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Mosquitoes are vectors that can cause serious negative impacts, including disease transmission, and altered ecosystem dynamics. In this research, phytochemical screening and toxicity of the essential oil derived from Murraya koenigii (L.) Spreng. leaves (Mk-EO) were evaluated against Aedes aegypti (Linn.) and Culex quinquefasciatus Say. The Mk-EO phytochemical screening revealed the presence of 16 chemicals, including Estragole (74.4%) as the main compound. Furthermore, the larvicidal activity showed a dose-dependent mortality rate across different instar larvae of filarial and dengue vectors. The action on larvae was vigorous at the peak dose (1000 ppm) of Mk-EO. The lethal concentration (LC 50) values against Ae. aegypti and Cx. quinquefasciatus were 503 and 513 ppm, respectively. Furthermore, the Mk-EO (500 ppm) significantly reduced the rate of α- and β-esterase activity and increased the rate of detoxifying enzymes glutathione-S-transferase (GST) and cytochrome P450 (CYP450). The repellent activity of Mk-EO was observed to be higher at 1000 ppm against Ae. aegypti (76%) and Cx. quinquefasciatus (76%) with a longer repellent period of up to 210 min. Severe midgut toxicity was observed, causing significant damage to midgut cells in both mosquito species. The screening against the non-target predators revealed that Mk-EO was harmless at a higher concentration (1200 ppm) compared to the chemical insecticide Temephos (1 ppm). Additionally, the in silico prediction of major metabolites of Mk-EO on the honey bee and aquatic models indicated minimal toxicity. Overall, Mk-EO serves as an environmentally friendly insecticidal agent against dengue and filarial vectors while also being nontoxic to aquatic predators. [Display omitted] • Chemical screening of essential oil from Murraya koenigii (Mk-EO) derived maximum peak area at Humulene. • Larvicidal activity of Mk-EO (1000 ppm) was maximum at the dose of 1000 ppm against dengue and filarial mosquito larvae. • Lethal dosage of Mk-EO (500 ppm) significantly affects the midgut enzymes of mosquito larvae. • Lethal dosage of Mk-EO (1000 ppm) displayed significant repellent activity up to 210 minutes. • Non-target screening against the mosquito predators displayed that the Mk-EO was harmless as compared to Temephos. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi, particularly Metarhizium and Beauveria species, are emerging as effective biocontrol agents for combating lepidopteran pests in agricultural and forestry settings. This review provides a comprehensive overview of their modes of action, secondary metabolites, extracellular enzymes, and infection mechanisms. These fungi employ various strategies, including the secretion of proteolytic enzymes, chitinolytic enzymes, esterases, and lipases, to penetrate the insect cuticle and initiate infection. The process involves spore recognition, adhesion, germination, and differentiation into infective structures. The impact of fungal strains on the insect immune system and the commercial availability of fungal pesticides are also discussed. Furthermore, the advancements in genetically engineered mycotoxins and the key challenges facing their implementation are addressed, in addition to listing future research directions. This review offers valuable insights for researchers involved in the development and application of entomopathogenic fungi for sustainable pest management practices. [Display omitted] • Exploration of the mode of actions of Metarhizium and Beauveria fungi, for targeting lepidopteran pests. • Examination of secondary metabolites contributing to the pathogenicity of entomopathogenic fungi. • Analyze the mechanism of extracellular enzymes, in the degradation of insect cuticles. • Insights into the infection mechanisms employed by mycotoxins to invade lepidopteran hosts. [ABSTRACT FROM AUTHOR]

The present research targets to explore the chemical and mosquitocidal screening of Ficus auriculata (Lour.), on arthropod mosquito vectors Aedes aegypti (Linn.) and Anopheles stephensi (Linn.) and their toxicity screening on the mosquito predator. Crude leaf ethanolic extract of F. auriculata (Ex-Fa) was isolated and screened for major bio-active compounds through GC-MS (Gas Chromatography-Mass Spectrometry) and lethal dosages was examined for larvicidal, enzyme inhibition, repellent and non-target toxicological evaluation on the aquatic predator. Chemical screening showed highest peak area in Flavone (51.48%). Larvicidal activity of Ex-Fa deliver that the rate of mortality was detected in dose dependent and it was significant in second instar treated with the maximum dosage (300 ppm) in Ae. aegypti (93.2%) and An. stephensi (95.6%) respectively and mortality is significant in positive control Temephos-1 ppm (97.7%) in second instar. The sub-lethal concentration of Ex-Fa (150 ppm) delivered significant enzyme inhibition action on second instar of α-carboxylesterase (Ae. aegypti : 0.513 mg/protein; An. stephensi : 0.511 mg/protein), β-carboxylesterase (Ae. aegypti : 0.612 mg/protein; An. stephensi : 0.732 mg/protein). The level of detoxifying enzymes uplifted post treatment with Ex-Fa in glutathione s-transferase (GST) (Ae. aegypti : 0.654 μmol/mg; An. stephensi : 0.674 μmol/mg) and cytochrome P450 (CYP 450) (Ae. aegypti : 8.379 μmol/mg; An. stephensi : 8.412 μmol/mg) it is significant with the positive control galantamine (1 ppm). The repellent percentage was significant at the higher concentration (300 ppm) in Ae. aegypti (89.6%) and An. stephensi (90.2%) up to 210 min. The non-target toxicity assay delivered that the Ex-Fa are nontoxic to the aquatic predator Toxorhynchites splendens. The present research delivers that herbal based extracts are promoting sustainable and effective vector control strategies. • Chemical screening of Crude leaf ethanolic extract of F. auriculata (Ex-Fa) showed highest peak area in Flavone. • Larvicidal activity of Ex-Fa deliver significant mortality rate across Aedes aegypti and Anopheles stephensi. • Ex-Fa significantly alters the level of detoxifying enzymes (α- β-carboxylesterase, GST and CYP450). • The repellent percentage was significant at Ex-Fa (300 ppm) in both vectors. • The non-target toxicity assay delivered that the Ex-Fa are harmless against mosquito predator. [ABSTRACT FROM AUTHOR]

Satsuma myomphala is critically endangered through loss of natural habitats, predation by natural enemies, and indiscriminate collection. It is a protected species in Korea but lacks genomic resources for an understanding of varied functional processes attributable to evolutionary success under natural habitats. For assessing the genetic information of S. myomphala , we performed for the first time, de novo transcriptome sequencing and functional annotation of expressed sequences using Illumina Next-Generation Sequencing (NGS) platform and bioinformatics analysis. We identified 103,774 unigenes of which 37,959, 12,890, and 17,699 were annotated in the PANM (Protostome DB), Unigene, and COG (Clusters of Orthologous Groups) databases, respectively. In addition, 14,451 unigenes were predicted under Gene Ontology functional categories, with 4581 assigned to a single category. Furthermore, 3369 sequences with 646 having Enzyme Commission (EC) numbers were mapped to 122 pathways in the Kyoto Encyclopedia of Genes and Genomes Pathway database. The prominent protein domains included the Zinc finger (C2H2-like), Reverse Transcriptase, Thioredoxin-like fold, and RNA recognition motif domain. Many unigenes with homology to immunity, defense, and reproduction-related genes were screened in the transcriptome. We also detected 3120 putative simple sequence repeats (SSRs) encompassing dinucleotide to hexanucleotide repeat motifs from > 1 kb unigene sequences. A list of PCR primers of SSR loci have been identified to study the genetic polymorphisms. The transcriptome data represents a valuable resource for further investigations on the species genome structure and biology. The unigenes information and microsatellites would provide an indispensable tool for conservation of the species in natural and adaptive environments. [ABSTRACT FROM AUTHOR]

Abstract: Samia cynthia ricini is a commercial silk-producing insect that is now reared year-round in Korea, with the expectation of being utilized for diverse purposes. In this report, we present the complete mitochondrial genome (mitogenome) of S. c. ricini. The 15,384-bp long S. cynthia ricini mitogenome was amplified into 26 short fragments using three long overlapping fragments using primers designed from reported lepidopteran mitogenome sequences. The genome comprises 37 genes (13 protein-coding genes, two rRNA genes, and 22 tRNA genes), and one large non-coding region termed the A+T-rich region. The A/T content of the third codon position was 91.7%, which was 18.8% and 21.6% higher than those of first and second codon positions, respectively. The high A/T content in the genome is reflected in codon usage, accounting for 39.5% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlike a previous report on the start codon for the COI gene, the S. c. ricini COI gene commences with a typical ATT codon. A total of 221bp of non-coding sequences are dispersed in 17 regions, ranging in size from 1 to 54bp, which comprise 1.4% of the total genome. One of the non-coding sequence located between tRNAGln and ND2 (54bp) has 77% sequence homology with the 5′-sequence of the neighboring ND2 gene, suggesting partial duplication of the sequence during evolution. The 361-bp long A+T-rich region contains an 18bp-long poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, poly-A stretch and one tRNA-like sequence, as typically found in Lepidoptera including Bombycoidea. [Copyright &y& Elsevier]

In this review, we describe the experimental observations that led us to propose the Time Bomb model of ookinete midgut invasion and discuss potential implications of this model when considering malaria transmission-blocking strategies aimed at arresting parasite development within midgut cells. A detailed analysis of the molecular interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei parasites, as they migrate through midgut cells, revealed that ookinetes induce nitric oxide synthase (NOS) expression, remodeling of the actin cytoskeleton and characteristic morphological changes in the invaded epithelial cells. Parasites inflict extensive damage that ultimately leads to genome fragmentation and cell death. During their migration through the cytoplasm, ookinetes release a subtilisin-like protease (PbSub2) and the surface protein (Pbs21). The model proposes that ookinetes must escape rapidly from the invaded cells, as the responses mediating cell death could be potentially lethal to the parasites. In other words, the physical and/or chemical damage triggered by the parasite can be thought of as a ‘lethal bomb’. Once this cascade of events is initiated, the parasite must leave the cellular compartment within a limited time to escape unharmed from the ‘bomb’ it has activated. The midgut epithelium has the ability to heal rapidly by ‘budding off’ the damaged cells to the midgut lumen without losing its integrity. [Copyright &y& Elsevier]

Abstract: In this study, a bacterium Serratia marcescens PRC-5 that displayed strong chitinolytic activity on 0.5% colloidal chitin-containing agar medium was isolated from soil. The chitinase activity increased rapidly with a maximum level (6.14U/mL) on 4 days of incubation with swollen chitin (pH 5.0). Three active bands of chitinase isozymes were observed (53, 44, and 34kDa) on SDS-PAGE gel and there pI values ranged from pI 5.4 to 5.8 on 2D gels. The chitinase from the PRC-5 strain was also able to produce GlcNAc monomers on TLC plates. The chitinase of PRC-5 inhibited the mycelial growth of Rhizoctonia solani KACC40111, which indicates that it could be used as a biocontrol agent for phytophathogens. The chitinase isozyme N1, which had a molecular weight of 62kDa, was transferred from a native and SDS-PAGE gel onto an immunoblot and was probed using an anti-PrGV-chitinase. [Copyright &y& Elsevier]

Abstract: Two similar full-length cDNAs of biliprotein were isolated and shown to encode the two isoelectric forms of biliverdin-binding proteins (BPs): BP1 and BP2 in Pieris rapae. Sequence analysis of two cDNA clones shows that both BPs contain a 567-bp open reading frame which predicts a 189-amino acid protein and a 15-amino acid signal peptide. The calculated isoelectric points are pI=7.25 (BP1) and 6.74 (BP2), respectively. Comparison of two sequences of BP1 and BP2 reveals 12 base differences in the open reading frame, of which three nucleotide changes lead to two amino acid substitutions. The 5′-UTR from the two clones shows no difference, but an additional 45-bp fragment is inserted in the 3′-UTR of BP2 making its message a little longer than that of BP1. Northern blot analysis confirmed that the BP mRNAs are expressed from the late 4th instar to the adult stage with exception of prepupae and newly ecdysed pupae. While the BP1 transcript was prevalent in the larval stage, the BP2 transcript was abundant in the whole body only after the pupal stage in P. rapae. Both BPs were detected in a stage-specific pattern in the epidermis, testis, hindgut, wing, brain, and egg, with a lesser amount in the fat body. Two-dimensional gel electrophoresis and Western blot analyses revealed that BP1 was dominant in tissues from larvae, BP2 was dominant in tissues from pupal stages, and both BPs appeared in tissues from the adult stage, though BP2 was predominant. [Copyright &y& Elsevier]

Toll and IMD pathways regulate antimicrobial innate immune responses in insect model systems. The transcriptional activation of antimicrobial peptides (AMPs) confers humoral immunity in the host against invaded pathogens. The IKK kinase complex (IKKα, IKKβ, and the regulatory subunit IKKγ/NEMO) centrally regulates the NF-κB response to various stimuli. It triggers an appropriate antimicrobial immune response in the host. In this study, a Tm IKKβ (or Tm Ird5) homolog was screened from the RNA-seq database of the coleopteran beetle, Tenebrio molitor. A single exon characterizes the Tm IKKβ gene, and the open reading frame (ORF) comprises of 2112 bp that putatively encodes a polypeptide of 703 amino acid residues. Tm IKKβ contains a serine/threonine kinase domain and is phylogenetically close to Tribolium castaneum IKKβ homolog (Tc IKKβ). TmIKKβ transcripts were highly expressed in the early pupal (P1) and adult (A5) stages. Among the tissues, TmIKKβ showed higher expression in the integument of the last instar larvae and the fat body and hemocytes of 5-day-old adults. TmIKKβ mRNA was upregulated post- E. coli challenge to the host. Moreover, RNAi-based TmIKKβ mRNA silencing increased host larvae' susceptibility against E. coli, S. aureus and C. albicans. TmIKKβ RNAi in the fat body led to a downregulation in mRNA expression of ten out of fourteen AMP genes, including TmTenecin1, -2, and -4 ; TmDefensin, and -like ; TmColeoptericinA, and -B ; and TmAttacin1a, - 1b, and -2 , suggesting the requirement of the gene in antimicrobial innate immune responses. Further, a decrease in the mRNA expression of NF-κB factors such as TmRelish, TmDorsal1 , and TmDorsal2 in the fat body of T. molitor larvae was observed post-microorganisms challenge. Thus, Tm IKKβ regulates antimicrobial innate immune responses in T. molitor. • We have newly identified IκB kinase β gene from the Tenebrio molitor , named TmIKKβ and domain analysis indicated that it contains one Serine/Threonine protein kinase catalytic domain, suggesting that TmIKKβ may regulate cell signaling by phosphorylation. • The temporal and spatial expression patterns of TmIKKβ transcripts and its induction patterns in responses to microbial challenges indicated that the TmIKKβ may have important roles in development as well as immune responses against microbial challenges. • We have investigated the effects of TmIKKβ RNAi on larval survivability, and expression patterns of antimicrobial peptide (AMP) genes and transcription factors for Toll and Imd pathways to understand the critical function of TmIKKβ in antimicrobial immune responses. [ABSTRACT FROM AUTHOR]

A bstract Scavenger receptors (SRs) constitute a family of membrane-bound receptors that bind to multiple ligands. The SR family of proteins is involved in removing cellular debris, oxidized low-density lipoproteins, and pathogens. Specifically, class C scavenger receptors (SR-C) have also been reported to be involved in phagocytosis of gram-positive and -negative bacteria in Drosophila and viruses in shrimp. However, reports are unavailable regarding the role of SR-C in antifungal immune mechanisms in insects. In this study, a full-length Tenebrio molitor SR-C ( Tm SR-C) sequence was obtained by 5′- and 3′-Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The TmSR-C full-length cDNA comprised 1671 bp with 5′- and 3′-untranslated regions of 23- and 107-bp, respectively. Tm SR-C encodes a putative protein of 556 amino acid residues that is constitutively expressed in all tissues of late instar larvae and 2-day-old adults, with the highest transcript levels observed in hemocytes of larvae and adults. TmSR-C mRNA showed a 2.5-fold and 3-fold increase at 24 and 6 h after infection with Candida albicans and β-glucan, respectively. Immunoassay with Tm SR-C polyclonal antibody showed induction of the putative protein in the cytosols of hemocytes at 3 h after inoculation of C. albicans . RNA interference (RNAi)-based gene silencing and phagocytosis assays were used to understand the role of Tm SR-C in antifungal immunity. Silencing of TmSR-C transcripts reduced the survivability of late instar larvae at 2 days post-inoculation of C. albicans , Escherichia coli , or Staphylococcus aureus . Furthermore, in TmSR-C -silenced larvae, there was a decline in the rate of microorganism phagocytosis. Taken together, results of this study suggest that Tm SR-C plays a pivotal role in phagocytosing not only fungi but also gram-negative and -positive bacteria in T. molitor . [ABSTRACT FROM AUTHOR]

Valuable insect models have tremendously contributed to our understanding of innate immunity and symbiosis. Bean bug, Riptortus pedestris , is a useful insect symbiosis model due to harboring cultivable monospecific gut symbiont, genus Burkholderia . Bean bug is a hemimetabolous insect whose immunity is not well-understood. However, we recently identified three major antimicrobial peptides of Riptortus and examined the relationship between gut symbiosis and host immunity. We found that the presence of Burkholderia gut symbiont positively affects Riptortus immunity. From studying host regulation mechanisms of symbiont population, we revealed that the symbiotic Burkholderia cells are much more susceptible to Riptortus immune responses than the cultured cells. We further elucidated that the immune-susceptibility of the Burkholderia gut symbionts is due to the drastic change of bacterial cell envelope. Finally, we show that the immune-susceptible Burkholderia symbionts are able to prosper in host owing to the suppression of immune responses of the symbiotic midgut. [ABSTRACT FROM AUTHOR]

Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes ( ATG ) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor ( TmATG8 ). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes . Unlike ds EGFP -treated control larvae, TmATG8- silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes . These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor . [ABSTRACT FROM AUTHOR]

Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5′-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6 h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus . These results indicate that TmMyD88 is required for survival against Staphylococcus infection. [ABSTRACT FROM AUTHOR]

Abstract: Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5′-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3′. The ‘helix–short helix–helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor. [Copyright &y& Elsevier]

Vertebrates rely on the most sophisticated adaptive immunity to defend themselves against various pathogens. This includes immunologic memory cells, which mount a stronger and more effective immune response against an antigen after its first encounter. Unlike vertebrates, invertebrates' defense completely depends on the innate immunity mechanisms including humoral and cell-mediated immunity. Furthermore, the invertebrate equivalent of the memory cells was discovered only recently. Since the discovery of transgenerational immune priming (TGIP) in crustaceans, numerous findings have proven the IP in invertebrate classes such as insects. TGIP can be induced through maternal priming pathways such as transcriptional regulation of antimicrobial peptides, and also paternal IP including the induction of proPO system activity. We appraise the diversity and specificity of IP agents to provide sustained immunologic memory in insects, particularly T. molitor in the review. An understanding of IP (more so TGIP) response in T. molitor will deepen our knowledge of invertebrate immunity, and boost the mass-rearing industry by reducing pathogen infection rates. • Unlike mammals, solo arm of defense mechanism in invertebrates is innate immunity, particularly, antimicrobial peptide production. • The ability of being immunized against a preexisting elicitor, was a matter of uncertainty until a wile ago. • This phenomenon known as "immune priming", can be transferred to progenies by both parents. • The diversity and specificity of immune priming in insects more precisely, Tenebrio molitor has been investigated in numbers of studies. • Understanding of immune priming in insects can provide us with novel strategies and applications at the field during T. molitor mass rearing. [ABSTRACT FROM AUTHOR]

Abstract: Apolipophorin-III (apoLp-III), a hemolymph protein that facilitates lipid transport in aqueous media in insects was recently shown to play a role in insect immune activation. Here, we report another novel possible function of apoLp-III in insects. To identify genes affected by apoLp-III expression in larvae, we decreased endogenous apoLp-III mRNA in Hyphantria cunea (Hc) through RNA interference; subsequently, we observed lower levels of antioxidant enzymes, including manganese superoxide dismutase (MnSOD), glutathione S-transferase, and immune proteins. Knockdown of Hc apoLp-III led to decreased MnSOD expression in fat body tissues and elevated superoxide anion levels in Hc fat body cells, suggesting that Hc apoLp-III is involved in the action and/or expression of antioxidant enzymes, especially MnSOD. [Copyright &y& Elsevier]

Abstract: Among the three zinc finger-containing glycine-rich RNA-binding proteins, named AtRZ-1a, AtRZ-1b, and AtRZ-1c, in the Arabidopsis thaliana genome, AtRZ-1a has previously been shown to enhance cold and freezing tolerance in Arabidopsis. Here, we determined and compared the functional roles of AtRZ-1b and AtRZ-1c in Arabidopsis and Escherichia coli under cold stress conditions. AtRZ-1b, but not AtRZ-1c, successfully complemented the cold sensitivity of E. coli BX04 mutant cells lacking four cold shock proteins. Domain deletion and site-directed mutagenesis showed that the zinc finger motif of AtRZ-1b is important for its complementation ability, and that the truncated N- and C-terminal domains of AtRZ-1b and AtRZ-1c harbor the complementation ability. Despite an increase in transcript levels of AtRZ-1b and AtRZ-1c under cold stress, overexpression or loss-of-function mutations did not affect seed germination or seedling growth of Arabidopsis under cold stress conditions. AtRZ-1b and AtRZ-1c proteins, being localized to the nucleus, have been shown to bind non-specifically to RNA sequences in vitro, in comparison to AtRZ-1a that is localized to both the nucleus and the cytoplasm and binds preferentially to G- or U-rich RNA sequences. Taken together, these results demonstrate that the three AtRZ-1 family members showing different cellular localization and characteristic nucleic acid-binding property have a potential to contribute differently to the enhancement of cold tolerance in Arabidopsis and E. coli. [ABSTRACT FROM AUTHOR]

Abstract: Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733. The heavy chain (HC) and light chain (LC) genes of mAb CO17-1A were cloned under the control of P10 and Polyhedrin promoters in the pFastBac™ dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. Confocal immunofluorescence and Western blot analyses confirmed expression of mAb CO17-1A in baculovirus-infected insect cells. The optimum conditions for mAb expression were evaluated at 24, 48, and 72 h after the virus infection at an optimum virus multiplicity of infection of 1. Expression of mAb CO17-1A in insect cells significantly increased at 72 h after infection. HPLC analysis of glycosylation status revealed that the insect-derived mAb (mAbI) CO17-1A had insect specific glycan structures. ELISA showed that the purified mAbI from cell culture supernatant specifically bound to SW948 human colorectal cancer cells. Fluorescence-activated cell sorting analysis showed that, although mAbI had insect specific glycan structures that differed from their mammalian counterparts, mAbI similarly interacted with CD64 (FcγRI) and Fc of IgG, compared to the interactions of mammalian-derived mAb. These results suggest that the baculovirus insect cell expression system is able to express, assemble, and secrete biofunctional full size mAb. [Copyright &y& Elsevier]

Abstract: The 15,360-bp long complete mitogenome of Caligula boisduvalii possesses a gene arrangement and content identical to other completely sequenced lepidopteran mitogenomes, but different from the common arrangement found in most insect order, as the result of the movement of tRNAMet to a position 5''-upstream of tRNAIle. The 330-bp A+T-rich region is apparently capable of forming a stem-and-loop structure, which harbors the conserved flanking sequences at both ends. Dissimilar to what has been seen in other sequenced lepidopteran insects, the initiation codon for C. boisduvalii COI appears to be TTG, which is a rare, but apparently possible initiation codon. The ATP8, ATP6, ND4L, and ND6 genes, which neighbor another PCG at their 3'' end, all harbored potential sequences for the formation of a hairpin structure. This is suggestive of the importance of such structures for the precise cleavage of the mRNA of mature PCGs. Phylogenetic analyses of available sequenced species of Bombycoidea, Pyraloidea, and Tortricidea supported the morphology-based current hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As previously suggested, Bombycidae (Bombyx mori and B. mandarina) and Saturniidae (Antheraea pernyi and C. boisduvalii) formed a reciprocal monophyletic group. [Copyright &y& Elsevier]

Abstract: The complete 16,434-bp nucleotide sequence of the mitogenome of the bumble bee, Bombus ignitus (Hymenoptera: Apidae), was determined. The genome contains the base composition and codon usage typical of metazoan mitogenomes. An unusual feature of the B. ignitus mitogenome is the presence of five tRNA-like structures: two each of the tRNA Leu(UUR) -like and tRNA Ser(AGN) -like sequences and one tRNA Phe -like sequence. These tRNA-like sequences have proper folding structures and anticodon sequences, but their functionality in their respective amino acid transfers remained uncertain. Among these sequences, the tRNA Leu(UUR) -like sequence and the tRNA Ser(AGN) -like sequence are seemingly located within the A+T-rich region. This tRNA Ser(AGN) -like sequence is highly unusual in that its sequence homology is very high compared to the tRNA Met of other insects, including Apis mellifera, but it contains the anticodon ACT, which designates it as tRNA Ser(AGN) . All PCG and rRNAs are conserved in positions observed most frequently in insect mitogenome structures, but the positions of the tRNAs are highly variable, presenting a new arrangement for an insect mitogenome. As a whole, the B. ignitus mitogenome contains the highest A+T content (86.9%) found in any of the complete insects mt sequences determined to date. All protein-coding sequences started with a typical ATN codon. Nine of the 13 PCGs have a complete termination codon (all TAA), but the remaining four genes terminate with the incomplete TA or T. All tRNAs have the typical clover-leaf structures of mt tRNAs, except for tRNA Ser(AGN) , in which the DHU arm forms a simple loop. All anticodons of B. ignitus tRNAs are identical to those of A. mellifera. In the A+T-rich region, a highly conserved sequence block that was previously described in Orthoptera and Diptera was also present. The stem-and-loop structures that may play a role in the initiation of mtDNA replication were also found in this region. Phylogenetic analysis among three corbiculate tribes, represented by Melipona bicolor (Meliponini), A. mellifera (Apini), and B. ignitus (Bombini), showed the closest relationship between M. bicolor and B. ignitus. [Copyright &y& Elsevier]

Abstract: Transferrin in insects is known as an iron transporter, an antibiotic agent, a vitellogenin, and a juvenile hormone-regulated protein. We show here a novel functional role for insect transferrin. Stresses, such as iron overload, bacterial or fungal challenge, cold or heat shock, wounding, and H2O2 or paraquat exposure, cause upregulation of the beetle Apriona germari transferrin (AgTf) gene in the fat body and epidermis, and they cause increased AgTf protein levels. RNA interference (RNAi)-mediated AgTf reduction results in rapid induction of apoptotic cell death in the fat body during exposure to heat stress. The observed effect of AgTf RNAi indicates that AgTf inhibits heat stress-induced apoptotic cell death, suggesting a functional role for AgTf in defense and stress responses in the beetle. [Copyright &y& Elsevier]

Two hemolymph proteins were isolated from the wax moth, Galleria mellonella, larvae by a two-step procedure consisting of acid extraction and reversed phase (RP)-HPLC. One was an apolipophorin III (apoLp-III) previously characterized as a lipopolysaccharide (LPS) binding protein in the hemolymph of G. mellonella. The other was confirmed to be a new protein with a molecular mass of 23,768.69 Da, referred to as Gm protein-24. The full-length cDNA of Gm protein-24 was cloned from the fat body. The cDNA structure showed that it is a 219-residues protein derived from the precursor of 236 amino acids. The effects of apoLp-III and Gm protein-24 have been tested on the insect humoral immunity. ApoLp-III enhanced the activity of antibacterial peptide such as cecropin but Gm protein-24 had no effect on cecropin activity. On the other hand, Gm protein-24 and apoLp-III were both involved in the activation of prophenoloxidase (PPO) cascade, which has been regarded as a critical immune reaction in insect hemolymph. Of note, the Gm protein-24 was a significantly stronger activator of PPO cascade than apoLp-III. [Copyright &y& Elsevier]

Apolipophorin-III (apoLp-III) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium. Recently, apoLp-III in Galleria mellonella larvae was shown to play an unexpected role in insect immune activation. We identified the cDNA sequence of Hyphantria cunea apoLp-III by oligonucleotide-primed amplification, and 5′- and 3′-RACE PCR. Since H. cunea has an unusually low level of apoLp-III in the hemolymph, a recombinant apoLp-III was overexpressed using a baculovirus expression system to investigate its biological activity. Recombinant apoLp-III and/or Escherichia coli were injected into the hemocoel of last instar larvae, and the expression of antimicrobial peptide from fat body was determined by Northern blot. Injection of apoLp-III as well as E. coli induced slight up-regulation of its transcription rate in fat body, whereas the expression of antimicrobial peptide was dramatically induced by the injection of apoLp-III and E. coli. H. cunea hemocytes had apoLp-III in the granules and expressed its transcript, albeit at a much lower level than in the fat body. Upon bacterial injection, a subpopulation of hemocytes showed degranulation and degradation. Local discharge of apoLp-III from hemocytes caused by the injection of E. coli might be related to the immune response through an unknown mechanism. [Copyright &y& Elsevier]