Cadherin AdCad1 in Alphitobius diaperinus larvae is a receptor of Cry3Bb toxin from Bacillus thuringiensis

Bacillus thuringiensis (Bt) Cry proteins are used as components of biopesticides or expressed in transgenic crops to control diverse insect pests worldwide. These Cry toxins bind to receptors on the midgut brush border membrane and kill enterocytes culminating in larval mortality. Cadherin proteins have been identified as Cry toxin receptors in diverse lepidopteran, coleopteran, and dipteran species. In the present work we report a 185 kDa cadherin (AdCad1) from larvae of the lesser mealworm (Alphitobius diaperinus) larvae as the first identified receptor for Cry3Bb toxin. The AdCad1 protein contains typical structural components for Cry toxin receptor cadherins, including nine cadherin repeats (CR9), a membrane-proximal extracellular domain (MPED) and a cytosolic region. Peptides corresponding to the CR9 and MPED regions bound Cry3Bb toxin with high affinities (23 nM and 40 nM) and significantly synergized Cry3Bb toxicity against A. diperinus larvae. Silencing of AdCad1 expression through RNA interference resulted in highly reduced susceptibility to Cry3Bb in A. diperinus larvae. The CR9 peptide fed with toxin to RNAi-treated larvae restored Cry3Bb toxicity. These results are evidences that AdCad1 is a functional receptor of Cry3Bb toxin and that exogenously fed CR9 peptide can overcome the effect of reduced AdCad1expression on Cry3Bb toxicity to larvae.     

Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach,Diploptera punctata

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca²⁺ and cAMP, which suggests that Dippu-AstR can act through the Gα_q and Gα_s protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.     

Complete mitochondrial genome of Pycnonotus xanthorrhous (Passeriformes,Pycnonotidae) and phylogenetic consideration

The complete mitochondrial genome (mitogenome) of Pycnonotus xanthorrhous was sequenced via next generation sequencing. The full length of the circular genome is 16,952 bp. It consists of 37 typical animal mitochondrial genes including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) and 2 ribosomal RNA (rRNA) genes. P. xanthorrhous also contains one control region (CR) and one pseudo-control region, and shares the identical gene arrangements with sequenced Pycnonotus spp. which differs from the typical vertebrates gene order. Phylogenetic analyses indicates that Passerida sensu stricto contains three major clades and the core Sylvioidea form a monophyletic group. Furthermore, we investigated the evolution of control region within this lineage and revealed the multiple independent origins of duplicate control region.     

Cloning and preliminary functional studies of the JAM-A gene in grass carp (Ctenopharyngodon idellus)

Grass carp (Ctenopharyngodon idellus) is a very important aquaculture species in China and other South-East Asian countries; however, disease outbreaks in this species are frequent, resulting in huge economic losses. Grass carp hemorrhage caused by grass carp reovirus (GCRV) is one of the most serious diseases. Junction adhesion molecule A (JAM-A) is the mammalian receptor for reovirus, and has been well studied. However, the JAM-A gene in grass carp has not been studied so far. In this study, we cloned and elucidated the structure of the JAM-A gene in grass carp (GcJAM-A) and then studied its functions during grass carp hemorrhage. GcJAM-A is composed of 10 exons and 9 introns, and its full-length cDNA is 1833 bp long, with an 888 bp open reading frame (ORF) that encodes a 295 amino acid protein. The GcJAM-A protein is predicted to contain a typical transmembrane domain. Maternal expression pattern of GcJAM-A is observed during early embryogenesis, while zygote expression occurs at 8 h after hatching. GcJAM-A is expressed strongly in the gill, liver, intestine and kidney, while it is expressed poorly in the blood, brain, spleen and head kidney. Moreover, lower expression is observed in the gill, liver, intestine, brain, spleen and kidney of 30-month-old individuals, compared with 6-month-old. In a GcJAM-A-knockdown cell line (CIK) infected with GCRV, the expression of genes involved in the interferon and apoptosis pathways was significantly inhibited. These results suggest that GcJAM-A could be a receptor for GCRV. We have therefore managed to characterize the GcJAM-A gene and provide evidence for its role as a receptor for GCRV.     

Positive feedback regulation of prothoracicotropic hormone secretion by ecdysteroid – A mechanism that determines the timing of metamorphosis

When insect larvae have fully grown, prothoracicotropic hormone (PTTH) is released from the brain, triggering the initiation of metamorphic development through stimulation of ecdysteroid secretion by the prothoracic glands. The present study analyzes the mechanism that regulates the occurrence of this PTTH surge. In the silkworm Bombyx mori, the PTTH surge occurs on day 6 of the fifth instar and is preceded by a small rise in hemolymph ecdysteroid titer, which occurs late on day 5. We therefore hypothesized that this rise of ecdysteroid titer is involved in the induction of the PTTH surge. To test this hypothesis, two experiments were conducted. First, a small amount of 20-hydroxyecdysone was injected on day 4, two days before the expected day of the PTTH surge, to simulate the small rise in hemolymph ecdysteroid titer on day 5. This injection led to a precocious surge of PTTH the next day. Next, the hemolymph ecdysteroid titer on day 5 was artificially lowered by injecting ecdysteroid-22-oxidase, which inactivates 20-hydroxyecdysone. After this treatment, the PTTH surge did not occur on day 6 in 80% of the animals. These results indicate that a small rise of the hemolymph ecdysteroid titer plays a critical role in the induction of the PTTH surge. Since basal ecdysteroidogenic activity of the prothoracic glands increases with larval growth, a circulating level of ecdysteroids may convey information about larval maturity to the brain, to coordinate larval growth and metamorphosis. This is the first report in invertebrates to demonstrate positive feedback regulation of the surge of a tropic hormone by a downstream steroid hormone.     

The immune signaling pathways of Manduca sexta

Signal transduction pathways and their coordination are critically important for proper functioning of animal immune systems. Our knowledge of the constituents of the intracellular signaling network in insects mainly comes from genetic analyses in Drosophila melanogaster. To facilitate future studies of similar systems in the tobacco hornworm and other lepidopteran insects, we have identified and examined the homologous genes in the genome of Manduca sexta. Based on 1:1 orthologous relationships in most cases, we hypothesize that the Toll, Imd, MAPK-JNK-p38 and JAK-STAT pathways are intact and operative in this species, as are most of the regulatory mechanisms. Similarly, cellular processes such as autophagy, apoptosis and RNA interference probably function in similar ways, because their mediators and modulators are mostly conserved in this lepidopteran species. We have annotated a total of 186 genes encoding 199 proteins, studied their domain structures and evolution, and examined their mRNA levels in tissues at different life stages. Such information provides a genomic perspective of the intricate signaling system in a non-drosophiline insect.     

Transposition burst of mariner-like elements in the sequenced genome of Rhodnius prolixus

Transposable elements (TEs) are widespread in insect's genomes. However, there are wide differences in the proportion of the total DNA content occupied by these repetitive sequences in different species. We have analyzed the TEs present in R. prolixus (vector of the Chagas disease) and showed that 3.0% of this genome is occupied by Class II TEs, belonging mainly to the Tc1-mariner superfamily (1.65%) and MITEs (1.84%). Interestingly, most of this genomic content is due to the expansion of two subfamilies belonging to: irritans himar, a well characterized subfamily of mariners, and prolixus1, one of the two novel subfamilies here described. The high amount of sequences in these subfamilies suggests that bursts of transposition occurred during the life cycle of this family. In an attempt to characterize these elements, we performed an in silico analysis of the sequences corresponding to the DDD/E domain of the transposase gene. We performed an evolutionary analysis including network and Bayesian coalescent-based methods in order to infer the dynamics of the amplification, as well as to estimate the time of the bursts identified in these subfamilies. Given our data, we hypothesized that the TE expansions occurred around the time of speciation of R. prolixus around 1.4 mya. This suggestion lays on the “Transposon Model” of TE evolution, in which the members of a TE population that are replicative active are present at multiple loci in the genome, but their replicative potential varies, and of the “Life Cycle Model” that states that when present-day TEs have been involved in amplification bursts, they share an ancestral copy that dates back to this initial amplification.     

Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin

Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that ⁷⁷⁰DYWL⁷⁷³ of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around ⁷⁷⁰DYWL⁷⁷³ of ECL 4 in the ABCC2.     

Ins and Outs of Interpreting Lipidomic Results

Membrane lipids are essential for life; however, research on how cells regulate cell lipid composition has been falling behind for quite some time. One reason was the difficulty in establishing analytical methods able to cope with the cell lipid repertoire. Development of a diversity of mass spectrometry-based technologies, including imaging mass spectrometry, has helped to demonstrate beyond doubt that the cell lipidome is not only greatly cell type dependent but also highly sensitive to any pathophysiological alteration such as differentiation or tumorigenesis. Interestingly, the current popularization of metabolomic studies among numerous disciplines has led many researchers to rediscover lipids. Hence, it is important to underscore the peculiarities of these metabolites and their metabolism, which are both radically different from protein and nucleic acid metabolism. Once differences in lipid composition have been established, researchers face a rather complex scenario, to investigate the signaling pathways and molecular mechanisms accounting for their results. Thus, a detail often overlooked, but of crucial relevance, is the complex networks of enzymes involved in controlling the level of each one of the lipid species present in the cell. In most cases, these enzymes are redundant and promiscuous, complicating any study on lipid metabolism, since the modification of one particular lipid enzyme impacts simultaneously on many species. Altogether, this review aims to describe the difficulties in delving into the regulatory mechanisms tailoring the lipidome at the activity, genetic, and epigenetic level, while conveying the numerous, stimulating, and sometimes unexpected research opportunities afforded by this type of studies.     

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90–180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P < 0.01) and increase the number of IRS2 positive cells and Ob-R positive cells in hippocampus (P < 0.01). ② The expressions of mitochondrial energy-relative proteins, VDAC1 and cytC, were higher in cold-exposure group than in control group with both immunohistochemical staining and Western blot (P < 0.01). ③ Chronic cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P < 0.01). In conclusion, chronic cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance.     

Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning,expression and substrate specificity analysis

Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3′- and 5′-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (l-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.