Chao-Liang Liu

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages.

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.

Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli.

Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for comprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in Escherichia coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of expression conditions, particularly the culture temperature of PPO-transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. Additional Cu(2+), either added into the culture medium during PPO induction or directly mixed with the purified rPPO preparations, was necessary to produce Cu(2+) associated proenzymes. Cu(2+) associated PPOs showed obvious enzyme activities after activation by either ethanol or cetylpyridinium chloride, or by AMM1 (a pupal protein fraction containing native serine proteases for PPO activation). Dose responses for association of individual purified Drosophila rPPOs with Cu(2+) showed that Drosophila rPPO1 and rPPO3 had relatively higher affinity for Cu(2+) than rPPO2 did. Surprisingly, however, high concentration of Cu(2+) (2 mM) completely inhibited PPO activity. Each rPPO had similar activity when dopamine or l-DOPA was the substrate. However, rPPO1 alone had very high activity if l-tyrosine was used as a substrate. After activation by ethanol or 2-propanol, Km and Vmax of the three rPPOs changed as shown in the following: rPPO2<rPPO3<rPPO1. If activated by ethanol, the Km and Vmax of each rPPO were lower than by 2-propanol. Due to the difficulty in obtaining functional PPOs via traditional purification methods, the method established in this study will be helpful to produce active insect recombinant PPOs for the study of PPO properties and functions in the future.

Serpin-15 from Bombyx mori inhibits prophenoloxidase activation and expression of antimicrobial peptides.

Serine protease inhibitors (SPIs) play a key role in physiological responses by controlling protease activities. In this study, we studied the biochemical functions of serpin-15, an SPI, from Bombyx mori (Bmserpin-15). Recombinant Bmserpin-15 was expressed in Escherichia coli cells and used to raise rabbit anti-Bmserpin-15 polyclonal antibodies. Bmserpin-15 mRNA and protein expression was detected in all tested tissues, particularly in the fat body and silk gland. After challenge with four different microorganisms (Escherichia coli, Beauveria bassiana, Micrococcus luteus and B. mori nuclear polyhedrosis virus), the expressions of Bmserpin-15 mRNA and protein were induced significantly, particularly by B. bassiana and M. luteus. Recombinant Bmserpin-15 inhibited prophenoloxidase activation, but did not affect phenoloxidase activity, in B. mori hemolymph. Injection of recombinant Bmserpin-15 into B. mori larvae reduced significantly the transcript levels of antimicrobial peptides in fat body. Our results suggested that Bmserpin-15 plays an important role in the innate immunity of B. mori.

The complete mitochondrial genome of the common cutworm, Spodoptera litura (Lepidoptera: Noctuidade)

Abstract The complete mitochondrial genome (mitogenome) of Spodoptera litura (Lepidoptera: Noctuidae) was determined to be 15,374 bp (GenBank accession No. KF543065), including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A + T-rich region. It has the typical gene organization and order of mitogenomes from lepidopteran insects. The AT skew of this mitogenome was slightly positive and the nucleotide composition was also biased toward A + T nucleotides (81.03%). All PCGs were initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which was initiated by CGA. Four of the 13 PCGs harbor the incomplete termination codon by T. All the tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA, with the exception of trnS1 (AGN). The A + T-rich region of the mitogenome was 326 bp in length.

Inhibitors of eicosanoid biosynthesis influencing the transcripts level of sHSP21.4 gene induced by pathogen infections, in Antheraea pernyi.

Small heat shock proteins (sHSPs) can regulate protein folding and protect cells from stress. To investigate the role of sHSPs in the silk-producing insect Antheraea pernyi response to microorganisms, a sHsp gene termed as Ap-sHSP21.4, was identified. This gene encoded a 21.4 kDa protein which shares the conserved structure of insect sHsps and belongs to sHSP21.4 family. Ap-sHSP21.4 was highly expressed in fat body and up-regulated in midgut and fat body of A. pernyi challenged with Escherichia coli, Beauveria bassiana and nuclear polyhedrosis virus (NPV), which was determined by quantitative real-time PCR. Meanwhile, knock down of Ap-sHSP21.4 with dsRNA result in the decrease at the expression levels of several immune response-related genes (defensin, Dopa decarboxylase, Toll1, lysozyme and Kazal-type serine protease inhibitor). Additionally, the impact of eicosanoid biosynthesis on the expression of Ap-sHSP21.4 response to NPV was determined using qPCR, inhibitors of eicosanoid biosynthesis significantly suppress Ap-HSP21.4 expression upon NPV challenge. All together, Ap-sHSP21.4 was involved in the immunity of A. pernyi against microorganism and possibly mediated by eicosanoids pathway. These results will shed light in the understanding of the pathogen-host interaction in A. pernyi.

The complete mitochondrial genome of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

Abstract The complete mitochondrial genome (mitogenome) of Plutella xylostella (Lepidoptera: Plutellidae) was determined (GenBank accession No. KM023645). The length of this mitogenome is 16,014 bp with 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and an A + T-rich region. It presents the typical gene organization and order for completely sequenced lepidopteran mitogenomes. The nucleotide composition of the genome is highly A + T biased, accounting for 81.48%, with a slightly positive AT skewness (0.005). All PCGs are initiated by typical ATN codons, except for the gene cox1, which uses CGA as its start codon. Some PCGs harbor TA (nad5) or incomplete termination codon T (cox1, cox2, nad2 and nad4), while others use TAA as their termination codons. The A + T-rich region is located between rrnS and trnM with a length of 888 bp.

Eicosanoids mediate sHSP 20.8 gene response to biotic stress in larvae of the Chinese oak silkworm Antheraea pernyi.

Small heat shock proteins (sHSPs) can regulate protein folding and protect cells from stress. To investigate the role of sHSPs in the silk-producing insect Antheraea pernyi (A. pernyi; Lepidoptera: Saturniidae), cDNA encoding HSP20.8 in A. pernyi, termed Ap-sHSP20.8, was identified as a 564 bp ORF. The translated amino acid sequence encoded 187 residues with a calculated molecular mass of 20.8 kDa and an isoelectronic point (pI) of 5.98; the sequence showed homology to sHSP chaperone proteins from other insects. Ap-sHSP20.8 mRNA transcript expression was abundant in the midgut and fat body and found to be both constitutive and inducible by infectious stimuli. Therefore, Ap-sHSP20.8 may play important roles in A. pernyi immune responses under biotic stress. Furthermore, we found that eicosanoids could mediate the induction of Ap-sHSP20.8 in the fat body and midgut. Our findings show that sHSPs may be promising molecules to target in order to cripple immunity in insect pests.

Characterization of the Complete Mitochondrial Genome of Cerura menciana and Comparison with Other Lepidopteran Insects.

The complete mitochondrial genome (mitogenome) of Cerura menciana (Lepidoptera: Notodontidae) was sequenced and analyzed in this study. The mitogenome is a circular molecule of 15,369 bp, containing 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and a A+T-rich region. The positive AT skew (0.031) indicated that more As than Ts were present. All PCGs were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which was initiated by CAG. Two of the 13 PCGs contained the incomplete termination codon T or TA, while the others were terminated with the stop codon TAA. The A+T-rich region was 372 bp in length and consisted of an ‘ATAGA’ motif followed by an 18 bp poly-T stretch, a microsatellite-like (AT)8 and a poly-A element upstream of the trnM gene. Results examining codon usage indicated that Asn, Ile, Leu2, Lys, Tyr and Phe were the six most frequently occurring amino acids, while Cys was the rarest. Phylogenetic relationships, analyzed based on the nucleotide sequences of the 13 PCGs from other insect mitogenomes, confirmed that C. menciana belongs to the Notodontidae family.

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.

Comparative Mitochondrial Genome Analysis of Eligma narcissus and other Lepidopteran Insects Reveals Conserved Mitochondrial Genome Organization and Phylogenetic Relationships

In this study, we sequenced the complete mitochondrial genome of Eligma narcissus and compared it with 18 other lepidopteran species. The mitochondrial genome (mitogenome) was a circular molecule of 15,376 bp containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and an adenine (A) + thymine (T) − rich region. The positive AT skew (0.007) indicated the occurrence of more As than Ts. The arrangement of 13 PCGs was similar to that of other sequenced lepidopterans. All PCGs were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which was initiated by the CGA sequence, as observed in other lepidopterans. The results of the codon usage analysis indicated that Asn, Ile, Leu, Tyr and Phe were the five most frequent amino acids. All tRNA genes were shown to be folded into the expected typical cloverleaf structure observed for mitochondrial tRNA genes. Phylogenetic relationships were analyzed based on the nucleotide sequences of 13 PCGs from other insect mitogenomes, which confirmed that E. narcissus is a member of the Noctuidae superfamily.