MicroRNAs (miRNA) participate in regulating diverse biological pathways by 
translational repression in animals. They have attracted increasing attention 
recently. However, little work has been done on the miRNA genes in 
agriculturally important pests. Because the transcripts of most miRNA genes are 
the products of type-II RNA polymerase, pri-miRNA has a poly(A) tail and appears 
in expressed sequence tags (EST). We developed a computational pipeline to 
identify miRNA genes from insect ESTs. First, 980,697 ESTs from 63 insects were 
collected and used to search the nr database. The ESTs which did not share 
significant similarities with any known protein-coding genes were treated as 
non-coding ESTs. Next, known mature miRNAs were used to align with non-coding 
ESTs. The ESTs which contain the sequence of mature miRNA were treated as 
candidate ESTs. Finally, putative precursors were extracted flanking the mature 
miRNA region in candidate ESTs and evaluated by the Triplet-SVM algorithm. As a 
result, 86 miRNAs from 30 insect species were found based on a strict criterion 
while 330 miRNAs from 51 species were found based on a loose criterion. 
Evolution analysis indicated that mir-467, mir-297 and mir-466 were the highest 
conserved miRNA families in insects. To confirm the reliability of putative 
insect miRNAs, the expression profile of nine predicted miRNAs in Locusta 
migratoria was investigated. Eight miRNAs were successfully detected by RT-PCR. 
Most miRNAs were expressed ubiquitously at all examined tissues and 
developmental stages whereas Lmi-mir-509 was specifically expressed in the 
thorax of the 2nd, 4th and 5th instars and adult locust. In all, our work 
reported an efficient computational strategy for predicting miRNA genes from 
insect ESTs and presented tens of miRNAs in diverse insect species which are 
expected to participate in many important physiological processes.