a 95% confidence interval.Venn diagrams (http://bioinfogp.cnb.csic.es/tools/venny/)were used to determine proteins in common and exclusive during the different evaluation periods.Heat maps were generated using XLSTAT-Biomed (https://www.xlstat.com/en/solutions/biomed).

Compositional changes of macromolecules (total proteins, lipids and carbohydrates)
Significant differences for each of the macromolecules analysed were observed for the different sampling periods-before aestivation, early aestivation, and mid-late aestivation.During the before aestivation period, significant differences were found between the five collection times; therefore, the data are presented for each collection time.No significant differences were observed, however, between the two collections done during each of the aestivation sampling periods (early aestivation, mid aestivation, mid-late aestivation and late aestivation).
Data from the two sampling weeks for each aestivation period were, therefore, pooled.Values given in the text are the mean values; see supplementary material for associated standard deviations.
Glycogen levels at week 44 (before aestivation) were significantly higher than for all the other time points (F (8,20) = 31.440p < 0.0001) (Figure 1).The glycogen content almost doubled from 28 μg/mg DW (dry weight) to 51 μg/mg DW from week 43 to 44.After this increase, glycogen levels decreased to between 6 and 12 μg/mg DW during weeks 45 to 47. Once the insects entered aestivation (week 48), an increase in glycogen levels were observed until mid-late aestivation (week 20 and 22), with levels reaching 42 μg/mg DW.During the late aestivation period (week 24 and 26), glycogen levels decreased to 29 μg/mg DW.
During the five weekly collections made in the before aestivation period, sugar content decreased significantly from 166 μg/mg DW (week 43) to 50 μg/mg DW (week 47).This was followed by a slight increase and then a decrease as the aestivation period started (early (weeks 48 and 50) and mid (weeks 11 and 13)), but levels were not significantly different from weeks 45 and 46, before aestivation.By mid-late aestivation, sugar content increased significantly again to 110 μg/mg DW.A decrease in sugar content occurred from mid-late (weeks 20 and 22) to the late aestivation period (weeks 24 and 26).
Prior to the aestivation period, total lipid content increased significantly reaching a peak of 153 μg/mg DW by week 45 (F (8,21) = 23.229p < 0.0001) (Figure 1).Lipid content started to decrease gradually as the insects progressed into and through aestivation.By mid-late aestivation (weeks 20 and 22), lipid content was 97.7 μg/mg DW.This was followed by a significant decrease in lipid content in late aestivation (weeks 24 and 26) samples to 56 μg/mg DW.
Figure 1 also illustrates an overview of changes in glycogen, sugar and lipid content over the entire sampling period (weeks 43 to 26) with highlighted sections indicating when the most significant changes occurred.During week 45 (before aestivation), an increase in lipid content occurred, with a decrease in sugar and glycogen content.During the early aestivation period (weeks 48 and 50), the lipid content stabilized, while increases in glycogen and sugar content occurred.A significant increase in sugar and glycogen content was observed from the mid to mid-late aestivation periods (weeks 11 to 22), with a nonsignificant decrease in lipid content during that same time period.
During the mid-late to late period.a decrease in total lipids, sugar and glycogen content was observed.

Soluble proteins comparison
The soluble protein extraction method yielded different compositional profiles of different proteins for each of the sampling periods (before, early until late).No significant differences between the proteins identified for each weekly collection during the before aestivation period, as well as the two weekly collections during aestivation, were observed.Additionally, no significant differences were observed across the samples collected during the mid, mid-late and late aestivation periods.Therefore, the data for the five collections in the before aestivation period were pooled, as well as all the mid, mid-late and late aestivation periods (six collections in total).An overview comparing the different proteins for the different sampling periods, before (pooled); early (pooled); and mid until late (pooled) is illustrated in Figure 2 as a Venn chart.The chart illustrates that 281 proteins were exclusively found in before samples, 48 proteins were in common between before and early samples, 38 were exclusive to early samples, 230 were common between before and mid until late aestivation samples, and 89 proteins were in common across all the sampling periods (before, early until late).No proteins were found to be exclusive to the mid until late aestivation period.
The deduced functions of the annotated genes for M. diplopterus allowed for the different proteins present during the sampling period to be placed into functional groups.Comparison of the functional Although the total protein content decreased significantly over time as seen under analysis of the macromolecules, the levels of the individual proteins that make up the total protein content changed over time.This is evident in the gel images generated in the soluble protein assays (Figure 4).
The gel image indicates two bands that became darker over time (as the insect moved into aestivation).Darkening and thickening of the bands imply that the concentration of protein increased.Gel section digestion and identification of proteins in the isolated band ($80-85 kDA in size), revealed that from the early aestivation period (weeks 48 and 50) to the mid aestivation period (weeks 11 and 13), 14 proteins were common to both periods, but 15 were unique to the early period, and the number unique to the mid aestivation period increased to 46 (Figure 5).Analysis of isolated gel sections indicated F I G U R E 2 Venn diagram illustrating the proteins that were exclusive to, and in common between, the before, early and mid until late aestivation periods.281 proteins were exclusive to before aestivation samples, 48 proteins were in common in before and early aestivation samples, 38 were exclusive to early samples, 230 were in common between before and mid until late aestivation, and 89 proteins were in common during the whole sampling period.
that the mid aestivation samples contained a significantly higher number of heat shock proteins compared to the early aestivation samples.
Indicating that although the individual proteins of the posttranslational modification group decreased the abundance of individual proteins increased from early to mid.
Comparing the aestivation periods with each other revealed 86 proteins unique to the early aestivation period, 230 proteins in common between mid until late aestivation periods and 89 in common between all four aestivation periods with (Figure 6).These 89 proteins were also present during the before aestivation period (see Figure 2).A heat map (Figure 7) was generated to visualize the abundance or intensity of expression of these common proteins over time, based on some of the identified proteins.These included the heat shock proteins and others involved in energy storage, synthesis and transport.
During the before aestivation period (weeks 44 to 47), Acyl-CoA dehydrogenase/oxidase, fructose-bisphosphate aldolase, enolase, glyceraldehyde 3-phosphate dehydrogenase and fatty acid synthase all had high levels of expression (Figure 7).This was particular to weeks 44 and 45 for Acyl-CoA dehydrogenase/oxidase and fructosebisphosphate aldolase, which decreased in expression after that, but had high levels of expression again by late aestivation (week 24).For the latter three, enolase, glyceraldehyde 3-phosphate dehydrogenase and fatty acid synthase, high expression continued into early aestivation (weeks 48 and 50) but decreased after that.The differences in expression of these various proteins before and during aestivation may be related to the interconversions in lipids, sugars and glycogen, presented above.
Expression of the Hsp70 family is confined to the aestivation period (Figure 7), as very low intensity of expression occurs before aestivation.Hsp90 also increases in expression during late aestivation.
In addition, an unknown protein was expressed during aestivation, and requires characterization.Of the unique heat shock proteins (not included in Figure 7) identified during mid until late aestivation, smHsp20 families were amongst the highest in abundance.During early aestivation, heat shock proteins which form part of the Hsp10 and 60 families were identified as unique when compared to mid until late aestivation.

DISCUSSION
Aestivation involves an ancient signalling and regulatory system enabling organisms to suppress growth and development during periods in which water, nutrients, and energy levels are limited, and is considered a form of less severe dormancy, where physiological changes can be reversed rapidly (Storey & Storey, 2012).Organisms F I G U R E 6 Venn diagram illustrating the proteins that were exclusive to, and in common between, the aestivation periods early, mid, mid-late and late aestivation of Macchiademus diplopterus.86 proteins were exclusive to early aestivation samples, 230 proteins were common to mid, mid-late and late aestivation and 89 were in common in all four aestivation periods.Energy usage during aestivation is minimized through reduction in metabolic rate, but this also results in a reduction in the normal synthesis and degradation of macromolecules (carbohydrates, lipids and proteins).Therefore, other preservation strategies require upregulation to ensure survival.These mechanisms include enhanced antioxidant defences and increased chaperone proteins (e.g.heat shock proteins) to manage different stress responses (Kültz, 2005).
The biochemical regulation of organisms during aestivation has two main options to regulate low metabolic activity.One is through reversible controls suppressing cell function, examples including the inhibition of enzyme and functional protein activity.The other option is through compositional changes of selected proteins due to differential transcription, translation or degradation, for example, the up-regulation of the urea cycle enzymes, antioxidant enzymes, iron binding enzymes and heat shock proteins (Ip & Chew, 2010;Ramnanan et al., 2009;Storey & Storey, 2007, 2011).During extended periods of dormancy, the ability of oxidatively damaged macromolecules to degrade and resynthesize is low due to the suppression of these energy consuming processes.Through elevated levels of antioxidants, the oxidative damage to macromolecules could be limited, as with increased levels of elevated chaperones, which limit the accumulation of misfolded proteins during long-term dormancy.
Before aestivation, M. diplopterus feeds on host plants and can fly to aestivation sites in which they seek shelter during the aestivation period when a low metabolic state is implemented for survival.As M. diplopterus moves into and through aestivation its energy reserves, with regard to glycogen, sugars and lipids change.The decrease in glycogen observed during the period before the insect enters aestivation could be a result of energy usage for migratory flight to aestivation sites.A rapid decrease in glycogen could occur through either direct utilization as an energy source during a cold shock or through flight (Amiri & Bandani, 2013).This is due to the ability of glycogen and sugars to be interconverted during temperature changes.
The increase in glycogen content during the early to mid-aestivation period, in conjunction with a decrease in sugar content could have been due to inter-conversion taking place at this time and is also reflected in the changes in the functional groups and the increase of proteins involved in energy production.During the mid to mid-late aestivation period, an increase in the sugar levels occurred.This could be a result of a stress response in which adaption to high temperatures (during summer) was needed, and the production of trehalose and sugar alcohols was induced from glycogen (Storey, 1997).
The importance of body fat during dormancy is evident in the increase in lipid content in M. diplopterus during the weeks before the onset of aestivation.Macchiademus diplopterus appears to create a large lipid reserve before entering aestivation, probably as an energy source for survival during its prolonged period of low metabolic activity.
The same phenomenon is seen in another Hemipteran pest of graminaceous plants, Eurygaster integriceps Put.(Hemiptera: Scutellidae), a member of a complex of species that make up a group generally known as Sunn pests (Critchley, 1998).Macchiademus diplopterus and E. integriceps display similar behaviour and in that, the latter also undergoes an obligatory diapause period, during which it ceases to feed and remains in sheltering sites.In E. integriceps, however, the diapause period is made up of two phases: aestivation during late summer and autumn, and hibernation during winter, collectively termed as the overwintering period (Brown, 1962a;Brown, 1962b;Critchley, 1998).Bashan et al. (2002) found that an increase in stored lipids in prediapause adult E. integriceps occurred in spring, which would then be used to support their energy requirements during aestivation and hibernation.During the overwintering period of E. integriceps, approximately 25% of the insect's stored fat reserves is consumed, and a large portion of the population may die if these fat reserves are inadequate (Critchley, 1998).The higher levels of lipids within the insect's body fat are due to its higher capacity for lipid synthesis than glycogen synthesis (Amiri & Bandani, 2013).An insect that contains higher levels of lipids has a more significant energy reserve, which could also be used for post aestivation or diapause activity (Ellers & Van Alphen, 2002).During aestivation, it is crucial that the organism conserves energy and rations the use of stored fuels.It needs to retain body water and has to dispense nitrogenous end products and stabilize organs and cells for extended periods (Storey & Storey, 2012).Lipid accumulation is, therefore, also important in aestivation or diapause termination, which may indicate why the lipid levels in M. diplopterus started to decrease significantly during the late aestivation period.Macchiademus diplopterus has a long flight from wheat fields to aestivating sites in which the conversion of carbohydrates to lipids takes place for energy storage during aestivation.This could be why there was an increase in both glycogen and lipids before entering aestivation as the insects are actively flying around, but simultaneously preparing for energy conservation.
Although macromolecule analysis indicated that total protein content of M. diplopterus, before and during aestivation decreased, analysis of individual soluble proteins indicated that changes occurred that also support the hypometabolic state of aestivation.
The increase in abundance of acyl-CoA desaturase which occurred during late aestivation, coincides with the onset of winter.The enzyme Δ 9-acyl-CoA desaturase has an essential role in temperature adaptation for insects exposed to low temperatures by increasing the ratio of unsaturated to saturated fatty acids in cell membranes to maintain the liquid crystalline phase (Kayukawa et al., 2007).An increase in Δ 9acyl-CoA desaturase expression, therefore, has a role in cold hardiness.(Kikuchi et al., 2017).Fatty acid synthase is a multi-enzyme protein that catalyses fatty acid synthesis.During extended periods of nonfeeding, lipid metabolism is essential as an energy source (Gilbert, 1967).
The identification of proteins at the different stages before and during aestivation shows how M. diplopterus can become more tolerant to thermal stresses such as heat and cold treatments as the aestivation period progresses.The increase in heat shock proteins (HSP) such as smHsp20, Hsp60, 70, 83 and 90 during the mid and mid-late aestivation periods, compared to the early aestivation period is significant.The importance of Hsp70 and 90 in an organism's thermal response is well documented in literature (Guo & Feng, 2018;Lindquist & Craig, 1988).Heat shock proteins are identified and related to their molecular weight, for example, 70 kDa HSP is referred to as Hsp70.These Hsp70s consist of HSC70 (heat shock cognate 70) and stress-inducible isoforms that are upregulated with long-term cold exposure (Clark & Worland, 2008).These highly conserved proteins act as chaperones to stabilize and refold denatured proteins.In doing so, the insect can prevent the formation of cytotoxic aggregates (Fink, 1999;Hartl, 1996;Parsell & Lindquist, 1993).An increase in the expression of heat shock proteins is a reliable indicator of thermotolerance or thermal protection.In Drosophila melanogaster Meigen (Diptera: Drosophilidae), the development of thermo-tolerance is linked to the production of Hsp70, which is virtually absent from nonstressed cells (Lurie & Jang, 2007).
The level of expression of Hsp70 will influence the outcome of thermotolerance; too little (induced by either a too short stress treatment or lowering the temperature) will restrict the level of thermotolerance that develops (Lurie & Jang, 2007).Over expression of Hsp70 has resulted in problems with D. melanogaster lines in that treated lines were unable to tolerate the stress, compared to control lines.Overproduction of Hsp70 can, however, increase thermo-tolerance in some stages of development.Insects from warmer climates may have tighter control of HSP production to enable them to limit unnecessary production of HSPs, which could be an energy costly process.Thermotolerance has also been correlated with induction of HSP in insect pests after exposure to non-lethal temperatures (Neven, 2000).The presence of Hsp70 induced by heat conditioning treatment in codling moth, Cydia pomonella (Lepidoptera: Tortricidae), is related to the survival rate of the insect after application of a quarantine heat treatment (Lurie & Jang, 2007).Accumulation of Hsp70 followed thermo-resistance to high Regulators through epigenetic mechanisms (e.g.DNA methylation and histone modification) of transcriptional suppression provide a mechanism for gene silencing during periods of hypometabolism for functions such as development and differentiation (Fraga et al., 2007;Storey & Storey, 2007;Storey & Storey, 2012).During a cell stress response, mRNA's are reprogrammed and recruited to stress granules which are preserved and made available for rapid translation when organisms are exiting their hypometabolic state.These stress granules are another area for research in aestivating organisms (Kedersha & Anderson, 2009;Storey & Storey, 2012).Specific mRNA is preserved over winter in the fat cells of larvae of Eurosta solidaginis Fitch (Diptera: Tephritidae) and Gynaephora groenlandica (Lepidoptera: Lymantriidae) which allows for a rapid recovery to original levels when an increase in temperature occurs (Levin et al., 2003).Thus, indicating not one element or protein is solely responsible for the insects' survival and adaption to thermal and desiccation stresses, but an array of components working in synergy is able to achieve this.
Further research is required to better understand the environmental or genetic triggers that enable the insect to initialize the production of heat shock proteins, etc., as defence mechanisms, allowing them to tolerate a variety of stresses.The large number of soluble proteins isolated during this study, but that could not be characterized, need to be identified, as they may also shed some light on other mechanisms and proteins involved in the physiology of aestivation in M. diplopterus.
Analysis of mechanisms involved in transcriptional and translational control will provide insight into the regulation of aestivation and the mechanisms insects use to control their metabolic arrest and extend their life span during unfavourable conditions (Storey & Storey, 2012).This study has indicated that multiple factors play a role in preparing and protecting M. diplopterus against thermal and desiccation stresses which it may encounter during its aestivation cycle.The changes observed in abundance or intensity of heat shock proteins is but one of the components that enable M. diplopterus to adapt for survival.The increased levels of Hsp70 and 90 observed here, as aestivation progressed, now provide concrete evidence for the mechanisms behind the increase in thermal tolerance in aestivating M. diplopterus reported by Okosun (2012).Increasing thermal tolerance in aestivating M. diplopterus makes development of a thermal phytosanitary treatment challenging, as a single temperature that will be effective throughout aestivation must be developed using insects at their most tolerant state, which is difficult to ascertain.No universal thermal treatment will be effective to control M. diplopterus throughout the aestivation period, which is when they affect fruit and can be found contaminating export fruit consignments.
throughout aestivation, in order to obtain samples of insects representing five different stages: (based on calendar weeks) (1) before the aestivation cycle started (weeks 43 to 47; 5 collections); (2) early in the cycle (week 48 and 50; 2 collections); (3) midway through the aestivation cycle (week 11 and 13; 2 collections); (4) mid until late (week 20 and 22; 2 collections) and (5) late in the aestivation cycle (week 24 and 26; 2 collections).Aestivating M. diplopterus insects were collected from shelter sites under the bark of blue gum trees (Eucalyptus globulus Labill.) and from corrugated cardboard bands tied around the base of fruit trees.Six replicates, collections consisting of 40 insects per replication were made each sampling week.After collection, insects were placed in a À 80 C freezer until samples were freeze-dried to remove moisture and preserve content for analysis.Extractions from the freeze-dried (Beta 1-8LD plus, CHRIST, Germany) samples were used to determine the levels of macromolecules (proteins, lipids, sugars and glycogen) and soluble proteins present at each sampling point over the aestivation period.A 10 mg of sample was required for analyses, which was equivalent to 10 insects; therefore, 10 grain chinch bugs were used per replicate for determination of macromolecules (6 replicates; n = 60 insects per sampling week) and soluble proteins (3 replicates; n = 30 insects per sampling week), and the excess stored for future use.
Jackson BJLC365); 0.1% FA and solvent B: ACN, 0.1% FA.The multistep gradient for peptide separation was generated at 300 nL/min as follows: time change 6 min, gradient change: 3.5-9% Solvent B, time change 45.5 min, gradient change 9-24.6%Solvent B, time change 2 min, gradient change 24.6-38.7%Solvent B, time change 2.1 min, gradient change 38.7-52.8%Solvent B, time change 0.4 min, gradient change 52.8%-85.4%.The gradient was then held at 85.4% solvent B for 10 min before returning it to 3.5% solvent B for 15 min to condition the column.The mass spectrometer was operated in positive ion mode with a capillary temperature of 320 C. The applied electrospray voltage was 1.95 kV.
The peptide sequences were searched against Oncopeltus fasciatus [Dallas] downloaded from Baylor College of Medicine sequencing centre.Raw files were processed using Progenesis QI for Proteomics (Non-linear Dynamics, UK) software and regulated proteins ( p-value <0.05 and fold change ≥2), containing at least two unique peptides, were reported.Relative quantification was conducted using Progenesis QI for Proteomics (Nonlinear Dynamics, UK).Data processing included peak picking, run alignment, and normalization (singly charged spectra were removed from the processing pipeline).Relative quantification was based on four biological replicates per condition using non-conflicting peptides.A protein with a fold change ≥2 with a corresponding p-value of <0.05 was considered regulated.Database interrogation was performed with Byonic Software (Protein Metrics, USA) using the Oncopeltus faciatus database sourced from Baylor College of Medicine sequencing centre (www.hgsc.bcm.edu).The functions of annotated genes for M. diplopterus were hypothesized based on homology with gene sequences from other available sources.Annotations were provided by an online source OrthoDB (https://www.orthodb.org).Deductions are based on the recognition of gene sequence similarities which indicate shared ancestry(Kriventseva et al., 2019).

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I G U R E 1 Comparison of changes in mean glycogen, sugar, total lipid and total protein content (μg/mg DW) in Macchiademus diplopterus samples collected before and during the aestivation cycle (November 2016 -June 2017).The rectangles indicate when the most significant changes occurred.DW = dry weight.groups at different times during the sampling period indicates if an increase or decrease in activity or abundance occurred.Proteins identified for each sampling period could theoretically be involved in more than one functional group.This was taken into account when calculating the percentage of each group.Figure 3 illustrates the percentage of different functional groups present in the before, early and mid until late aestivation samples.A decrease in abundance was observed for various functional groups as the insects entered aestivation.The highest percentage of proteins observed during the before aestivation period were involved in energy production and conversion (approx.27%).Entering into the early stages of aestivation this functional group decreased by approx.7% in abundance.A 7% increase was also observed for proteins involved in post-translation modification as the insect progressed into early aestivation.The early aestivation insects contained a larger number of uncharacterized proteins (32%) compared to the 11% observed before the insect entering aestivation.Proteins involved in functions such as replication, recombinant and repair, cell motility and proteins involved in the cytoskeleton, which were present before aestivation, were not detected during the early aestivation period.Moving from early aestivation into the mid until late period, overall, a decrease in abundance of functional groups occurred, rather than in the number of individual proteins identified.A 7% increase in the abundance of proteins involved in energy production and conversion took place, with a 16% decrease in uncharacterized proteins.During week 48 and 50 proteins which were high in abundance included malate dehydrogenase, which is involved in energy production and conversion.Also during the transition from early aestivation to mid until late aestivation, a 4% decrease in individual proteins occurred in the posttranslational modification group, which contains proteins such as heat shock proteins.This indicates a decrease in the number of individual proteins but not necessarily their individual abundance.
Percentages of functional groups present in the soluble proteins extracted from Macchiademus diplopterus collected before entering aestivation (weeks 43 to 47 pooled), during early aestivation (weeks 48 and 50 pooled) in November and December 2016 and during the mid until late (weeks 11 until 26 pooled) from December 2016 to June 2017.The before aestivation period contains functional groups from 648 proteins; and the early aestivation period, functional groups from 175 proteins; and the mid until late period, functional groups from 319 proteins.Some gene products have more than one function.The functional group energy production, conversion and transport includes lipid and carbohydrate transport and metabolism.An example of proteins from energy production group included Acetyl-CoA carboxylase and fatty acid synthase; Cytoskeleton = Actin; Posttranslational modification, protein turnover, chaperones = Heat shock protein 70 family and small heat shock protein HSP20.
entering and maintaining a hypometabolic state during aestivation require biochemical changes that ensure conservation of energy, F I G U R E 5 Venn diagram illustrating the unique and common proteins from gel bands cut at $80-85kDA of Macchiademus diplopterus samples collected during early aestivation (weeks 48 and 50) and mid aestivation (weeks 11 and 13).

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I G U R E 7 Heat map illustration of intensity of expression of proteins in common, identified from Macchiademus diplopterus samples collected before and during aestivation (weeks 44-26) from November 2016 to June 2017.
temperatures in D. melanogaster.In studies conducted on C. pomonella, a decrease in Hsp70 was linked to disappearance of thermotolerance induced by a 35 C pre-treatment and the sensitivity of the insect to 50 C quarantine treatment(Yin et al., 2006).Research conducted byKinene et al. (2019) indicated the potential of the Hsp90 gene to increase whiteflies, Bemisia tabaci (Hemiptera: Aleyrodidae), chances of survival against heat stress and other biotic factors.The increase, therefore, in Hsp70 and Hsp90 observed in M. diplopterus, is an indication that the insect has an increased ability to tolerate thermal stresses more effectively.The activity of various proteins and enzymes is involved in the defence mechanisms that protect M. diplopterus against thermal stresses.The changes in proteins involved in post-translation modifications seen during aestivation also indicate adaptability.Posttranslational modification is one of the most important strategies to environmental adaption through the crucial role it plays in regulating protein function and controlling several fundamental features of cell biology, such as cell signalling, protein turnover, cell-cell interactions, and cell differentiation Zhang et al. (2016).