| The red imported fire ant,Solenopsis invicta Buren,is one of the most devastating invasive social insects in the world.Its rapid response to environmental changes has enabled itself to spread successfully around the globe.The fire ants have a highly social division of labor in two forms of workers of fire ants.Individuals within their colonies cooperate with each other and are responsible for various tasks.Studies showed that the workers perform diverse tasks in their life cycle,which are generally associated with morphology and age,but its regulatory mechanisms of division of labor are unclear.Foraging gene in insects,which encode cGMP-dependent protein kinase(PKG),regulates growth and development,division of labor and behaviors in social insects.The functions of for gene and PKG in the fire ants has remained unclear until now.In our study,gene sequence of the Solenopsis invicta foraging(Sifor)and it encoding amino acid structure were analyzed.we used the real-time quantitative PCR(RT-qPCR)and Insect Protein Kinase G(PKG)Total Activity Assay Kit to study relative Sifor expression and PKG activity among different developmental stages,or adult castes and different parts of bodies.We also analyzed the spatial-and temporal-distribution of Sifor gene expression and PKG activity in workers according to body size and age.Finally,RNA interference(RNAi)and additional chemicals were used to verify the regulatory role of Sifor gene and PKG in the behavioral swift of nurse and forager workers.In addition,the levels of Sifor gene expression in foragers following changes in temperature and food availability were analyzed using RT-qPCR.The main results are as follows:1.Characterization of Sifor gene and its encoding amino acid.There were three Sifor gene alternative spliceosome in the red imported fire ants,and the completeness of the open reading frame(ORF)and the corresponding amino acid sequence were analyzed.1579bp nucleotide sequences at the 3’end of the three spliceosomes of Sifor gene were 100%identical and the C-terminus of amino acid that encoded by three Sifor genes were 525aa 100%coincident.Howevere,the nucleotide sequence of three Sifor genes near the 5’end and its encoding amino acid sequence in N-terminal were highly different.The basic physical and chemical character and structure of the genes’amino acid sequences were predicted and the results showed that the structure had a high similarity to each other.Moreover,functional domains of the amino acid sequences were conserved.There was closest kinship in evolution by phylogenetic analysis between amino acid sequence of the SiFOR and FOR which was from the ant Atta cephalotes.The amino acid sequence which encoded by for genes and their homologs among insect species were highly conserved and all of them belong to the class of PKG I.2.Temporal and spatial Expression of Sifor gene and PKG activityThe temporal and spatial differentiation of Sifor gene expression and PKG activity in fire ants was investigated using RT-qPCR and PKG Total Activity Assay Kit.In workers and female alates of fire ants,the highest level of Sifor gene was detected in pupal stage,followed by adult,and then the lowest expression level in egg and larval stages.In the male alates,Sifor gene expression was mainly expression in the adult and pupal stages,significantly higher than in egg and larval stages.However,PKG activity in fire ant workers was highest in the pupal stage,in which its activity was lowest at the egg,larval and pupal stages.The enzymic activity in developmental period of female alates and male alates was similar to the tendency in workers.In addition,Sifor gene expression and PKG activity in adults of all castes were caste special with a descending order of workers>male alates≥ female alates.Moreover,the level of Sifor gene expression and PKG activity was identified higher in the heads than thorax and abdomen in the castes of workers,female alates and male alates.We systematically analyzed the temporal and spatial distribution of Sifor gene and PKG enzyme in all castes of fire ants suggesting the expression of Sifor gene mainly in the stages of pupae and adults,the action of PKG mainly in the stage of adults in all castes,especially in workers.3.Analysis of Sifor gene expression and PKG activity with workers’ polymorphic and ageWe compared the expression patterns of Sifor gene among major-,medium-,and minorcastes of workers.In foragers,Sifor gene expressed most highly in minor workers,in which it was 1.35 ± 0.07～1.86 ± 0.22 times than that in major workers.Sifor expression of medium workers was 1.00 ± 0.06～1.16 ± 0.03 times higher than that of major ones,and Sifor was lowest expressed in major foragers.However,Sifor gene expression was not significantly different among major-,medium-and minor-nursing workers.In addition,the expression level of Sifor gene in minor forms of foragers was 3 to 6 times significantly higher than those of the same forms of nurses.Similarly,the Sifor gene expression in medium-and majorforms of foragers were 2 to 4 times significantly higher than those of the same forms of nurses.PKG activity in the three forms of foragers was consistent with Sifor gene.Also,PKG activity was highest in the minor forms of foragers(343.39 ± 17.39～437.55 ± 22.12 U/g),followed by medium-foragers(257.75 ± 2.71～367.27 ± 14.41 U/g),then the lowest in major foragers(221.75 ± 5.98～287.04 ± 18.30 U/g).However,there was no significant difference in PKG activity among three groups of foragers,ranging from 178.60 ± 6.30 to 235.54 ± 11.52 U/g.PKG activity in the same forms of foragers were significantly higher than that of nurses.The expression of Sifor gene and PKG activity were investigated in the workers over time(1 to 40 day).The Sifor gene expression over aging in workers had a single-peak model,in which it appeared more highly at the time of 19～25 day.Sifor gene expression remained lowlier at 1～19 day and decreased slowly with aging and higher than that at early time.The temporal trends of PKG activity were similar to Sifor gene ones.These results indicated that Sifor gene expression and PKG activity in workers are correlated with body size and involved with division of labor of workers over age.4.Role of Sifor gene in behavioral shift between subcastes of nursing and foraging workersWe confirmed that fire ants exhibited different behaviors,Sifor gene expression and PKG activity between nursing and foraging workers.Foragers walked more quickly than nurses and meanwhile had no preference to both larval and adult cues,but nurses more likely preferred to care for larvae to adults.PKG activity in foragers was significantly higher than that in nurses.Meanwhile,the genotypic expression of Sifor gene was higher in the heads of foragers than that in the nurses.The fire ant workers were determined based on previous experiments.The interference system of dsSifor application was successfully established suggesting dsSifor concentration(0.5～1.5 μg/μL)and optimal interference time(48～72 h).At the same time the 8-Br-cGMP concentration(2～4 mM)and optimal period(72～96 h)were also determined for PKGenhancement test in fire ant workers.Subsequently,we study the role of Sifor gene and PKG in behavior fine-tuning between foragers and nurses using RNA interference and pharmacological methods we did previously.Foragers walked more slowly in the treatment than in control and more strongly preferred larval cue to adult cues once Sifor in the foraging form of workers was knock downed by dsRNA.Nurses walked significantly faster in the treatment than in the control and their preference for larval cues weakened 20%compared to control while the nursing form of workers were treated with the compound of 8-Br-cGMP.The results revealed that two castes of phenotypes,nursing and foraging,showed not only behavioral and physiological differentiation,but also genetic expression of Sifor gene.Furthermore,Sifor was proved to play an important role in division of labor of fire ant workers.5.Temperature-and food-dependent Sifor gene expression in foragers of the S.invictaThe behavioral plasticity and adaptability of insects to diverse habitats,is strongly associated with for genes in some insects.We used RT-qPCR to study levels of Sifor gene expression while foragers treated in the temperature change and food availability.Sifor expression levels were downregulated in the colonies at 18℃ and upregulated in the colonies at 35℃ while transferred to the colonies at the temperature of 26℃ as control.Moreover,Sifor expression levels decreased when food was deprived 30～102 h,but increased when food was restored in comparison with the control.The results showed that Sifor was linked,to a great extent,to temperature and,to some extent,with food availability,suggesting that Sifor may explain why fire ant foragers acclimatize to daily temperature fluctuations.Further research on for genes will generate more data for eliciting its role in ecological adaptation to more environmental change. |
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