Molecular Cloning, Sequence Analysis and Developmental Stage Expression of a Putative Septin Gene Fragment from Aedes Albopictus (diptera: Culicidae)

Septins belong to GTPases that are involved in vital cellular activities, including cytokinesis. Although present in many organisms, they are yet to be isolated from Ae-des albopictus. This study reports for the first time on a serendipitous isolation of a partial septin sequence from Ae. albopictus and its developmental expression profile. The Ae. albopictus partial septin sequence contains 591 nucleotides encoding 197 amino acids. It shares homol-ogy with several insect septin genes and has a close phylogenetic relationship with Aedes aegypti and Culex quinquefasciatus septins. The Ae. albopictus septin fragment was differentially expressed in the mosquito's developmental stages, with an increased expression in the adults.


INTRODUCTION
Septins are a group of P-loop guanosine triphosphate (GTP) binding proteins that were first discovered in the budding yeast Saccharomyces cerevisiae (Hartwell, 1971), with several homologues now identified in many animals and yeasts, but currently absent in higher plants (Pan et al., 2007;Nishihama et al., 2011).They form oligomeric filaments which polymerize into large paired septin filaments to further form higher order structures (Sirajuddin et al., 2007;DeMay et al., 2011).Septins are involved in cytokinesis (Hartwell, 1971), cytoskeletal regulation and membrane remodeling (Kinoshita, 2006;Berepiki & Read, 2013).Aberrations in septin genes have been implicated in several diseases such as cancer, male infertility and Parkinson disease (Peterson & Petty, 2010;Saarikangas & Barral, 2011).
Most studies on insect septins have focused on the fruit fly, Drosophila melanogaster, as a model organism, to understand the function of these genes.Although septins have been identified in some insects, at the time of this study none had been identified in the Asian tiger mosquito, Aedes albopictus.The identification of septins in this mosquito is essential due to the important roles they play; such discovery could provide useful information on the biology of this mosquito.Aedes albopictus is a vector of such diseases as dengue and chikungunya, and thus is a focus of chemical control.One of the ways in which it is able to survive insecticide applications is to metabolize the chemical compounds.Here, whilst trying to amplify a major insecticides metabolic enzyme, cytochrome P450 from Ae. albopictus, a septin gene fragment was serendipitously amplified.This gene was cloned and its phylogenetic relationship was inferred.The expression of this gene in the life stages of the mosquito was also studied.This is the first report on the cloning and expression of a septin gene fragment from Ae. albopictus.

MATERIALS AND METHODS
Extraction of total RNA and cDNA synthesis.Total RNA was extracted from the 4 th instar larvae (30 mg) of Ae. albopictus obtained from the Vector Control Research Unit, Universiti Sains Malaysia using the RNeasy ® Mini Kit (Qiagen ® ).Reverse transcription of 2 µg of total RNA using the M-MuLV Reverse Transcriptase and Oligo (dT) 18 from First strand cDNA synthesis kit (Fermentas ® ) was done at 37°C/1 h, followed by incubation at 70°C/5 min.The manufacturers' instructions of the products used in this study were followed unless stated otherwise.
Sequence and phylogenetic analysis.The nucleotide sequences were translated using the ExPASy translate tool (http://web.expasy.org/translate/)and used for Basic Local Alignment Search Tool (BLAST ® ) searches in the National Center for Biotechnology Information (NCBI) database.Phylogenetic analysis was done with comparison to other insect septins in Molecular Evolutionary Genetics Analysis version 5 (MEGA5) (Tamura et al., 2011).
Serial dilution of cDNA was used to construct standard curves for assessing the reaction efficiency for each of the gene specific primer pairs.The relative transcription ratio was compared to the control calibrator (4 th instar larvae) and computed (Pfaffl, 2001) after normalization with the reference genes.Fourth instar larvae were used as the control because the gene was cloned from this stage of the mosquito's development.At least three independent biological and technical replicates were done for each sample.The data was analyzed with a one-way analysis of variance using Genstat ® Release 9.2 (Payne et al., 2006) and means were separated using the least significant difference.

Phylogenetic tree
The phylogenetic relationship of the Ae.albopictus partial septin sequence was compared with other insect septins which were downloaded from the GenBank database and aligned by ClustalW (Gap opening penalty 10, Gap extension penalty 0.2, Delay divergent cutoff 30%), and the alignments were used to draw a neighbor-joining tree (Saitou & Nei, 1987) with 1 000 bootstraps (Felsenstein, 1985) (Fig. 2).The phylogenetic tree showed that the partial septin sequence of Ae. albopictus and septins of Ae. aegypti (partial gene sequence) and Cx.quinquefasciatus were closely related.It is inferred from the tree that the Ae.albopictus partial septin sequence is evolutionary closer to pnut septin gene than the other septin genes.The pnut gene was the first Drosophila septin gene to be discovered and is involved in cytokinesis (Neufeld & Rubin, 1994).Defects in this gene have lethal consequences (Neufeld & Rubin, 1994;Adam et al., 2000).In Drosophila, pnut null mutant larvae had a significantly reduced cell number, with multinucleated cells in such tissues like the imaginal discs and brain, and died after pupation (Neufeld & Rubin, 1994).Mutant embryos without maternal and zygotic pnut also developed abnormal actin cytoskeleton during the cellularization stage of embryogenesis and morphological defects during gastrulation (Adam et al., 2000).

Constitutive expression pattern
Septin expression in the life stages of the mosquito was significantly different, with an increased expres-sion in the adult stages when compared to the immature stages (p < 0.05, Fig. 3).The expression pattern among the immature stages was not significantly different.The highest expression of the septin gene fragment was detected in the 2-day old female, and this was not significantly different from the 2-day old male (p > 0.05, Fig. 3).Detection of the septin fragment in the developmental stages of Ae. albopictus is not surprising due to their well known cell division activities (Neufeld Rubin, 1994), which could be involved in transiting from one developmental stage to the other, particularly in the production of new cells associated with their metamorphic processes.The increased expression in the adult could be linked to adult-stage related functions of septin genes, such as oogenesis and spermatogenesis (Hime et al., 1996;O'Neill & Clark, 2013).Since septins play important developmental and structural roles in eukaryotic organisms, and are associated with physiological and morphological abnormalities when altered, the identification and elucidation of their roles in individual species could provide valuable biological information for the development of less fit individuals to control mosquito population.
In conclusion, a partial septin gene sequence was isolated from Ae. albopictus and its phylogeny with other insect septins was inferred.This gene was differentially expressed in the developmental stages of Ae. albopictus, with an elevated expression in the adult stage, which was not sex-specific.This is the first report on the isolation of a putative septin gene fragment from Ae. albopictus.It is a major step that could be used for further studies to elucidate the cellular and physiological roles of Ae. albopictus septins and their impact on mosquito biology and control.The phylogenetic tree was inferred using the neighbor-joining method.Aedes aegypti myosin was used as an outgroup.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1 000 replicates) is shown next to the branches.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

Figure 1 .
Figure 1.Nucleotide and deduced amino acid sequences of putative partial septin gene sequence from Aedes albopictus.The G1 motif is in bold and underlined.

Figure 2 .
Figure 2. Phylogenetic relationship of Aedes albopictus partial septin gene sequence with other insect septin genes (accession no. on the extreme right).The phylogenetic tree was inferred using the neighbor-joining method.Aedes aegypti myosin was used as an outgroup.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1 000 replicates) is shown next to the branches.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

Figure 3 .
Figure 3. Relative expression (± standard error) of septin fragment in Aedes albopictus at different life stages.The expression was normalized against β-actin and rpl8 and compared to the control (4th instar larvae).Asterisks represent a significant increase in the expression of the gene.