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1.
Insect Mol Biol ; 29(1): 48-55, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31294881

RESUMO

Phosphoserine phosphatase (PSP) catalyses the synthesis of l-serine via the phosphorylated pathway by facilitating the dephosphorylation of phosphoserine. A cDNA encoding PSP from the silkworm Bombyx mori (bmPSP) was isolated using reverse transcription-PCR and then sequenced. The resulting clone encoded 236 amino acids with a molecular weight of 26 150, exhibiting 14-60% sequence identity with other PSPs. The recombinant PSP was overexpressed in Escherichia coli and purified. Kinetic studies showed that bmPSP possessed activity toward l-phosphoserine, and Asp20, Asp22 and Asp204 in bmPSP were found to be critical for modulating bmPSP activity. Real-time PCR analysis provided evidence that the amount of bmpsp transcript was reduced in middle silk glands of a sericin-deficient silkworm strain. These findings revealed that bmPSP may play important roles in synthesizing one-carbon donors of l-serine, which is abundant in silk, as well as other cell metabolites in B. mori.


Assuntos
Bombyx/enzimologia , Monoéster Fosfórico Hidrolases/química , Serina/biossíntese , Sequência de Aminoácidos , Animais , Bombyx/genética , Bombyx/metabolismo , Clonagem Molecular , DNA Complementar/genética , Escherichia coli , Proteínas de Insetos/biossíntese , Proteínas de Insetos/metabolismo , Larva/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Seda
2.
Elife ; 82019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31591963

RESUMO

Unrelated genes establish head-to-tail polarity in embryos of different fly species, raising the question of how they evolve this function. We show that in moth flies (Clogmia, Lutzomyia), a maternal transcript isoform of odd-paired (Zic) is localized in the anterior egg and adopted the role of anterior determinant without essential protein change. Additionally, Clogmia lost maternal germ plasm, which contributes to embryo polarity in fruit flies (Drosophila). In culicine (Culex, Aedes) and anopheline mosquitoes (Anopheles), embryo polarity rests on a previously unnamed zinc finger gene (cucoid), or pangolin (dTcf), respectively. These genes also localize an alternative transcript isoform at the anterior egg pole. Basal-branching crane flies (Nephrotoma) also enrich maternal pangolin transcript at the anterior egg pole, suggesting that pangolin functioned as ancestral axis determinant in flies. In conclusion, flies evolved an unexpected diversity of anterior determinants, and alternative transcript isoforms with distinct expression can adopt fundamentally distinct developmental roles.


Assuntos
Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/biossíntese , Isoformas de Proteínas/biossíntese , Psychodidae/embriologia , Transcrição Genética , Animais , Embrião não Mamífero , Desenvolvimento Embrionário
3.
Elife ; 82019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31478483

RESUMO

Insects are the only known animals in which sexual differentiation is controlled by sex-specific splicing. The doublesex transcription factor produces distinct male and female isoforms, which are both essential for sex-specific development. dsx splicing depends on transformer, which is also alternatively spliced such that functional Tra is only present in females. This pathway has evolved from an ancestral mechanism where dsx was independent of tra and expressed and required only in males. To reconstruct this transition, we examined three basal, hemimetabolous insect orders: Hemiptera, Phthiraptera, and Blattodea. We show that tra and dsx have distinct functions in these insects, reflecting different stages in the changeover from a transcription-based to a splicing-based mode of sexual differentiation. We propose that the canonical insect tra-dsx pathway evolved via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factor to dedicated regulator of dsx).


Assuntos
Processamento Alternativo , Baratas/fisiologia , Hemípteros/fisiologia , Proteínas de Insetos/biossíntese , Ftirápteros/fisiologia , Desenvolvimento Sexual , Fatores de Transcrição/biossíntese , Animais , Baratas/genética , Hemípteros/genética , Proteínas de Insetos/genética , Ftirápteros/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Fatores de Transcrição/genética
4.
PLoS One ; 14(6): e0217493, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31216287

RESUMO

In insects, the gustatory system has a critical function not only in selecting food and feeding behaviours but also in growth and metabolism. Gustatory receptors play an irreplaceable role in insect gustatory signalling. Trichogramma chilonis is an effective biocontrol agent against agricultural insect pests. However, the molecular mechanism of gustation in T. chilonis remains elusive. In this study, we found that T. chilonis adults had a preference for D-fructose and that D-fructose contributed to prolong longevity and improve fecundity. Then, We also isolated the full-length cDNA encoding candidate gustatory receptor (TchiGR43a) based on the transcriptome data of T. chilonis, and observed that the candidate gustatory receptor gene was expressed from the larval to adult stages. The expression levels of TchiGR43a were similar between female and male. A Xenopus oocyte expression system and two-electrode voltage-clamp recording further verified the function analysis of TchiGR43a. Electrophysiological results showed that TchiGR43a was exclusively tuned to D-fructose. By the studies of behaviour, molecular biology and electrophysiology in T. chilonis, our results lay a basic fundation of further study on the molecular mechanisms of gustatory reception and provide theoretical basis for the nutritional requirement of T. chilonis in biocontrol.


Assuntos
Comportamento Alimentar/fisiologia , Frutose/metabolismo , Himenópteros/metabolismo , Proteínas de Insetos/biossíntese , Óvulo/metabolismo , Receptores de Superfície Celular/biossíntese , Animais , Regulação da Expressão Gênica/fisiologia , Himenópteros/genética , Proteínas de Insetos/genética , Receptores de Superfície Celular/genética
5.
Insect Biochem Mol Biol ; 111: 103177, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31228516

RESUMO

In the evolutionarily-derived termite subfamily Nasutitermitinae (family Termitidae), soldiers defend their nestmates by discharging polycyclic diterpenes from a head projection called the "nasus." The diterpenes are synthesised in the frontal gland from the precursor geranylgeranyl diphosphate (GGPP), which is generally used for post-translational modification of proteins in animals. In this study, we constructed a comprehensive gene catalogue to search for genes involved in the diterpene biosynthesis by assembling RNA sequencing reads of Nasutitermes takasagoensis, identifying eight gene copies for GGPP synthase (GGPPS). The number of gene copies is much larger in contrast to other related insects. Gene cloning by reverse transcription-PCR and rapid amplification of cDNA ends confirmed that seven GGPPS genes (NtGGPPS1 to NtGGPPS7) have highly variable untranslated regions. Molecular phylogenetic analysis showed that the NtGGPPS7 gene was grouped with homologs obtained from ancestral termites that have only a single copy of the gene, and the NtGGPPS6 gene was grouped with homologs obtained from a basal lineage of termitids, in which soldiers do not synthesise diterpenes. As the sister group to this clade, furthermore, a monophyletic clade included all the other NtGGPPS genes (NtGGPPS1 to NtGGPPS5). Expression analyses revealed that NtGGPPS7 gene was expressed in all the examined castes and tissues, whereas all the other genes were expressed only in the soldier head. These results suggest that gene duplication followed by subfunctionalisation of the GGPPS genes might have accompanied the evolution of chemical defence in the nasute termite lineage.


Assuntos
Farnesiltranstransferase/metabolismo , Proteínas de Insetos/metabolismo , Isópteros/enzimologia , Isópteros/genética , Animais , Farnesiltranstransferase/biossíntese , Farnesiltranstransferase/genética , Regulação Enzimológica da Expressão Gênica , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Filogenia , Análise de Sequência de RNA
6.
PLoS One ; 14(5): e0216800, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31107891

RESUMO

The reproductive ground plan hypothesis proposes that gene networks regulating foraging behavior and reproductive female physiology in social insects emerged from ancestral gene and endocrine factor networks. Expression of storage proteins such as vitellogenins and hexamerins is an example of this co-option. Hexamerins, through their role modulating juvenile hormone availability, are involved in caste determination in termites. The genome of the fire ant (Solenopsis invicta) encodes four hexamerin genes, hexamerin-like (LOC105192919, hereafter called hexamerin 1), hexamerin (LOC105204474, hereafter called hexamerin 2), arylphorin subunit alpha-like, and arylphorin subunit beta. In this study, a phylogenetic analysis of the S. invicta hexamerins determined that each predicted protein clustered with one of the orthologous Apis mellifera hexamerins. Gene expression analyses by RT-qPCR revealed differential expression of the hexamerins between queens and workers, and between specific task-allocated workers (nurses and foragers). Queens and nurses had significantly higher expression of all genes when compared to foragers. Hexamerin 1 was expressed at higher levels in queens, while hexamerin 2 and arylphorin subunit beta were expressed at significantly higher levels in nurses. Arylphorin subunit alpha-like showed no significant difference in expression between virgin queens and nurses. Additionally, we analyzed the relationship between the expression of hexamerin genes and S-hydroprene, a juvenile hormone analog. Significant changes in hexamerin expression were recorded in nurses, virgin queens, and foragers 12 h after application of the analog. Hexamerin 1 and arylphorin subunit alpha-like expression were significantly lower after analog application in virgin queens. In foragers, hexamerin 2 and arylphorin subunit beta were significantly lower after analog application, while in nurses expression of all genes were significantly lower after analog application. Our results suggest that in S. invicta hexamerin genes could be associated with reproductive division of labor and task-allocation of workers.


Assuntos
Formigas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Insetos/biossíntese , Hormônios Juvenis/farmacologia , Vitelogeninas/biossíntese , Animais , Formigas/genética , Feminino , Proteínas de Insetos/genética , Vitelogeninas/genética
7.
Arch Insect Biochem Physiol ; 101(2): e21553, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31004387

RESUMO

In this study, we identified and characterized a phosphoserine aminotransferase (bmPSAT) from Bombyx mori (B. mori) that is responsible for l-serine biosynthesis. A complementary DNA that encodes bmPSAT was cloned by reverse transcriptase polymerase reaction and sequenced. The presumed amino acid sequence revealed 47-87% identity with known PSATs from insects, humans, plants, and bacteria. Through phylogenetic analysis, we found that bmPSAT is evolutionary related to insect PSATs. Recombinant bmPSAT was produced in Escherichia coli by using a cold-shock promotor and purified to homogeneity. This enzyme utilizes phosphohydroxypyruvate and glutamate for transamination. bmPSAT messenger RNA (mRNA) was expressed at higher levels in several tissues of standard strain silkworm including the silk gland, whereas a sericin-deficient silkworm strain exhibited a diminished expression of bmPSAT mRNA in the silk gland. These findings indicate that bmPSAT may play an important role in synthesizing and supplying l-serine in the larva of B. mori.


Assuntos
Bombyx/enzimologia , Serina/biossíntese , Transaminases/química , Animais , Bombyx/genética , Bombyx/metabolismo , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/biossíntese , Proteínas de Insetos/metabolismo , Larva/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Transaminases/genética , Transaminases/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-31022468

RESUMO

Juvenile hormones (JH) regulate wide-ranging physiological and developmental processes in insects. However, molecular mechanisms underlying JH signaling remain to be determined. Vitellogenin (Vg) is primarily an egg-yolk protein, but recently proposed to serve many functions in insects. In the female American cockroach (Periplaneta americana), vitellogenin (Vg) genes are activated by JH III and suppressed by 20-hydroxyecdysone (20E) via cis-regulatory elements in a dose-dependent manner. In the present study, the upstream promoter region (935 bp) of Vg1 was cloned to elucidate the action of these hormones. A luciferase reporter assay identified an 81 bp region in the promoter region of Vg1 (-120 to -39 bp) that we found to be critical for JH III activation and 20E suppression. This 81 bp region contains a direct repeat separated by a 2-nucleotide spacer-designated Vg1HRE- that is similar to the Drosophila ecdysone response element direct repeat 4. Moreover, nuclear proteins isolated from nymphs, males, females, and Sf9 cells successfully bound to Vg1HRE, while binding was outcompeted by a 100-fold excess of cold probe or dephosphorylated nuclear protein extracts. In addition, binding was outcompeted by other ecdysone and JH response elements with similar half-site sequences (direct repeats) but to varying extents. Ultimately, we postulate that JH III indirectly activates Vg expression by interfering with or inhibiting the phosphorylation of nuclear proteins bound to Vg1HRE. Involvement of JH III in both induction of Vg1 and control of nuclear proteins binding to Vg1HRE suggest the latter to play an important role in JH signaling.


Assuntos
Regulação da Expressão Gênica , Proteínas de Insetos , Periplaneta , Sequências Repetitivas de Ácido Nucleico , Elementos de Resposta , Vitelogeninas , Animais , Feminino , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Hormônios Juvenis/genética , Hormônios Juvenis/metabolismo , Masculino , Ninfa , Periplaneta/genética , Periplaneta/metabolismo , Transdução de Sinais/genética , Vitelogeninas/biossíntese , Vitelogeninas/genética
9.
Dev Biol ; 450(1): 34-46, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30851270

RESUMO

Many organisms both undergo dramatic morphological changes during post-embryonic development and also regenerate lost structures, but the roles of epigenetic regulators in such processes are only beginning to be understood. In the present study, the functions of two histone modifiers were examined during metamorphosis and larval limb regeneration in the red flour beetle Tribolium castaneum. Polycomb (Pc), a member of Polycomb repressive complex 1 (PRC1), and Enhancer of zeste (E(z)), a member of Polycomb repressive complex 2 (PRC2), were silenced in larvae using RNA interference. In the absence of Pc, the head appendages of adults transformed into a leg-like morphology, and the legs and wings assumed a metathoracic identity, indicating that Pc acts to specify proper segmental identity. Similarly, silencing of E(z) led to homeotic transformation of legs and wings. Additional defects were also observed in limb patterning as well as eye morphogenesis, indicating that PcG proteins play critical roles in imaginal precursor cells. In addition, larval legs and antennae failed to re-differentiate when either Pc or E(z) was knocked down, indicating that histone modification is necessary for proper blastema growth and differentiation. These findings indicate that PcG proteins play extensive roles in postembryonic plasticity of imaginal precursor cells.


Assuntos
Membro Posterior/fisiologia , Proteínas de Insetos/biossíntese , Morfogênese , Complexo Repressor Polycomb 1/biossíntese , Complexo Repressor Polycomb 2/biossíntese , Regeneração , Tribolium/metabolismo , Animais , Proteínas de Insetos/genética , Larva/genética , Larva/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Tribolium/genética
10.
Sci Rep ; 9(1): 43, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631085

RESUMO

The mosquito Aedes aegypti is the primary vector for the fastest growing infectious disease in the world, dengue fever. Disease transmission heavily relies on the ability of female mosquitoes to locate their human hosts. Additionally, males may be found in close proximity to humans, where they can find mates. Host seeking behaviour of both sexes is dependent on adult sexual maturation. Identifying the molecular basis for the onset of host seeking may help to determine targets for future vector control. In this study, we investigate modulation of the host seeking behaviour and the transcript abundance of the main chemoreceptor families between sexes and across ages in newly-emerged mosquitoes. Attraction to human odour was assessed using a Y-tube olfactometer, demonstrating that both males and females display age-dependent regulation of host seeking. The largest increase in transcript abundance was identified for select chemosensory genes in the antennae of young adult Ae. aegypti mosquitoes and reflects the increase in attraction to human odour observed between 1 and 3 day(s) post-emergence in both males and females. Future functional characterisation of the identified differentially abundant genes may provide targets for the development of novel control strategies against vector borne diseases.


Assuntos
Aedes/fisiologia , Antenas de Artrópodes/fisiologia , Regulação da Expressão Gênica , Comportamento de Busca por Hospedeiro , Proteínas de Insetos/biossíntese , Mosquitos Vetores/fisiologia , Fatores Etários , Animais , Proteínas de Insetos/genética , Fatores Sexuais
11.
Artigo em Inglês | MEDLINE | ID: mdl-30641133

RESUMO

Arginine kinase (AK) plays a critical role in insect energy metabolism and has been proposed to be a potential insecticide target for commercial exploitation. In this study, the full length cDNA encoding a typical group 1 insect AK (FoAK) was isolated from the western flower thrips (WFT), Frankliniella occidentalis (Pergande). Sequence analysis showed that FoAK contains an open reading frame of 1068 nucleotides, which encods a protein of 355 amino acid residues including the signature sequence pattern of ATP-guanidino kinases. Genomic structure analysis showed that the coding region of FoAK contains five exons connected by four introns. RT-qPCR analysis revealed that the mRNA expression of FoAK was developmentally regulated with the lowest level in prepupal stage. Enzymatic activity analysis of the recombinant enzymes expressed in Escherichia coli showed that FoAK is highly stereo specific for L-arginine versus D-arginine and the apparent Michaelis constant for L-arginine (KmArg) is comparable to that of AKs from a variety of species. This research should enable further investigation of the function as well as in vitro screening for inhibitors of FoAK.


Assuntos
Arginina Quinase , Expressão Gênica , Proteínas de Insetos , Espécies Introduzidas , Tisanópteros , Animais , Arginina Quinase/biossíntese , Arginina Quinase/química , Arginina Quinase/genética , Proteínas de Insetos/biossíntese , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Tisanópteros/enzimologia , Tisanópteros/genética
12.
Biotechnol Appl Biochem ; 66(2): 209-215, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30471160

RESUMO

In this study, various constructs and hosts were used to produce high levels of cecropin B2 (cecB2). To mitigate cecB2's toxic inhibition of host cells, various cecB2 constructs were built. Results showed that the combination of a chitin-binding domain and an intein self-cleavage motif in front of cecropin B2, without a His-tag, was best for cecB2 expression. E. coli ER2566 was the best host, and 2YT was the best medium for cultivation. Under these conditions, a cecB2 yield of 98.2 mg/L could be obtained after purification. The purified cecB2 expressed a wide antimicrobial effect on most Gram-negative strains, including multidrug-resistant Acinetobactor baumannii and Staphylococcus aureus. This study provides a systematic approach to the efficient production of the antimicrobial peptide (AMP) cecB2 via the recombinant E. coli process, which is expected to be an efficient way for the production of other AMPs.


Assuntos
Acinetobacter baumannii/crescimento & desenvolvimento , Peptídeos Catiônicos Antimicrobianos , Escherichia coli , Proteínas de Insetos , Proteínas Recombinantes de Fusão , Staphylococcus aureus/crescimento & desenvolvimento , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia
13.
Arch Biochem Biophys ; 661: 107-116, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30452894

RESUMO

The arylalkylamine N-acyltransferases (AANATs) are enzymes that catalyze the acyl-CoA-dependent formation of N-acylarylalkylamides: acyl-CoA + arylalkylamine → N-acylarylalkylamides + CoA-SH. Herein, we describe our study of a previously uncharacterized AANAT from Bombyx mori: Bm-iAANAT3. Bm-iAANAT3 catalyzes the direct formation of N-acylarylalkylamides and accepts a broad range of short-chain acyl-CoA thioesters and amines as substrates. Acyl-CoA thioesters possessing an acyl chain length >10 carbon atoms are not substrates for Bm-iAANAT3. We report that Bm-iAANAT3 is a "versatile generalist", most likely, functioning in amine acetylation - a reaction in amine inactivation/excretion, cuticle sclerotization, and melanism. We propose a kinetic and chemical mechanism for Bm-iAANAT3 that is consistent with our steady-state kinetic analysis, dead-end inhibition studies, determination of the pH-rate profiles, and site-directed mutagenesis of a catalytically important amino acid in Bm-iAANAT3. These mechanistic studies of Bm-iAANAT3 will foster the development of novel compounds targeted against this enzyme and other insect AANATs for the control of insect pests.


Assuntos
Arilalquilamina N-Acetiltransferase/química , Bombyx , Expressão Gênica , Proteínas de Insetos/química , Acetilação , Animais , Arilalquilamina N-Acetiltransferase/biossíntese , Arilalquilamina N-Acetiltransferase/genética , Bombyx/enzimologia , Bombyx/genética , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato
14.
Insect Biochem Mol Biol ; 104: 11-19, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30423422

RESUMO

With the advent of next-generation sequencing, it is now possible to rapidly identify the entire repertoire of olfactory genes likely to be involved in chemical communication of an insect species. It remains, however, a challenge to identify olfactory proteins, such as odorant receptors and odorant-binding proteins (OBPs), vis-à-vis the odorants they detect. It has been reported that exposing the olfactory system to a physiologically relevant odorant alters the transcript levels of odorant receptor(s) involved in the detection of the tested odorant. We applied this paradigm in an attempt to identify putative OBPs from the scarab beetle Holotrichia oblita involved in the reception of plant-derived kairomones. Twenty-nine OBP genes were identified in the H. oblita transcriptome, 20 of which were enriched in antennae compared with nonolfactory tissues. Of these, 2 OBP genes, HoblOBP13 and HoblOBP9, were upregulated upon exposure to one of the female attractants (E)-2-hexenol and phenethyl alcohol; none of the OBP transcripts changed upon exposure to methyl anthranilate, which does not attract H. oblita females. Binding assays showed that HoblOBP13 and HoblOBP9 have high affinity for (E)-2-hexenol and phenethyl alcohol, respectively. RNAi treatment showed that transcripts of both HoblOBP13 and HoblOBP9 declined in a time-course manner 24-72 h postinjection. OBP-dsRNA-treated female beetles showed significantly lower attraction to (E)-2-hexenol and phenethyl alcohol than did water-injected beetles and those treated with GFP-dsRNA. We, therefore, concluded that HoblOBP13 and HoblOBP9 are essential for H. oblita reception of the plant-derived kairomones (E)-2-hexenol and phenethyl alcohol.


Assuntos
Antenas de Artrópodes/metabolismo , Besouros , Regulação da Expressão Gênica/fisiologia , Proteínas de Insetos , Receptores Odorantes , Transcriptoma/fisiologia , Animais , Besouros/genética , Besouros/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Receptores Odorantes/biossíntese , Receptores Odorantes/genética
15.
Insect Biochem Mol Biol ; 104: 65-72, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30503224

RESUMO

Insect metamorphosis produces reproductive adults and is commonly accompanied with the direct or indirect development of wings. In some winged insects, the imago is altered by life history changes. For instance, in scale insects and mealybugs, reproductive females retain juvenile features and are wingless. The transcription factor E93 triggers metamorphosis and plays in concert with the juvenile hormone pathway to guarantee the successful transition from juvenile to adult. We previously provided evidence of an atypical down-regulation of the juvenile hormone pathway during female development in the Japanese mealybug. Here, we further investigate how E93 is involved in the production of neotenic wingless females, by identifying its isoforms, assessing their expression patterns and evaluating the effect of exogenous juvenile hormone mimic treatment on E93. This study identifies three E93 isoforms on the 5' end, based on Japanese mealybug cDNA and shows that female development occurs with the near absence of E93 transcripts, as opposed to male metamorphosis. Additionally, while male development is typically affected by exogenous juvenile hormone mimic treatments, females seem to remain insensitive to the treatment, and up-regulation of the juvenile hormone signaling is not observed. Furthermore, juvenile hormone mimic treatment on female nymphs did not have an obvious effect on E93 transcription, while treatment on male prepupae resulted in depleted E93 transcripts. In this study, we emphasize the importance in examining atypical cases of metamorphosis as complementary systems to provide a better understanding on the molecular mechanisms underlying insect metamorphosis. For instance, the factors regulating the expression of E93 are largely unclear. Investigating the regulatory mechanism of E93 transcription could provide clues towards identifying the factors that induce or suppress E93 transcription, in turn triggering male adult development or female neoteny.


Assuntos
Hemípteros/embriologia , Proteínas de Insetos/biossíntese , Hormônios Juvenis/metabolismo , Metamorfose Biológica/fisiologia , Caracteres Sexuais , Transdução de Sinais/fisiologia , Animais , Feminino , Hemípteros/genética , Proteínas de Insetos/genética , Hormônios Juvenis/genética , Masculino , Pupa
16.
Insect Biochem Mol Biol ; 104: 82-90, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30578824

RESUMO

Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diseases of public health significance. The discovery of new metabolic targets is crucial for improving mosquito control strategies. We recently demonstrated that glucose oxidation supports ammonia detoxification in A. aegypti. Pyruvate kinase (PK, EC 2.7.1.40) catalyzes the last step of the glycolytic pathway. In most organisms, one or more allosteric effectors control PK activity. However, the kinetic properties and structure of PK in mosquitoes have not been previously reported. In this study, two alternatively spliced mRNA variants (AaPK1 and AaPK2) that code for PKs were identified in the A. aegypti genome. The AaPK1 mRNA variant, which encodes a 529 amino acid protein with an estimated molecular weight of ∼57 kDa, was cloned. The protein was expressed in Escherichia coli and purified. The AaPK1 kinetic properties were identified. The recombinant protein was also crystallized and its 3D structure determined. We found that alanine, glutamine, proline, serine and fructose-1-phosphate displayed a classic allosteric activation on AaPK1. Ribulose-5-phosphate acted as an allosteric inhibitor of AaPK1 but its inhibitory effect was reversed by alanine, glutamine, proline and serine. Additionally, the allosteric activation of AaPK1 by amino acids was weakened by fructose-1,6-bisphosphate, whereas the allosteric activation of AaPK1 by alanine and serine was diminished by glucose-6-phosphate. The AaPK1 structure shows the presence of fructose-1,6-bisphosphate in the allosteric site. Together, our results reveal that specific amino acids and phosphorylated sugars tightly regulate conformational dynamics and catalytic changes of AaPK1. The distinctive AaPK1 allosteric properties support a complex role for this enzyme within mosquito metabolism.


Assuntos
Aedes/enzimologia , Frutosedifosfatos/química , Glucose-6-Fosfato/química , Proteínas de Insetos/química , Piruvato Quinase/química , Aedes/genética , Regulação Alostérica/fisiologia , Processamento Alternativo/fisiologia , Animais , Feminino , Frutosedifosfatos/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose-6-Fosfato/metabolismo , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Cinética , Domínios Proteicos , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
Appl Microbiol Biotechnol ; 103(3): 1417-1427, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30554389

RESUMO

Periplanetasin-2 is a 15-mer antimicrobial peptide (AMP), derived from the American cockroach Periplaneta americana. This novel AMP exhibits potent antibacterial effect against several pathogenic bacteria including Escherichia coli. Distinct from the targeting cell membrane, which is the general antibacterial mechanism of AMP, periplanetasin-2 exerts its antibacterial activity via apoptosis-like death, which is physiologically and mechanistically similar to eukaryotic apoptosis. E. coli cells treated with periplanetasin-2 showed features of apoptosis in a concentration-dependent manner, such as membrane depolarization, DNA fragmentation, caspase-like protein activation, and phosphatidylserine externalization. These physiological changes were attenuated by pretreatment with the reactive oxygen species (ROS) scavenger, which demonstrates that periplanetasin-2 induced apoptosis-like death in E. coli by generating ROS. In addition, periplantasin-2-induced apoptotic death was affected by SOS response components. In the absence of RecA, an essential protein for SOS response, apoptosis did not occur and the antibacterial activity of periplanetasin-2 was decreased. In contrast, deletion of the SOS gene dinF caused higher ROS accumulation and apoptotic features were detected. Collectively, these results indicate that the antibacterial mechanism of periplanetasin-2 is ROS-induced apoptosis-like death, which requires RecA for proceeding it, and the role of DinF is assumed to contribute to the ROS defense SOS response.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/genética , Escherichia coli/fisiologia , Proteínas de Insetos/farmacologia , /genética , Antibacterianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Fragmentação do DNA/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Recombinases Rec A/genética , /efeitos dos fármacos
18.
Insect Biochem Mol Biol ; 104: 58-64, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30550974

RESUMO

Glutamate-gated chloride channels (GluCls) are found only in invertebrates and mediate fast inhibitory neurotransmission. The structural and functional diversity of GluCls are produced through assembly of multiple subunits and via posttranscriptional alternations. Alternative splicing is the most common way to achieve this in insect GluCls and splicing occurs primarily at exons 3 and 9. As expression pattern and pharmacological properties of exon 9 alternative splices in invertebrate GluCls remain poorly understood, the cDNAs encoding three alternative splice variants (9a, 9b and 9c) of the PxGluCl gene from the diamondback moth Plutella xylostella were constructed and their pharmacological characterizations were examined using electrophysiological studies. Alternative splicing of exon 9 had little to no impact on PxGluCl sensitivity towards the agonist glutamate when subunits were singly or co-expressed in Xenopus oocytes. In contrast, the allosteric modulator abamectin and the chloride channel blocker fipronil had differing effects on PxGluCl splice variants. PxGluCl9c channels were more resistant to abamectin and PxGluCl9b channels were more sensitive to fipronil than other homomeric channels. In addition, heteromeric channels containing different splice variants showed similar sensitivity to abamectin (except for 9c) and reduced sensitivity to fipronil than homomeric channels. These findings suggest that functionally indistinguishable but pharmacologically distinct GluCls could be formed in P. xylostella and that the upregulated constitutive expression of the specific variants may contribute to the evolution of insecticide resistance in P. xylostella and other arthropods.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Canais de Cloreto , Éxons , Proteínas de Insetos , Resistência a Inseticidas , Ivermectina/análogos & derivados , Pirazóis/farmacologia , Animais , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/biossíntese , Canais de Cloreto/genética , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Resistência a Inseticidas/efeitos dos fármacos , Resistência a Inseticidas/genética , Ivermectina/farmacologia , Mariposas/genética , Mariposas/metabolismo
19.
PLoS One ; 13(12): e0202829, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30557388

RESUMO

Bactrocera cucurbitae (melon flies) are prominent invasive pests in southern China. To screen for a stable reference gene in melon flies suitable for comparing tissue samples subjected to different conditions in four categories (temperature, insect stage, days of age and gender), the expression of 12 candidate reference genes under different treatment conditions was analyzed by real-time fluorescent quantitative PCR. The results obtained from a comprehensive analysis with geNorm, NormFinder, BestKeeper and RefFinder software showed that the most stable reference gene was RPL60, and the least stable reference gene was actin-5. We used a heat shock protein gene (HSP-90) to verify the results, and the conclusion was consistent. When the reference gene RPL60 was used as the reference gene, the relative expression of HSP-90 was essentially constant with the prolongation of treatment time. When actin-5 was used, HSP-90 expression changed markedly with treatment time. The results of this study can be used for further research on gene expression inBactrocera cucurbitae.


Assuntos
Actinas/genética , Regulação da Expressão Gênica , Genes de Insetos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Insetos/genética , Reação em Cadeia da Polimerase em Tempo Real , Tephritidae/genética , Actinas/biossíntese , Animais , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Insetos/biossíntese , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Tephritidae/metabolismo
20.
Biomed Res Int ; 2018: 5806179, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30402485

RESUMO

Introduction: In Madagascar, malaria control relies on the countrywide use of long lasting insecticide treated bed nets (LLINs) and on indoor residual spraying (IRS) in the central highland area as well as a small area on the eastern coast. We tested insecticide resistance mechanisms of Anopheles funestus from Tsararano, a malaria endemic village in the coastal health district of Marovoay. Methods: Insecticide susceptibility bioassays were done in July 2017 on first-generation Anopheles funestus (F1) to assess (i) the susceptibility to permethrin (0.05%), deltamethrin (0.05%), DDT (4%), malathion (5%), fenitrothion (1%), and bendiocarb (0.1%); (ii) the effect of preexposure to the piperonyl butoxide (PBO) synergist; and (iii) the enzymatic activities of cytochrome P450, esterases, and glutathione S-transferases (GST). Results: Our results demonstrated that An. funestus was phenotypically resistant to pyrethroids and bendiocarb, with a mortality rate (MR) of 33.6% (95%CI: 24.5-43.7%) and 86% (95%CI: 77.6-92.1%), respectively. In contrast, An. funestus were 100% susceptible to DDT and organophosphates (malathion and fenitrothion). Preexposure of An. funestus to PBO synergist significantly restored the susceptibility to bendiocarb (MR=100%) and increased the MR in the pyrethroid group, from 96% (95%CI: 90.0-98.9%) to 100% for deltamethrin and permethrin, respectively (χ 2 = 43, df = 3, P< 0.0001). Enzymatic activities of cytochrome P450 and α-esterases were significantly elevated among An. funestus compared with the IPM reference strain (Mann-Whitney U= 30, P<0.0001; U = 145.5, P <0.0001, respectively). No significant differences of ß-esterases activities compared to the IPM reference strain were observed (Mann-Whitney U = 392.5, P = 0.08). Conclusion: In Tsararano, despite the absence of an IRS programme, there is evidence of high levels of insecticide resistance to pyrethroids and bendiocarb in An.


Assuntos
Anopheles/crescimento & desenvolvimento , Resistência a Medicamentos/efeitos dos fármacos , Inseticidas/farmacologia , Fenilcarbamatos/farmacologia , Piretrinas/farmacologia , Animais , Anopheles/enzimologia , Proteínas de Insetos/biossíntese , Madagáscar
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