Biochemical and Molecular Studies Related to Phase Change in Gregarious and Solitarious Desert Locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae)

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 11(5)pp: 149161 (2018) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Entomology Journal publishes original research papers and reviews from any entomological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, development, genetics, systematics, morphology, evolution, control of insects, arachnids, and general entomology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
The desert locust, S. gregaria is a phylogenetically heterogeneous insect group within the family Acrididae that demonstrated a distinct ability to change phases from solitary to gregarious in response to population density (Kang et al., 2004).Uvarov (1921) formulate the term phase to illustrate the taxonomic status of different morphological forms of migratory locusts, Locusta migratoria and Locustana pardalina, where the concept of phase polymorphism was put firstly in these species.After that, this concept extended to S. gregaria (Uvarov, 1966(Uvarov, , 1977)).He described "solitaria", one of the two extreme phases, as isolated and relatively sedentary individuals, while "gregaria" is common crowded individuals and swarming population.Undoubtedly, the local population density induces the expression of graded changes in a group of traits that include colouration, morphometry, anatomy, egg mass, food selection, nutritional physiology and reproductive physiology (Pener, 1991;Pener and Yerushalmi, 1998;Simpson et al., 1999Simpson et al., , 2005;;Tanaka, 2001Tanaka, , 2006;;Ferenz and Seidelmann, 2003;Kang et al., 2004).Several investigations revealed that these changes evolved by external factors (Uvarov, 1966(Uvarov, , 1977;;Pener, 1991;Wiesel et al., 1996).Here, we supposed that local population density induced internal changes which may regulate and correlate the transformation processes.Consequently, we aimed to present preliminary information about levels of total proteins and DNA level, as indicators of differential internal bio-chemicals and molecular expression.Quantitative and qualitative analysis of both total protein and DNA levels were investigated in two extreme phases, gregarious and solitarious S. gregaria, in addition to, their transient stages.

Experimental Insect:
The gregarious insects were reared and handled under the crowded conditions for several consecutive generations at Plant Protection Research Institute, Agricultural Research Center, Dokki, Giza.The insects showed pronounced gregarious phase characteristics according to Hunter-Jones (1961).The solitarious phase of S. gregaria was derived from the gregarious stock colony.They were reared in the separated cages.Only one individual was kept in a small wooden cage (10×10×10 cm) in diameter.They were isolated from each other for several generations, five generations in this current study, under laboratory conditions.Locusts of the solitarious phase were reared singly, but at the time of the mating, they were paired.Locusts raised under these conditions showed pronounced solitarious phase characteristics (Simpson et al., 1999).

Biochemical Studies:
The experimental adults were collected as the fresh molting adults from the long-term gregarious and solitarious stock colony and their intermediate stages.For biochemical analysis, five healthy fresh molting adults were collected from each generation and kept under freezing conditions at -5°C.

Quantitative Protein Analysis:
The half gram of body tissue sample was weighed and homogenized with 1ml of the extraction buffer.The homogenate was diluted by adding about 100ml of PMSF (phenylmethylsulfonyl fluoride).Samples were ground using liquid N 2 and sonicated with ice water for about 30 minutes.Samples were centrifuged for about 15 min at 12000 rpm.The soluble protein content of the supernatant was determined by the method of Lowry et al. (1951) using bovine albumin as standard.The concentrations of total protein of all samples were measured using spectrophotometer (UV absorbance at 280 nm).

Qualitative Protein Analysis:
Samples which were prepared to estimate the total protein levels were used in electrophoresis analysis.A part from the supernatants was withdrawn carefully using automatic pipettes and transformed to a new clear Eppendorf tube and kept frozen at -70 o C till needed.SDS-PAGE was performed in 12% acrylamide slab gel according to the method of Laemmli (1970) with some modifications (Sambrook et al., 1989).

Quantitative DNA Analysis:
DNA was extracted by using the gSYNC TM kit.DNA concentrations were measured using spectrophotometer (UV absorbance at 260 nm).

Fraction DNA Pattern:
For molecular analysis of DNA, Polymerase Chain Reaction (PCR) amplification was carried out for extracted DNA of different phases.RAPD primer (OPA-7 5'-GAAACGGGTG -3') was used to determine the internal molecular variation between different DNA patterns.The reaction condition was performed according to Saiki et al. (1988), where the PCR mixture was in a total volume of 25µl contained 1µg of total DNA, 50 pmol of the primer, 0.2 mM deoxynucleoside triphosphates, 2.5 µl of the Taq polymerase enzyme, 2.5 µl of 10 X enzyme buffer and 2.5 µl MgCl 2 .
The amplification reaction was performed in the following settings: DNA denatured firstly at 94°C/5min then for 1min, after which for 50°C/2min (base annealing temperature of first three cycles).Finally, extension or synthesis of new strands was at 72°C/3min, and was continued until the base annealing temperature reached the final condition of 49 °C and the samples were stored at 4°C.Under the final conditions, the amplification was continued for 30 cycles.The amplification products were analyzed on 1% agarose gel electrophoresis was performed according to Sambrook et al. (1989).
Similarity index (SI) and genetic distance (Gd) values were used to compare patterns within as well as between generations.SI reflects the extent of band sharing and calculated as: SI = 2 N ab / (N a + N b ) and Gd = 1 -SI.Where N ab is the number of bands common to individuals a and b.N a and N b are the total numbers of bands in the individual a and b, respectively.The value produced by this index ranges from zero, respecting no bands sharing, to one, respecting complete identity (Nei and Li, 1979).

Statistical Analysis:
The data were subjected to statistical analysis using a software SPSS (2005) test program.The significance of the main effects was determined by ONE-way analysis of variance (ANOVA) and followed by post-hoc analysis using LSD-test.The significance of various generations was evaluated at P < 0.05.RESULTS

Quantitative Analysis of Protein 1.Protein Levels:
Protein levels were illustrated in table (1).Data showed a significant reduction in protein levels between each phase and their transient generations (P<0.05).In long-term gregarious S. gregaria, protein content was about 16.33±0.14mg protein/10ml.However, protein content was about 7.95±0.21mg protein/10ml in long-term isolated individuals (the 5 th isolated generation).This content decreased gradually and became the most minimum level at the 3 rd isolated generation.

Qualitative Analysis of Protein:
SDS patterns banding of soluble tissue protein of long-term gregarious, solitarious, and transient isolated generations were illustrated in figure (1) and table  (2).According to relative frequency (Rf), a maximum detected protein bands were 52 bands.These bands detected at molecular weight ranged from 25 to 225.4 kDa and Rf (0.744-0.035).The total protein bands were divided into 8, 11, 8, 9, 8 and 12, respectively, with long-term gregarious, 1 st , 2 nd , 3 rd , 4 th isolated generations and longterm isolated S. gregaria, 5 th isolated generation.The maximum number of protein bands, detected in lane 1 (1 st isolated generation) and lane 5 (long-term isolated generation), was 11 and 12, respectively.
However, the minimum number of protein bands was detected in lane 0 (longterm gregarious S. gregaria), lane 2 (2 nd isolated generation) and lane 4 (4 th isolated generation) and was 8 bands for each previous generations.Furthermore, the SDS protein pattern revealed each one of the two extreme phases and their transient generations with some unique bands.The extracted protein of long-term gregarious individuals resolved into 8 bands.These bands observed in no.6,13,20,28,33,41,46 and 50 and their Rf values were ranging from 0.08-0.748.Also, their Mw was 165.72-30.292kDa.In between these previous bands, there are six unique bands, bands no.6, 20, 33, 41, 46 and 50.In contrast, bands no. 13 and 28 detected in other isolated-generations.A band no. 13 detected also in both of 1 st and 3 rd isolatedgenerations as well as band 28 detected in both of 2 nd and 3 rd isolated-generations.
Densitometry scanning of SDS revealed that the highest concentration of protein, in long-term gregarious S. gregaria, was detected in a band no. 13 (Mw 98.497 kDa), where the protein concentration of this band was 26.93 %.However, band no.27, with Mw 60.539 kDa, was the highest concentration in 1 st isolated generation and amounted 19.24 %.On the other hand, in the 2 nd isolated generation, the highest protein concentration was detected in the band no.28 and amounted 26.10 % with Mw 59.894 kDa.In contrast, in the 3 rd isolated generation, the highest concentration of protein, which was amounted 20.36%, was detected in the band no. 1 (Mw 225.4 kDa).In case of 4 th isolated generation, the highest concentration of protein was detected in the band no.19 amounted 24.82 % with Mw of 80.898 kDa.Furthermore, band no.35, in the long-term solitarious generation, had the highest concentration (17.51 %) with Mw 48.503 kDa.
Generally, data in the table (2) concluded that the isolation condition of S. gregaria induced appearance and disappearance or sometimes creation new protein bands of the different isolated generations.

Quantitative Analysis DNA:
In long-term gregarious S. gregaria, the DNA content had the highest concentration, which was about 7.75±0.30µg/µL.In contrast, in long-term solitarious S. gregaria DNA content was about 4.4±0.28µg/µL(table 3).Furthermore, there was a successive significant reduction (P<0.05) between different generations.In transient isolated generations, DNA contents were 2.65±0.19,1.63± 0.11, 1.25± 0.13, and 2.25±0.14µg/µlrespectively, with 1 st , 2 nd , 3 rd , and 4 th isolated generation.Similarity index (SI) and genetic distance (Gd) values among long-term gregarious, 1 st isolated generation, 2 nd isolated generation, 3 rd isolated generation, 4 th isolated generation, and long-term solitarious samples using primer OPA-7 were recorded in the table (5).The similarity index values showed the major drop from 0.30, 0.30, 0.30, 0.29, to 0.13 between long-term gregarious sample and samples 1 st isolated generation, 2 nd isolated generation, 3 rd isolated generation, 4 th isolated generation, and long-term solitarious, respectively.This finding suggests major changes in DNA structure and sequence with the change from gregarious to solitarious phase.Carlisle et al. (1987) assessed that protein synthesis is necessary for the maintenance of body growth and reproduction.Also, he illustrated many factors, which have been implicated in the control of protein synthesis, enhancing the variety of biological aspects of insects.Baker et al. (2010) reported that each type of protein has a specific biological role; consequently, this role enhanced DNA to secrete enzymes, which act as catalysts to the produced specific type of protein, whereas this produced protein was responsible for a specific biological process.

DISCUSSION
Quantitative analysis of protein concluded that the isolation condition of S. gregaria induced the depression of protein content of these individuals.Protein content was higher in the gregarious S. gregaria, compared with the long-term solitarious generation.This result was in agreement with some authors as Ott et al. (2012).They reported the reduction of the protein level of solitary individuals, compared with gregarious S. gregaria.
Also, in the present investigation, polyacrylamide gel electrophoresis of SDS was used to separate different extracted proteins.That helped in understanding the biological process and factors that happened inside the cells of living organisms, which are related to the phase polymorphism of S. gregaria.The obtained results showed that each generation has its own characteristic protein pattern.Distinct differences are observed when all generations are compared to each other and with long-term gregarious S. gregaria.These protein patterns recorded diversity between different generations, especially in their molecular weight and number of bands.A maximum increase of the soluble protein bands is observed in both 1 st isolated generation and the long-term solitarious generation, as a result of an appearance of new bands.There is an appearance of new bands, disappearance of some bands and reappearance of bands during the isolation condition.This may be due to appearing of new peptide in extracted proteins with the disappearance of others.
Indeed, these new peptides may change the chemical and physical structure of extracted proteins and undoubtedly these peptides can also change the solubility of these proteins; therefore, there were variations in the amounts of protein extractable from the long-term gregarious S. gregaria and its isolated generations although the same amount of protein has been applied to the gel.These findings were in agreement with some authors as Ayali et al. (1996aAyali et al. ( , 1996b)), Rahman et al. (2002) and Clynen et al. (2002).They reported that some pronounced differences could be detected in peptide profiles of the brain, corpora cardiac and the hemolymph of both isolated and crowd-reared locusts (S. gregaria and L. migratoria).
Furthermore, the obtained results in this investigation declared that the extracted protein bands that were detected, may be related to phase polymorphism.This was concluded as these bands disappeared from other transient phases and the long-term isolated generation.These findings agree with Hirschberger et al. (1999) who reported that some proteins are expressed in respect to the phase state when the hemolymph protein pattern of S. gregaria was analyzed using 2D-gel electrophoresis.Rahman et al. (2002) showed some differences in the peptide pattern of the hemolymph of solitary and gregarious animals of S. gregaria by using an HPLC analysis of hemolymph extracts.They reported that some peptides and proteins associated with phase polymorphism are designated as 'phase-related peptide' (PRP).Moreover, Rahman et al. (2003a) discovered a peptide with a potential molecular marker of phase transition as it is present in higher concentrations in the gregarious phase than in the solitarious one.The peptide level decreased with the successive generations of solitary reared animals, detected by comparing the peptide of the hemolymph of successive isolated generations.Amel et al. (2011) reported that peptide concentration is higher in the gregarious female's accessory glands than in the solitarious, and these results suggest a role in phase polyphenism.
Indeed, Knowledge of the expression profile of the internal genetic, related to phase change, is increasingly important in understanding biological processes.Therefore, this investigation illustrated the quantitative and qualitative analysis of DNA content, extracted from both of the two extreme phases of S. gregaria and isolated generations.Quantitative analysis of DNA level declared that the isolation condition of S. gregaria induced a significant reduction of DNA level.This level was at the maximum in the long-term gregarious S. gregaria, compared with the longterm 5 th isolated generation.This finding was in agreement with some authors as Kang et al. (2004) who illustrated EST (expressed sequence tag) datasets and their distributions among cDNA libraries.They reported a higher level in EST datasets in gregarious than solitarious in some parts of the body such as head, midgut, and hind-leg.
Furthermore, the analysis of DNA by agarose gel PCR amplification technique was used to separate different extracted DNA.This helped in understanding the biological process and factors that happened inside the cells of living organisms, which are related to the phase polymorphism of S. gregaria.The obtained results showed that each generation has its own characteristic DNA pattern.Distinct differences are observed when all generations are compared to each other, and with long-term gregarious S. gregaria.It has been noticed that there are 6 bands detected in long-term gregarious S. gregaria.
Two bands out of the 6 long-term gregarious S. gregaria bands were specifically from the long-term gregarious S. gregaria generation.In contrast, the long-term solitarious S. gregaria generation had 9 bands.Five bands out of these 9 detected bands were specific bands to the long-term solitarious S. gregaria generation.However, other bands, detected in the transient stages (1 st , 2 nd , 3 rd , isolated generation), disappeared from long-term gregarious and solitarious generations.Furthermore, all DNA profiles were different, especially in mobility and number of bands.The maximum increase of DNA bands is observed in the last longterm isolated generations, the 4 th and 5 th generation, as a result of the appearance of new bands.
Generally, the appearance of new bands, disappearance and reappearance of bands during the isolation condition may be due to appear of new peptides in the extracted DNA.These new peptides may be related to phase change or enhancement of phase polymorphism.The highest genetic distance 0.87, which reflects the highest degree of change in DNA structure and sequence, was recorded between the genomes of the long-term gregarious sample and long-term solitarious sample using OPA-7.These findings were in agreement with some authors as Claeys et al. (2003), who detected some neuroparsin precursor in the different amount in gregarious and solitarious S. gregaria, may be related to phase polymorphism.These neuroparsin precursors are Scg-NPP1 and Scg-NPP2, which transcripts in several parts of the body such as fat body, gut, accessory glands, gonads and brain of female and male.These transcripts are generally more abundant in solitarious than in gregarious individuals.This finding constitutes the first indication of phase-dependent transcriptional regulation of Scg-NPP1and Scg-NPP2 gene expression.Also, Kang et al. (2004) generated 76,012 ESTs from the whole body and dissected organs in the two phases of L. migratoria to establish the molecular mechanisms of the phase change.They identified 532 genes as phase-related by comparing 12,161 unigene clusters.Furthermore, PCR analysis in this investigation declared that each stage (long-term gregarious, transient and long-term solitarious) had a specific genome.Franz et al. (1998) studied brain area-specific gene expression of gregarious S. gregaria, and used differential display PCR without making a comparison with solitary animals.They illustrated 7 specific different areas: one from the midbrain, 3 from the thoracic and 3 from optic lobes ganglia.Also, Rahman et al. (2003b) reported that there are specific solitarious genes (SSG) and gregarious specific genes (GSG).They concluded that the two identified genes can now be used as novel molecular markers for a more specific characterization of the gene regulation events involved in locust phase transformation.Ma et al. (2011) illustrated some critical target genes related to behavioral phase changes in L. migratoria.They reported a relationship between genes and behavior in phase transition.Wang and Kang (2013) reported that the phase change in locusts is a continuous, accumulative, and easily reversible process and is involved in behavioral and physiological traits in response to changes in population density.Furthermore, genes and metabolites have a critical role in phase transition in desert locust.Also, Ernst et al. (2015) showed the important role of DNA, which is involved in phase polymorphism of L. migratoria and S. gregaria.
In conclusion, quantitative and qualitative analysis of protein and DNA revealed that the variation of population density induced internal changes.This was responsible for a specific biological process and external changes.Therefore, phase polymorphism evolved from internal changes followed by external changes, but not vice versa.Consequently, more detailed studies of these internal changes may be considered a valuable tool for the control of these internal factors.Therefore, a new strategy to control S. gregaria and a new environment-friendly method can be reached.
generations with visible changes in the number and size of amplified DNA fragments.The amplification pattern of RAPD-PCR of the genomic DNA revealed 35 different fragments.The fragments ranged from 366 bp (band No 15) to 3484 bp (band No 1).Bands 4 and 6 were common in all samples with Rf values 0.58 and 0.64, respectively.The Long-term gregarious sample was identified by the presence of 2 unique bands (366 and 860 bp).The sample of 4 th isolated generation was recognized by one unique band with molecular weight 828 bp.On the other hand, five unique bands were distinguished sample of long-term solitarious with molecular weight548, 797, 1263, 2328, and 3484 bp.

Table 1 :
Protein levels of the two extreme phases of S. gregaria and its transient isolated generations.
*Means bearing different letters within the column are significantly different (P<0.05)ANOVA, LSD test

Table 3 :
DNA Changes in DNA profile in two extreme phases and their transient generations were shown in the table (4) and figure (2).The generated RAPD profile revealed differences among long-term gregarious, solitarious, and transient isolated levels of long-term gregarious, solitarious S. gregaria, and transient isolated generations.No. of generation Conc. of DNA (µg/µl) 2.1.Qualitative analysis of DNA: