Features of Protein-Peptide and Carbohydrate Composition of Supernatants From Tenebrio Molitor Larvae After Cold Acclimation

The paper describes the chromatographic studies on the molecular-mass distribution of proteins and peptides of supernatants, derived from Tenebrio molitor larvae during cold acclimation. Supernatants of non-acclimated larvae of T. molitor had the highest amount of peptide fractions. It has been shown that cold-acclimated T. molitor larvae comprised the low-molecular peptide fractions with MW of (540 ± 20) – (2.255 ± 85) Da, and high-molecular peptides with MW of (4.675 ± 225) – (6.595 ± 550) Da were characteristic for non-acclimated larvae. The content of sugars and polyols in supernatants of cold-acclimated and non-acclimated T. molitor larvae was determined. It has been found that glucose concentration in non-acclimated T. molitor larvae was in 1.34 times higher than in cold-acclimated ones. Аcclimated T. molitor larvae had more hydrophilic and less hydrophobic proteins as compared to non-acclimated ones. As well it has been established that non-acclimated T. molitor larvae had low concentrations of polyols (sorbitol and mannite), which were absent in acclimated insects.

The research aim was to study peptide and carbohydrate composition of supernatants derived from T. molitor larvae by gel-permeation and high performance liquid chromatography, as well as to investigate the protein distribution on their hydrophobicity extent after cold acclimation of T. molitor larvae by reverse phase chromatography.

Materials and methods
The research was performed in the larvae (n = 6) of T. molitor mealworm of last ages, kept at 25°C in transparent containers filled with wheat and oat bran.Before the experiment the insects were acclimated at 5...7°C for three weeks.To derive the proteins and peptides, the larvae were homogenized in 0.6% NaCl in 0.1 M Na-phosphate buffer (pH 7.4) supplemented with protease inhibitor phenylmethylsulfonyl fluoride (Sigma, USA) putting six insects per 2 ml of buffer.The homogenate was centrifuged for 10 min at 1,800g then the supernatant was centrifuged for 60 min at 100,000g.The supernatant was then used for studies.
Protein concentration in the samples was determined by the Bradford method [19].
Peptide composition of supernatants of T. molitor larvae was quantitatively and qualitatively analyzed using the gel-permeation chromatography in the 400×16 mm column filled with polyvinyl gel TSK-Gel Toyopearl HW-40 Fine (Toyo Soda Manufacturing Co, Japan), the use of which allows to separate polypeptide molecules with MW 100-12,000 Da.
The samples of 0.2 ml were injected through a loop injector into the column and eluting phosphate buffered saline (18  Da (all the substances were of Sigma production) were used as the molecular weight markers.
Aliquots derived from the supernatants were passed through membrane filters Chromafil GF/PET-45/25 ZF-S (Macherey-Nagel, Germany), and then the content of sugars and sugar alcohols were examined and revealed at 75°C by high performance liquid chromatography using Smartline chromatograph (Knauer, Germany) in the 300 × 8 mm column Erokat H (Knauer) filled with cross-linked polystyrene copolymers with a pore size of 10 µm.
The sample of 0.015 ml was introduced into the column.Water of the first purification degree with 0.5 ml/min flow rate was used as mobile phase.Content of sugars in supernatants was determined by RI Detector 2,300 refractometric detector (Knauer).To process and calculate the results the Clarity Chrom software (Knauer) was applied.There were used glucose, sucrose, fructose, lactose, sorbitol, mannite (all substances of Sigma production) as standards.
To investigate T. molitor larvae protein hydrophobicity by reverse phase high performance liquid chromatography we have used a liquid chromatograph Shimadzu LC-2010 (Shimadzu, Japan) and the 250×4.6 mm column Vydac C4 (The Separations Group, USA) filled with spherical silica gel having a pore size of 300 D.
Proteins were gradient eluted from the column with two mobile phases (MP) reagents of Fluka Chemie (Switzerland) (MP A -90% of buffer solution at pH 4.2, 10% of acetonitrile and MP B -10% of buffer solution at pH 4.2, 90% of acetonitrile) according to the following program: first 8 mins elution was performed with buffer A; from 9 to 35 mins buffer B amount was increased from 0 to 80%; from 36 to 40 min, the concentration of buffer B was not changed.
To prepare buffer solution we have used 0.026 M lithium dihydrogenphosphate solution, which was brought to concentrated phosphoric acid to pH 4.2.Mobile phase flow rate made 0.75 ml/min, column temperature was 30°C.
Proteins were identified at 280 nm wavelength with a UV detector SPD-M6A (Shimadzu).
The experimental data were statistically processed using the non-parametric Mann-Whitney criterion.The differences between the samples were considered as significant at p < 0.05.
Adaptation of living organisms to low temperatures is complex and multi-step process, accompanied with the changes in protein spectrum, structural rearrangements in molecules of some proteins, as well as qualitative and quantitative modifications of cryoprotectants, sugars and polyols.Therefore, it is of interest to study the adaptation features of cold-resistant species to low temperatures.
As reported widely the mealworm T. molitor larvae (Tenebrionidae family) is a convenient model to investigate molecular mechanisms of cold resistance of living organisms.As we have earlier shown using SDS-PAGE electrophoresis [13] they were capable to produce the proteins with MW of 5-10, 30 and 65 kDa during two weeks of cold acclimation.Antifreeze proteins [3,12,16,17] providing hypothermia of T. molitor larvae under low temperature conditions wre also studied.Nevertheless, the information about the changes of the whole spectrum of watersoluble pro-teins and peptides, their structural features, as well as qualitative and quantitative composition of sugars and polyols during cold acclimation are not comprehesive yet.
Using gel-permeation chromatography, we obtained molecular weight distributions of proteins and peptides from supernatants derived from cold-acclimated and non-acclimated T. molitor larvae (Fig. 1, Table 1).Substances with MW # 7,000 Da were defined as peptides and substances with MW $ 12,000 Da were considered as proteins.
Analysis of the experimental data presented in Fig. 1, showed that qualitative composition of low-molecular substances of protein-peptide nature from T. molitor larvae during cold acclimation was significantly changed.A decrease in peptide fractions number was observed in T. mollitor larvae acclimated for three weeks.A high molecular weight fraction of proteins with MW $ 12,000 Da and low molecular weight fractions with MW (2.255 ± 85), (1.525 ± 115), (1.105 ± 115), (825 ± 55) and (540 ± 20) Da were the main protein-peptide fractions of supernatants derived from cold-acclimated and non-acclimated T. molitor larvae.
Figure 1 shows that supernatants derived from cold acclimated T. molitor larvae contain six low molecular weight peptide fractions with MW (540 ± 20) -(2.255 ± 85) Da.It should be noted that cold acclimation T. molitor larvae resulted in disapperance of four high-molecular peptide fractions with MW (6.595 ± 550), (6.040 ± 280), (5.325 ± 195)  Comparing the chromatograms of protein-peptide fractions distribution for supernatants derived from acclimated and non-acclimated T. molitor larvae, we have revealed that the total contents of all the components slightly differed.In particular, the content of substances absorbing in UV region of spectrum in coldacclimated insects was by 1.11 times higher than in non-acclimated ones.
Существенно изменялось распределение белково-пептидных компонентов в процессе акклимации.Сравнительный анализ хроматографических данных белково-пептидных фракций из личинок T. molitor показал, что количество белковой фракции (пик Pr) с м. м. $12 000 Да у cold-acclimated ones.In non-acclimated T. molitor insects we have found an increase in the number of peptides with low MW: Analysis of the findings enable to assume that the change in protein-peptide composition of the supernatants derived from T. molitor larvae, during lowtemperature acclimation is associated either with synthesis of protein and peptide components de novo, or structural modifications of existing proteins.
Retention time of proteins in a column allowed to establish, that the amount of hydrophobic proteins in non-acclimated T. molitor larvae was higher than in cold-acclimated ones (Fig. 3).Proteins retained in the column filled with hydrophobic substance during maximum time were hydrophobic proteins and the proteins with a minimal retention time were hydrophilic.A larger amount of hydrophilic proteins in acclimated T. molitor insects compared to non-acclimated ones was revealed.The findings were consistent with the reported data on diminishing the degree of protein hydrophobicity in cold-resistant organisms following low temperature adaptations [4,15].
Accumulation of natural cryoprotective agents (sugars and sugar alcohols) had been known to be one of the mechanisms of cold adaptation in animals and plants, therefore we investigated the content of sugars and sugar alcohols in supernatants, derived from T. molitor larvae using high-performance liquid chromatography (Table 2).
Table 2 demonstrates, that in non-acclimated T.molitor larvae the glucose level is 1.34 times higher than in acclimated ones.Moreover, low concentrations of sugar alcohols (sorbitol and mannite) were observed in non-acclimated T. molitor larvae, and absent in coldacclimated insects.Change in the carbohydrate composition during acclimation of T. molitor larvae may indicate to either transformation of metabolic processes of sugars and sugar alcohols formation or possible formation of complex components such as carbohydrateprotein compounds in insects during acclimation.It allows preservation and optimizations of protein functions, as well as stabilization of macromolecules under stress conditions.
The analysis of experimental data enabled us to assume that in the mechanism of the complex and multistep cold adaptation a key role was played by the rearrangements in protein exchange, including enzymes, cold shock proteins, as well as regulatory proteins and peptides.To confirm this hypothesis there is a need in additional studies using protein synthesis inhibitor (e. g., cycloheximide), which will be the goal of further experiments.

Conclusions
It has been shown that low-temperature adaptation of T. molitor larvae was accompanied with the changes within spectrum of protein and peptide substances in their tissues occurred.There was an in increase in the amount of protein fractions with MW of 12.000 Da, a reduced Литература (2.255 ± 85) Da in 8; (1.525 ± 115) Da in 2.3; (1.105 ± 115) Da in 1.3, and (825 ± 55) Da in 1.22 times.