The increase in absorbance at 490 nm was measured using a microplate reader (Thermo Labsystems)

The increase in absorbance at 490 nm was measured using a microplate reader (Thermo Labsystems). either normal or bacteria-challenged by an and mixture. A total of 54,821,138 clean reads (4.93 Gb) were yielded by Illumina sequencing, which were assembled into 89,842 unigenes. Of the 89,842 unigenes, based on a similarity search with known genes in other insects, 24 putative genes related to the proPO system were identified. Eight of the identified genes encoded for peptidoglycan recognition receptors, two encoded for prophenoloxidases, three encoded for prophenoloxidase-activating enzymes, and 11 encoded for Rabbit Polyclonal to SERPINB12 serine proteinase inhibitors. The expression levels of these identified genes were investigated by qRT-PCR assay, which were consistent with expected activation process of the proPO system, and their activation functions were confirmed by the measurement of phenoloxidase activity in bacteria-infected larvae after proPO antibody blockage, suggesting these candidate genes might have potentially different roles in the activation of proPO system. Collectively, this study has provided the comprehensive transcriptomic data of an insect and some fundamental basis toward achieving understanding of the activation mechanisms and immune functions of the proPO system in is a worldwide insect Etodolac (AY-24236) vector that can transport numerous pathogenic organisms, including parasites, viruses, bacteria, and even antibiotic-resistant bacteria (Tan 1997; Sasaki 2000; Davari 2012; Schuster 2013; Wei 2013). These pathogens can cause more than 100 serious diseases in human and animals, such as salmonellosis, typhoid fever, cholera, infantile diarrhea, and amoebic dysentery (Scott 2014). In addition to the public health threat, the housefly can suppress milk and egg production in livestock and poultry farming, as well as reduce food conversion. Confusingly, this species can resist infections and maintain its growing prosperity even living in an environment full of pathogens. However, until recently, little has been known about the molecular mechanism of housefly immune response to these pathogens (Li 2013; Liu 2012; Scott 2014; Tang 2014). Insects rely on their innate immune system as a defense against pathogens because they lack an acquired immune system (Kingsolver and Hardy 2012). The prophenoloxidase-activating system (proPO system) can produce melanin within a few minutes after pathogen invasion and participate in host innate immune responses, including killing, eliminating, or inhibiting invading pathogens (Rao 2010; Qian 2013). The proPO system has been extensively investigated in various insect species, such as (An 2013), (An 2011a), (Tindwa 2013), (An 2011b; Wang 2011, 2014), and (Chen 2014), and Etodolac (AY-24236) the activation cascade has also been preliminarily summarized as follows (Cerenius and S?derh?ll 2004; Cerenius 2008, 2010). Generally, invaders are recognized by pattern recognition proteins of the host, such as peptidoglycan recognition proteins (PGRPs) or -1,3-glucan recognition proteins, and then a cascade reaction of serine proteases is initiated in which many serine proteases are involved, including prophenoloxidase (proPO), prophenoloxidase-activating enzymes (PAPs), serine protease inhibitors (Serpins), and serine protease homologs (SPHs). Once activated, proPO is released into the plasma and converted into phenoloxidase (PO) via restrictive proteolysis. PO is the last and most important Etodolac (AY-24236) component of the proPO system, which oxidizes phenol into benzoquinone that is then polymerized into insoluble melanin by nonenzymatic reactions. The melanin is deposited at the injury site or on the invading pathogens to induce the blackening and healing of wound (Tindwa 2013). Studies on the contribution of melanization to the survival of dipterans obtained variable results. Two reports on (Leclerc 2006) and (Schnitger 2007) revealed that the proPO system exhibits no bactericidal activity, but recent work has demonstrated that requires proPO activation to survive microbial infections (Binggeli 2014). Therefore, it is crucial Etodolac (AY-24236) to identify the genes related to the proPO system in other dipterans, such as for elucidating the activation mechanisms and immune functions of this system. Although there were a few reports about PO (Sun 2008), PO inhibitors (Tsukamoto 1992), and proPO sequence (“type”:”entrez-protein”,”attrs”:”text”:”AAR84669″,”term_id”:”40362984″,”term_text”:”AAR84669″AAR84669) in the past, current knowledge of this proPO system is limited compared with antimicrobial peptides and pattern recognition proteins in (Wang 2006; Fu 2009; Ai 2013; Sun 2014). Etodolac (AY-24236) In addition, no report is available on the PGRPs, PAPs, and Serpins of larvae and identified a few cDNA segments of PAP and proPO (Li 2010). However, the SSH library yielded limited genomic resources (Qiu 2012); therefore, the main components and the activation mechanisms of the proPO system in remain unclear. For the past few years, the high-throughput technology RNA-Seq has been used to produce millions of short cDNA reads and.