Saima Kausar

Serpin-14 negatively regulates prophenoloxidase activation and expression of antimicrobial peptides in Chinese oak silkworm Antheraea pernyi

Abstract Genes encoding proteins of serpins superfamily are widely distributed in invertebrates. In insects, serpins play important roles in regulating immune responses and other physiological processes. Here, we report the cloning and characterization of cDNA of Apserpin‐14 from Chinese oak silkworm (Antheraea pernyi). The Apserpin‐14 gene contains 1206 bp open reading frame, encoding a predicted 401 amino acid residue protein. We expressed the recombinant Apserpin‐14 protein in Escherichia coli and then purified protein was used to prepare rabbit anti‐Apserpin‐14 polyclonal antibodies. Quantitative real‐time PCR analysis revealed that mRNA level of Apserpin‐14 was highest in the fat body, whereas, among developmental stages the 5th instar and pupal stage showed greatest expression. Furthermore, Escherichia coli, Beauveria bassiana, Micrococcus luteus and nuclear polyhedrosis virus challenge enhanced Apserpin‐14 transcript in both the fat body and hemocyte. Recombinant Apserpin‐14 added to hemolymph inhibited spontaneous melanization and suppressed prophenoloxidase activation stimulated by M. luteus, but did not affect phenoloxidase (PO) activity. Injection of recombinant Apserpin‐14 protein into A. pernyi larvae significantly reduced the transcript levels of antimicrobial peptides in the fat body, while its depletion by double stranded RNA enhanced their expression. We concluded that Apserpin‐14 likely involved in regulation of proPO activation and production of antimicrobial peptides, implying its important role in the innate immune system of A. pernyi. HighlightsFull‐length cDNA of Serine Protease Inhibitor14 was identified in Antheraea pernyi.Apserpin‐14 expressed ubiquitously in all tested tissues and developmental stages.Apserpinn‐14 mRNA in fat body and hemocyte up regulate after microbial challenge.Apserpin‐14 regulates prophenol oxidase activation.Apserpin‐14 was found to regulate antimicrobial peptides.

Mitochondrial genome of Diaphania indica (saunders) (Lepidoptera: Pyraloidea) and implications for its phylogeny

The cucumber moth, Diaphania indica (Saunders) (Lepipdoptera: Pyralidae) is an economically important insect pest of cucurbits and is widely distributed in tropical and sub-tropical regions. Herein, we obtained entire mitochondrial genome (mitogenome) sequence of D. indica using polymerase chain reaction amplification and sequencing method. The complete mitogenome of D. indica is 15,367bp long, including 13 protein coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and an A+T rich element. Both the AT skew and GC skew are slightly negative in the newly sequenced mitogenome. All of the 12 PCGs initiate with canonical start codon (ATN), except for coxI that initiate with CGA. The A+T rich element of D. indica is 467bp long and contains many features common to Pyraloidea insects. In addition, we reconstructed phylogenetic relationships among the eight superfamiles of lepidopterans by employing Neighbor joining (NJ) and Maximum likelihood (ML) methods, and both yielded identical topologies. The phylogeny results supported the monophyly of lepidopteran superfamilies. Furthermore, phylogenetic tree showed that D. indica belongs to the Pyraloidea superfamily.

Comparative mitochondrial genome analysis of Daphnis nerii and other lepidopteran insects reveals conserved mitochondrial genome organization and phylogenetic relationships

In the present study, the complete sequence of the mitochondrial genome (mitogenome) of Daphnis nerii (Lepidoptera: Sphingidae) is described. The mitogenome (15,247 bp) of D.nerii encodes13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and an adenine (A) + thymine (T)-rich region. Its gene complement and order is similar to that of other sequenced lepidopterans. The 12 PCGs initiated by ATN codons except for cytochrome c oxidase subunit 1 (cox1) gene that is seemingly initiated by the CGA codon as documented in other insect mitogenomes. Four of the 13 PCGs have the incomplete termination codon T, while the remainder terminated with the canonical stop codon. This mitogenome has six major intergenic spacers, with the exception of A+T-rich region, spanning at least 10 bp. The A+T-rich region is 351 bp long, and contains some conserved regions, including ‘ATAGA’ motif followed by a 17 bp poly-T stretch, a microsatellite-like element (AT)9 and also a poly-A element. Phylogenetic analyses based on 13 PCGs using maximum likelihood (ML) and Bayesian inference (BI) revealed that D. nerii resides in the Sphingidae family.

Role of Antheraea pernyi serpin 12 in prophenoloxidase activation and immune responses

Serine protease inhibitors play a key role in the immune system of invertebrates by controlling proteolytic cascades. Besides its importance, the knowledge on immune functions of serpins in most of insects is fragmentary. In the present study, we identified serpin-12 from Antheraea pernyi encoding a predicted 402 amino acid residue protein (Apserpin-12). We expressed the recombinant protein in Escherichia coli and the purified protein was used for the synthesis of rabbit anti-Apserpin-12 polyclonal antibodies and functional studies. Quantitative real-time ploymerase chain reaction (qRT-PCR) analysis revealed that the knock-down of Apserpin-12 enhanced the prophenoloxidase (PPO) cascade stimulated by Micrococcus luteus in hemolymph, whereas addition of recombinant Apserpin-12 protein along with same elicitor led to down-regulate PPO activation. Following different microbial challenge (E. coli, Beauveria bassiana, M. Luteus, and nuclear polyhedrosis virus), the expression of Apserpin-12 mRNA was induced significantly. Furthermore, the Apserpin-12 double-stranded RNA administration elicited the expression of antimicrobial peptides, while the treatment with recombinant protein suppressed their expression. Tissue profile of Apserpin-12 indicated that it is expressed in all examined tissues, that is, hemolymph, malpighian tubules, midgut, silk gland, integument, and fat body with variation in their transcript levels. We concluded that Apserpin-12 may regulate PPO activation and inhibit the production of antimicrobial peptides in A. pernyi, suggesting important role in its immune system.

Identification of a small pacifastin protease inhibitor from Nasonia vitripennis venom that inhibits humoral immunity of host (Musca domestica)

ABSTRACT Nasonia vitripennis is an important natural enemy of many flies. Pacifastin protease inhibitors (PPIs) play an important role in development and innate immunity of insects. In this study, cDNA sequences of a small pacifastin protease inhibitor in N. vitripennis (NvSPPI) was characterized and its open reading frame (ORF) contains 243bp. Real‐time quantitative PCR (RT‐qPCR) results revealed that NvSPPI mRNA were detected specifically in the venom apparatus, while they were expressed at low levels in other tissues tested. In the venom apparatus, NvSPPI transcript was highly expressed on the fourth day post eclosion and then declined gradually. The NvSPPI gene was recombinantly expressed utilizing a pGEX‐4T‐2 vector, and the recombinant products fused with glutathione S‐transferase were purified. Inhibition of recombinant GST‐NvSPPI to three serine protease inhibitors (trypsin, chymotrypsin, and protease K) were tested and results showed that recombinant NvSPPI could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST‐NvSPPI on the phenoloxidase (PO) activity and prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by recombinant NvSPPI; however, there was no significant inhibition on the PO activity. Our results suggested that NvSPPI could inhibit PPO activation in host hemolymph and trypsin activity in vitro. HIGHLIGHTSNvSPPI is detected specifically in the venom apparatus of N. vitripennis.NvSPPI can inhibit trypsin activity in vitro.NvSPPI can inhibit PPO activation in host (Musca domestica) hemolymph.

Identification and function of cAMP response element binding protein in Oak silkworm Antheraea pernyi

Cyclic AMP response element binding (CREB) proteins participate in the regulation of many biological processes. However, little is known about their role in immune regulation in the Oak silkworm (Antheraea pernyi). In this study, a CREB gene was identified in A. pernyi and its role in immune regulation was investigated. ApCREB shares conserved signature motifs with other CREB proteins, and includes a typical leucine zipper domain, specific DNA-binding site, nuclear localisation signal (NLS) and cAMP-dependent protein kinase phosphorylation site. Recombinant ApCREB was expressed in Escherichia coli and used to raise rabbit anti-ApCREB polyclonal antibodies. ApCREB mRNA was detected in all examined tissues, with maximum expression in the midgut and integument. Following exposure to four pathogenic microorganisms (Beauveria bassiana, Escherichia coli, Micrococcus luteus, and Antheraea pernyi nuclear polyhedrosis virus), expression of ApCREB was up-regulated by B. bassiana, E. coli and ApNPV, down-regulated by M. luteus. RNA interference of ApCREB affected mRNA expression levels of antimicrobial peptide genes attacin-1, cecropin B, defensin-1, gloverin, and lebocin-2. These findings demonstrate that ApCREB is a CREB homologue that may be involved in innate immunity in A. pernyi.

Suppressor of cytokine signaling 2-12 regulates antimicrobial peptides and ecdysteroid signaling pathways in Bombyx mori (Dazao)

Suppressors of cytokine signaling (SOCS) are a potent negative regulator of diverse cytokine-related responses to maintain various physiological processes in animals. Here, we obtained the SOCS2-12 gene sequence of Bombyx mori (Dazao) (BmSOCS2-12) from the National Center for Biotechnology Information (NCBI) to study its expression profile in different tissues, as well as in the immune tissues following larval exposure to pathogens. Further, we investigated the role of BmSOCS2-12 in producing antimicrobial peptides (AMPs) and as a regulator of ecdysteroid signaling transduction. The quantitative real-time PCR analysis revealed unequal transcript levels of BmSOCS2-12 in the different tissues, however the genes expression was highest in those of fat body and hemocyte. The challenge with pathogens significantly upregulated the transcript level of BmSOCS2-12 in both fat body and hemocyte when compared with the control. By contrast, recombinant BmSOCS2-12 protein injections strongly suppressed the expression of AMPs, while the knockdown of BmSOCS2-12 by double-stranded RNA enhanced their production. Administration of 20-hydroxyecdysone significantly downregulated the BmSOCS2-12 expression in fat body, and the depletion of BmSOCS2-12 enhanced the transcript levels of 20-hydroxyecdysone-responsive genes at 48 h. Altogether, BmSOCS2-12 may have multiple functional roles in the physiology of B. mori (Dazao), since it negatively regulates the expression of AMPs and ecdysteroid signaling transduction.

Suppressor of cytokine signaling 6 can enhance epidermal growth factor receptor signaling pathway in Bombyx mori (Dazao)

&NA; The SOCS (Suppressor of cytokine signaling) family members are a potential negative regulator of cytokine signaling pathway and play a key role to maintain immunological functions in animals. SOCS‐6 is an important member of the SOCS family, however the functions of this gene have rarely been explored among eukaryotes. Herein, we cloned and expressed SOCS‐6 gene from Bombyx mori (Dazao) (BmSOCS‐6), and anti‐rabbit antibodies were prepared using purified recombinant BmSOCS‐6 protein. Under normal physiological conditions, the BmSOCS‐6 expression was observed at varied levels in six tissues, with most greatly expressed in fat body and hemocytes. After immune challenge with viral, fungal and bacterial pathogens, the BmSOCS‐6 showed distinctly varied expression patterns in tissue, time and microbe dependent manner. By contrast, recombinant BmSOCS‐6 protein strongly enhanced the expression of epidermal growth factor receptor (EGFR) pathway related genes, while the depletion of BmSOCS‐6 by double stranded RNA suppressed their production. Altogether we concluded that BmSOCS‐6 may improve the efficiency of EGFR signaling pathway in B. mori (Dazao). HighlightsA BmSOCS‐6 gene was firstly identified from B. mori (Dazao).BmSOCS‐6 ubiquitously expressed in different tissue as well as in developmental stages.BmSOCS‐6 transcript in fat body and hemocytes was up‐regulated following pathogen challenge.BmSOCS‐6 was involved in the regulation of epidermal growth factor receptor pathway.

PREDATOR-PREY RATIOS OF MACROINVERTEBRATES IN WHEAT AND SUGARCANE AGROECOSYSTEMS

*The present study on selected species of coleopteran, hymenopterans, arachnids and hemipterans was conducted to evaluate prey-predator ratios (p/p) based on the logistic abundance of species on wheat-weeds and sugarcane-weeds agroecosystems in Faisalabad District. Theoretically, these interactions have significant impact on the structure and dynamics of an agro-ecosystem. Polynomial regression was applied on the relative abundance of selected predators with each of the prey species. In wheat-weeds agro-ecosystem coleopterans and hymenopterans predators showed more R 2 = 0.70 with aphids and P. perpusilla. Whereas in sugarcane-weeds agro-ecosystem coleopterans, arachnids and only single species of hymenopterans showed more R 2 = 0.70 with aphids and P. perpusilla. By using such inferences species specific biological