Muhammad Nadeem Abbas

Characterization of the Complete Mitochondrial Genome of Leucoma salicis (Lepidoptera: Lymantriidae) and Comparison with Other Lepidopteran Insects

The complete mitochondrial genome (mitogenome) of Leucoma salicis (Lepidoptera: Lymantriidae) was sequenced and annotated. It is a circular molecule of 15,334 bp, containing the 37 genes usually present in insect mitogenomes. All protein-coding genes (PCGs) are initiated by ATN codons, other than cox1, which is initiated by CGA. Three of the 13 PCGs had an incomplete termination codon, T or TA, while the others terminated with TAA. The relative synonymous codon usage of the 13 protein-coding genes (PCGs) was consistent with those of published lepidopteran sequences. All tRNA genes had typical clover-leaf secondary structures, except for the tRNASer(AGN), in which the dihydrouridine (DHU) arm could not form a stable stem-loop structure. The A + T-rich region of 325 bp had several distinctive features, including the motif ‘ATAGA’ followed by an 18 bp poly-T stretch, a microsatellite-like (AT)7 element, and an 11-bp poly-A present immediately upstream of tRNAMet. Relationships among 32 insect species were determined using Maximum Likelihood (ML), Neighbor Joining (NJ) and Bayesian Inference (BI) phylogenetic methods. These analyses confirm that L. salicis belongs to the Lymantriidae; and that Lymantriidae is a member of Noctuoidea, and is a sister taxon to Erebidae, Nolidae and Noctuidae, most closely related to Erebidae.

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.

Cathepsin L-like protease can regulate the process of metamorphosis and fat body dissociation in Antheraea pernyi

ABSTRACT Cathepsins are a group of protease, located in lysosome and play a vital role in physiological process. Here, we reported cathepsin L‐like protease (Ap‐cathL), which contained an open reading frame of 1155 bp and encoding 385 amino acid residues protein. The I29 inhibitor domain and peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) putative conserved domains were detected in Ap‐cathL. Quantitative real‐time PCR (qRT‐PCR) analysis revealed that Ap‐cathL highly expressed in the fat body and midgut. The high expression during the molting stage, pupal stage and following 20E (20‐hydroxyecdysone) treatment indicated that it maybe involved in the process of molting and metamorphosis. In addition, depletion of Ap‐cathL influenced the expression of apoptosis pathway related genes. The protease inhibitor and RNA interference experiments showed that Ap‐cathL was involved in the fat body dissociation of A. pernyi. These results suggest that Ap‐cathL may involve in the process of metamorphosis and fat body dissociation of A. pernyi. HighlightsA cathepsin L‐like protease (Ap‐cathL) was first characterized from A. pernyi.The spatial and temporal expression profiles of Ap‐cathL were investigated.20E (molting hormone) induces Ap‐cathL expression in the fat body.Ap‐cathL RNAi influenced the expression of apoptosis‐related genes.Ap‐cathL functions in the fat body dissociation via inhibitor and RNAi experiments.

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.

Cathepsin O is involved in the innate immune response and metamorphosis of Antheraea pernyi

Cathepsins are key members of mammalian papain-like cysteine proteases that play an important role in the immune response. In this study, a fragment of cDNA encoding cathepsin O proteinase (ApCathepsin O) was cloned from Antheraea pernyi. It contains an open reading frame of 1170bp and encodes a protein with 390 amino acid residues, including a conserved I29 inhibitor domain and a peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) domain. Comparison with other previously reported cathepsin O proteins showed identity ranging from 45% to 79%. Quantitative real-time PCR (qRT-PCR) and Western blot analysis revealed that ApCathepsin O was highly expressed in the fat body; furthermore, the high expression during the pupal stage indicated that it might be involved during metamorphosis. After exposure to four different heat-killed pathogens (Escherichia coli, Beauveria bassiana, Micrococcus luteus, and A. pernyi nucleopolyhedrovirus), the expression levels of ApCathepsin O mRNA significantly increased and showed variable expression patterns. This indicates that ApCathepsin O is potentially involved in the innate immune system of A. pernyi. Interestingly, ApCathepsin O expression was upregulated after 20-hydroxyecdysone (20E) injection, which suggested that it might be regulated by 20E. In conclusion, ApCathepsin O is a protease that may play an important role in the innate immune response and metamorphosis of 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