不同杀虫剂处理对赤拟谷盗P450基因诱导表达特性影响

害虫细胞色素P450基因可被杀虫剂迅速诱导,然而当前对不同杀虫剂处理下赤拟谷盗P450基因诱导表达特性的研究较少。本研究首先通过序列比对选取了来自不同家族的8个赤拟谷盗P450基因CYP4G7、CYP4Q4、CYP4BR3、CYP12H1、CYP6BK11、CYP9D4、CYP9Z5和CYP345A1,然后采用四种不同杀虫剂氯氰菊酯、氟氯氰菊酯、氯菊酯和吡虫啉对赤拟谷盗20 d幼虫进行生物测定,再根据生测结果以四个药剂亚致死剂量分别处理幼虫,并采用荧光定量PCR分析8个P450基因的表达特性。结果表明,CYP4G7和CYP345A1可以分别被氯氰菊酯(分别上调1.97倍和2.06倍)、氟氯氰菊酯(2.00倍和2.03倍)和氯菊酯(1.73倍和1.81倍)显著诱导,而CYP4BR3和CYP345A1可以被吡虫啉(分别上调1.99倍和1.93倍)显著诱导。本研究结果表明赤拟谷盗P450基因的显著诱导与基因家族类型以及农药品种有关。     

Different effects of paternal trans-generational immune priming on survival and immunity in step and genetic offspring

Paternal trans-generational immune priming, whereby fathers provide immune protection to offspring, has been demonstrated in the red flour beetle Tribolium castaneum exposed to the insect pathogen Bacillus thuringiensis. It is currently unclear how such protection is transferred, as in contrast to mothers, fathers do not directly provide offspring with a large amount of substances. In addition to sperm, male flour beetles transfer seminal fluids in a spermatophore to females during copulation. Depending on whether paternal trans-generational immune priming is mediated by sperm or seminal fluids, it is expected to either affect only the genetic offspring of a male, or also their step offspring that are sired by another male. We therefore conducted a double-mating experiment and found that only the genetic offspring of an immune primed male show enhanced survival upon bacterial challenge, while phenoloxidase activity, an important insect immune trait, and the expression of the immune receptor PGRP were increased in all offspring. This indicates that information leading to enhanced survival upon pathogen exposure is transferred via sperm, and thus potentially constitutes an epigenetic effect, whereas substances transferred with the seminal fluid could have an additional influence on offspring immune traits and immunological alertness.     

Predicted versus expressed adipokinetic hormones, and other small peptides from the corpus cardiacum-corpus allatum: a case study with beetles and moths

This mass spectrometric study confines itself to peptide masses in the range of 500-1500Da. Adipokinetic hormones (AKHs) that are predicted from the genome of the red flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori, are shown to exist as expressed peptides in the corpora cardiaca (CC) of the respective species as evidenced by various mass spectrometric methods. Additionally, some related species were included in this study, such as the tenebrionid beetles Tribolium brevicornis and Tenebrio molitor, as well as the moths Spodoptera frugiperda, Spodoptera littoralis, Mamestra brassicae and Lacanobia oleracea, to investigate whether AKH peptides are structurally conserved in the same genus or family. Interestingly, the AKH peptide of T. brevicornis is identical to that of T. molitor but not to the ones of its close relative T. castaneum. Moreover, other peptides in T. brevicornis, such as various FXPRL amides (=pyrokinins), also match the complement in T. molitor but differ from those in T. castaneum. All the CC of beetles lacked the signal for the mass of the peptide corazonin. All moths have the nonapeptide Manse-AKH expressed in their CC. In addition, whereas the silk moth has the decapeptide Bommo-AKH as a second peptide, all other moths (all noctuids) express the decapeptide Helze-HrTH. In M. brassicae and L. oleracea a novel amidated Gly-extended Manse-AKH is found as a possible third AKH. The noctuid moth species also all express the same FLRF amide-I, corazonin, and a group-specific isoform of a gamma-PGN-(=gamma-SGNP) peptide. In L. oleracea, however, the latter peptide has a novel sequence which is reported for the first time, and the peptide is code-named Lacol-PK.     

Larval RNAi in Drosophila?

RNA interference (RNAi) has become a common method of gene knockdown in many model systems. To trigger an RNAi response, double-stranded RNA (dsRNA) must enter the cell. In some organisms such as Caenorhabditis elegans, cells can take up dsRNA from the extracellular environment via a cellular uptake mechanism termed systemic RNAi. However, in the fruit fly Drosophila melanogaster, it is widely believed that cells are unable to take up dsRNA, although there is little published data to support this claim. In this study, we set out to determine whether this perception has a factual basis. We took advantage of traditional Gal4/upstream activation sequence (UAS) transgenic flies as well as the mosaic analysis with a repressible cell marker (MARCM) system to show that extracellular injection of dsRNA into Drosophila larvae cannot trigger RNAi in most Drosophila tissues (with the exception of hemocytes). Our results show that this is not due to a lack of RNAi machinery in these tissues as overexpression of dsRNA inside the cells using hairpin RNAs efficiently induces an RNAi response in the same tissues. These results suggest that, while most Drosophila tissues indeed lack the ability to uptake dsRNA from the surrounding environment, hemocytes can initiate RNAi in response to extracellular dsRNA. We also examined another insect, the red flour beetle Tribolium castaneum, which has been shown to exhibit a robust systemic RNAi response. We show that virtually all Tribolium tissues can respond to extracellular dsRNA, which is strikingly different from the situation in Drosophila. Our data provide specific information about the tissues amenable to RNAi in two different insects, which may help us understand the molecular basis of systemic RNAi.     

TGFβ signaling in Tribolium: vertebrate-like components in a beetle

The cytokines of the TGFβ superfamily are highly conserved in evolution and elicit a diverse range of cellular responses in all metazoa. In Drosophila, the signaling pathways of the two TGFβ subfamilies, Activins and Bone Morphogenetic Proteins (BMPs), have been well studied. To address the question of whether the findings from Drosophila are representative of insects in general, we analyzed the components of TGFβ-signaling present in the genome of the beetle Tribolium castaneum. We were able to identify orthologs of the BMPs Decapentaplegic and Glass bottom boat, of the Activins Activinβ and Dawdle, as well as orthologs of the less well-known ligands Myoglianin and Maverick, together with orthologs of all TGFβ receptors and cytoplasmic signal transducers present in Drosophila. This indicates that the diversity of TGFβ signaling components is generally well conserved between Drosophila and Tribolium. However, the genome of the beetle—and of the bee Apis mellifera—lacks an ortholog of the Drosophila BMP Screw but does contain a vertebrate-like BMP10 homolog which is not found in Drosophila. Concerning BMP inhibitors, Tribolium displays an even more vertebrate-like ensemble of components. We found two orthologs of the vertebrate DAN family, Dan and Gremlin, and show embryonic expression of a vertebrate-like BAMBI ortholog, all of which are absent in Drosophila. This suggests that Tribolium might have retained a more ancestral composition of TGFβ signaling components and that TGFβ signaling underwent considerable change in the Drosophila lineage. Tribolium is an excellent model to study the function of these ancestral signaling components in insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two small heat shock protein genes in Apis cerana cerana: characterization,regulation, and developmental expression

In the present study, we identified and characterized two small heat shock protein genes from Apis cerana cerana, named AccHsp24.2 and AccHsp23.0. An alignment analysis showed that AccHsp24.2 and AccHsp23.0 share high similarity with other members of the α-crystallin/sHSP family, all of which contain the conserved α-crystallin domain. The recombinant AccHsp24.2 and AccHsp23.0 proteins were shown to have molecular chaperone activity by the malate dehydrogenase thermal aggregation assay. Three heat shock elements were detected in the 5′-flanking region of AccHsp24.2 and eleven in AccHsp23.0, and two Drosophila Broad-Complex genes for ecdysone steroid response sites were found in each of the genes. The presence of these elements suggests that the expression of these genes might be regulated by heat shock and ecdysone, which was confirmed by quantitative RT-PCR (RT-qPCR). The results revealed that the expression of the two genes could be induced by cold shock (4 °C) and heat shock (37 °C and 43 °C) in an analogous manner, and AccHsp24.2 was more susceptible than AccHsp23.0. In addition, the expression of the two genes was induced by high concentrations of ecdysone in vitro and in vivo. The accumulation of AccHsp24.2 and AccHsp23.0 mRNA was also detected in different developmental stages and tissues. In spite of the differential expression at the same stage, these genes shared similar developmental patterns, suggesting that they are regulated by similar mechanisms.     

Evaluation of inhibitor of apoptosis genes as targets for RNAi-mediated control of insect pests

Apoptosis has been widely studied from mammals to insects. Inhibitor of apoptosis (IAP) protein is a negative regulator of apoptosis. Recent studies suggest that iap genes could be excellent targets for RNA interference (RNAi)-mediated control of insect pests. However, not much is known about iap genes in one of the well-known insect model species, Tribolium castaneum. The orthologues of five iap genes were identified in T. castaneum by searching its genome at NCBI ( https://www.ncbi.nlm.nih.gov/ ) and UniProt ( https://www.uniprot.org/ ) databases using Drosophila melanogaster and Aedes aegypti IAP protein sequences as queries. RNAi assays were performed in T. castaneum cell line (TcA) and larvae. The knockdown of iap1 gene induced a distinct apoptotic phenotype in TcA cells and induced 91% mortality in T. castaneum larvae. Whereas, knockdown of iap5 resulted in a decrease in cell proliferation in TcA cells and developmental defects in T. castaneum larvae which led to 100% mortality. Knockdown of the other three iap genes identified did not cause a significant effect on cells or insects. These data increase our understanding of iap genes in insects and provide opportunities for developing iap1 and iap5 as targets for RNAi-based insect pest control.     

Larval RNA Interference in the Red Flour Beetle,Tribolium castaneum

The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity.In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.     

The chitin synthase involved in marine bivalve mollusk shell formation contains a myosin domain

Chitin is a key component in mollusk nacre formation. However, the enzyme complex responsible for chitin deposition in the mollusk shell remained unknown. We cloned and characterized the chitin synthase of the marine bivalve mollusk Atrina rigida. We present here the first chitin synthase sequence from invertebrates containing an unconventional myosin motor head domain. We further show that a homologous gene for chitin synthase is expressed in the shell forming tissue of larval Mytilus galloprovincialis even in early embryonic stages. The new data presented here are the first clear-cut indication for a functional role of cytoskeletal forces in the precisely controlled mineral deposition process of mollusk shell biogenesis.