RESUMO
It is difficult to produce chitin oligosaccharides by hydrolyzing untreated natural chitinous waste directly. In this study, two fungi Talaromyces allahabadensis Hi-4 and Talaromyces funiculosus Hi-5 from rotten black soldier fly were isolated and identified through multigene phylogenetic and morphological analyses. The chitinolytic enzymes were produced by solid state fermentation, and the growth conditions were optimized by combining single-factor and central composite design. The best carbon sources were powder of molting of mealworms (MMP) and there was no need for additional nitrogen sources in two fungi, then the maximum chitinolytic enzyme production of 46.80 ± 3.30 (Hi-4) and 55.07 ± 2.48 (Hi-5) U/gds were achieved after analyzing the 3D response surface plots. Pure chitin (colloidal chitin) and natural chitinous substrates (represented by MMP) were used to optimize degradation abilities by crude enzymes obtained from the two fungi. The optimum temperature for hydrolyzing MMP (40 °C both in two fungi) were lower and closer to room temperature than colloidal chitin (55 °C for Hi-4 and 45 °C for Hi-5). Then colloidal chitin, MMP and the powder of shrimp shells (SSP) were used for analyzing the products after 5-day degradation. The amounts of chitin oligosaccharides from SSP and MMP were about 1/6 (Hi-4), 1/17 (Hi-5) and 1/8 (Hi-4), 1/10 (Hi-5), respectively, in comparison to colloidal chitin. The main components of the products were GlcNAc for colloidal chitin, (GlcNAc)2 for MMP, and oligosaccharides with higher degree of polymerization (4-6) were obtained when hydrolyzing SSP, which is significant for applications in medicine and health products.
Assuntos
Quitinases , Dípteros , Talaromyces , Animais , Quitina/metabolismo , Fermentação , Filogenia , Pós , Talaromyces/metabolismo , Oligossacarídeos , Quitinases/genética , Insetos , Dípteros/metabolismoRESUMO
The black soldier fly larvae (BSFL), Hermetia illucens (Linnaeus), has been largely utilized for animal feed. Due to its interesting composition, BSFL has great potential to be further implemented in the human diet. Herein we compared the flour and protein extract composition based on their moisture, ash, amino acids, mineral, and protein content. To have wide knowledge on protein profile and behavior, SDS-page electrophoresis, Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) were used to give information about protein structure and thermal stability, respectively. The flour and protein extract contained respectively 37.3% and 61.1% of protein. DSC graph reported a glass transition temperature around 30 °C, recognizable by a shift in the curve, and an endothermic peak for solid melting at around 200 °C. FTIR analysis showed the main amide bands (A, B, I, II, III) for the flour and protein extract. The foam properties of BSFL protein extract were explored under different temperatures treatment, and the best foam stability was reached at 85 °C with 15 min of treatment. The data highlight the promising techno-functional properties of BSFL protein extract, and that the nutritional composition might be suitable for further use of BSFL as food fortification system.
Assuntos
Dípteros/metabolismo , Insetos Comestíveis/metabolismo , Proteínas de Insetos/química , Sequência de Aminoácidos , Animais , Coloides , Dípteros/embriologia , Insetos Comestíveis/embriologia , Manipulação de Alimentos , Alimentos Fortificados , Temperatura Alta , Proteínas de Insetos/isolamento & purificação , Larva/metabolismo , Valor Nutritivo , Estabilidade ProteicaRESUMO
The evolution of obligate ectoparasitism in blowflies (Diptera: Calliphoridae) has intrigued scientists for over a century, and surprisingly, the genetics underlying this lifestyle remain largely unknown. Blowflies use odors to locate food and oviposition sites; therefore, olfaction might have played a central role in niche specialization within the group. In insects, the coreceptor Orco is a required partner for all odorant receptors (ORs), a major gene family involved in olfactory-evoked behaviors. Hence, we characterized the Orco gene in the New World screwworm, Cochliomyia hominivorax, a blowfly that is an obligate ectoparasite of warm-blooded animals. In contrast, most of the closely related blowflies are scavengers that lay their eggs on dead animals. We show that the screwworm Orco orthologue (ChomOrco) is highly conserved within Diptera, showing signals of strong purifying selection. Expression of ChomOrco is broadly detectable in chemosensory appendages, and is related to morphological, developmental, and behavioral aspects of the screwworm biology. We used CRISPR/Cas9 to disrupt ChomOrco and evaluate the consequences of losing the OR function on screwworm behavior. In two-choice assays, Orco mutants displayed an impaired response to floral-like and animal host-associated odors, suggesting that OR-mediated olfaction is involved in foraging and host-seeking behaviors in C. hominivorax. These results broaden our understanding of the chemoreception basis of niche occupancy by blowflies.
Assuntos
Dípteros/fisiologia , Comportamento Alimentar , Comportamento de Busca por Hospedeiro , Proteínas de Insetos/metabolismo , Receptores Odorantes/metabolismo , Animais , Dípteros/metabolismo , Proteínas de Insetos/genética , Mutação , Filogenia , Receptores Odorantes/genética , OlfatoRESUMO
Micronucleoli are among the structures composing the peculiar scenario of the nucleolus in salivary gland nuclei of dipterans representative of Sciaridae. Micronucleolar bodies contain ribosomal DNA and RNA, are transcriptionally active and may appear free in the nucleoplasm or associated with specific chromosome regions in salivary gland nuclei. This report deals with an extreme case of nucleolar fragmentation/dispersion detected in the salivary gland of Schwenkfeldina sp. Such a phenomenon in this species was found to be restricted to cell types undergoing polyteny and seems to be differentially controlled according to the cell type. Furthermore, transcriptional activity was detected in virtually all the micronucleolar bodies generated in the salivary gland. The relative proportion of the rDNA in polytene and diploid tissues showed that rDNA under-replication did not occur in polytene nuclei suggesting that the nucleolar and concomitant rDNA dispersion in Schwenkfeldina sp. may reflect a previously hypothesised process in order to counterbalance the rDNA loss due to the under-replication. The chromosomal distribution of epigenetic markers for the heterochromatin agreed with early cytological observations in this species suggesting that heterochromatin is spread throughout the chromosome length of Schwenkfeldina sp. A comparison made with results from another sciarid species argues for a role played by the heterochromatin in the establishment of the rDNA topology in polytene nuclei of Sciaridae.
Assuntos
Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Dípteros/genética , Glândulas Salivares/citologia , Animais , Fragmentação do DNA , Replicação do DNA , DNA Ribossômico/metabolismo , Dípteros/metabolismo , Heterocromatina/metabolismo , Cromossomos Politênicos/metabolismo , RNA Ribossômico/metabolismo , Transcrição GênicaRESUMO
Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.
Assuntos
Dípteros/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Luciferases/metabolismo , Animais , Cromatografia por Troca Iônica , Dípteros/enzimologia , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/isolamento & purificação , Perfilação da Expressão Gênica , Luciferases/química , Luciferases/isolamento & purificação , Medições Luminescentes , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Espectrometria de FluorescênciaRESUMO
One of the most important contributions of forensic entomology is to assist criminal expertise to determine the postmortem interval, which depends on the duration of the immature stages of insects of forensic interest. On the other hand, the time of development of the different stages varies according to the species; therefore, its identification is essential. Currently, few studies have investigated the use of cuticular hydrocarbons, and none regarding fatty acids, as complementary taxonomic tools to expedite species identification. Therefore, we evaluated whether cuticular hydrocarbons together with fatty acids of eggs of flies of the family Calliphoridae, main group of forensic interest, can be used to distinguish species. The analyses were performed by chromatographic techniques. The results show that there are significant differences between the composition of cuticular hydrocarbons and fatty acids between species and, therefore, they can be used to provide a complementary taxonomic tool to expedite the forensic expertise.
Assuntos
Dípteros/metabolismo , Ácidos Graxos/metabolismo , Hidrocarbonetos/metabolismo , Óvulo/metabolismo , Escamas de Animais/metabolismo , Animais , Cromatografia , Análise Discriminante , Entomologia/métodos , Ciências Forenses , Especificidade da EspécieRESUMO
Antibiotic resistance is at dangerous levels and increasing worldwide. The search for new antimicrobial drugs to counteract this problem is a priority for health institutions and organizations, both globally and in individual countries. Sarconesiopsis magellanica blowfly larval excretions and secretions (ES) are an important source for isolating antimicrobial peptides (AMPs). This study aims to identify and characterize a new S. magellanica AMP. RP-HPLC was used to fractionate ES, using C18 columns, and their antimicrobial activity was evaluated. The peptide sequence of the fraction collected at 43.7 min was determined by mass spectrometry (MS). Fluorescence and electronic microscopy were used to evaluate the mechanism of action. Toxicity was tested on HeLa cells and human erythrocytes; physicochemical properties were evaluated. The molecule in the ES was characterized as sarconesin II and it showed activity against Gram-negative (Escherichia coli MG1655, Pseudomonas aeruginosa ATCC 27853, P. aeruginosa PA14) and Gram-positive (Staphylococcus aureus ATCC 29213, Micrococcus luteus A270) bacteria. The lowest minimum inhibitory concentration obtained was 1.9 µM for M. luteus A270; the AMP had no toxicity in any cells tested here and its action in bacterial membrane and DNA was confirmed. Sarconesin II was documented as a conserved domain of the ATP synthase protein belonging to the Fli-1 superfamily. The data reported here indicated that peptides could be alternative therapeutic candidates for use in infections against Gram-negative and Gram-positive bacteria and eventually as a new resource of compounds for combating multidrug-resistant bacteria.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dípteros/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Bactérias/efeitos dos fármacos , Fenômenos Químicos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The insulin pathway is an anabolic pathway that controls, amongst other things, glucose homeostasis. It is an evolutionarily conserved pathway. Disruptions in insulin pathway functions can lead to diabetic states. Diabetes, a very common occurrence in modern life, afflicts a significant portion of the population of developed and developing countries worldwide. Yet, few studies have addressed the evolution of diabetic states on a long-term basis. Here, we cultured three different insulin pathway signaling compromised flies (heteroallelic mutant combinations, akin to diabetes mellitus type II) and wild type control flies, for the extent of one generation in different isocaloric diets fed at libitum, with or without extra methionine added. All fly stocks have a homogenized genetic background. We measured weight, total lipid, and carbohydrate content of adults at two different time points, and survival of adults reared in some of the different diets. Results show that, despite the fact that all diet regimes allow survival of at least a fraction of flies to adulthood, life histories are significantly different. Higher protein content diets promote better survival compared to higher percentage lipid and carbohydrate diets, and added methionine promotes survival in moderately reduced protein content diets. In mutants, survival is significantly reduced, and added methionine generally has an effect, albeit a more modest one. Our results highlight the value of higher percentage protein diets, and differences in effects in "healthy" versus "diabetic" states. They also show that added methionine, proposed as a "sensor" for protein content in food for flies, leads to differential effects depending on the adequacy of the diet regime.
Assuntos
Dípteros/metabolismo , Insulina/metabolismo , Transdução de Sinais/fisiologia , Animais , Peso Corporal/fisiologia , Metabolismo dos Carboidratos/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Dieta/métodos , Proteínas Alimentares/metabolismo , Metabolismo dos Lipídeos/fisiologia , Metionina/metabolismoRESUMO
BACKGROUND: The horn fly Haematobia irritans is a blood-sucking ectoparasite responsible for substantial economic loss of livestock. Like other hematophagous arthropods species, the successful blood-feeding of H. irritans is highly dependent on the modulation of the host's hemostasis and immune system. Here, we evaluated the biological activity of hematobin (HTB), a protein recently identified in the H. irritans saliva, on macrophage biology. The goal was to understand the putative interactions between the components of H. irritans saliva and the early host immune responses. RESULTS: Thioglycolate-elicited peritoneal macrophages from BALB/c mice were stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) in the presence or absence of recombinant HTB. The presence of the salivary protein in the cultures inhibited nitric oxide production and decreased the inducible nitric oxide synthase (iNOS) expression induced by LPS plus IFN-γ. The tumor necrosis factor-α (TNF-α) and interleukin-12p40 (IL-12p40) levels were also reduced in the macrophages pre-incubated with HTB; these findings correlated to the decreased NF-κB expression. The biological activities described here were not associated with changes in annexin V binding to macrophages suggesting that HTB does not induce cell death. In addition, the activity of HTB seems to be specific to macrophages because no changes were observed in lymphocyte proliferation or cytokine production. CONCLUSIONS: We describe here the first bioactive salivary protein of H. irritans. We characterized its ability to modulate macrophage inflammatory response, and the results can help explain how horn flies modulate the host immune system to feed on blood.
Assuntos
Dípteros/metabolismo , Inflamação/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células Cultivadas , Citocinas , Dinoprostona , Regulação da Expressão Gênica/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico , Óxido Nítrico Sintase Tipo II , Baço/citologiaRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression through post-transcriptional regulation. Here, we report the identification and characterization of miRNAs in two closely related screwworm flies with different feeding habits: Cochliomyia hominivorax and Cochliomyia macellaria. The New World screwworm, C. hominivorax, is an obligatory parasite of warm-blooded vertebrates, whereas the secondary screwworm, C. macellaria, is a free-living organism that feeds on decaying organic matter. Here, the small RNA transcriptomes of adults and third-instar larvae of both species were sequenced. A total of 110 evolutionarily conserved miRNAs were identified, and 10 putative precursor miRNAs (pre-miRNAs) were predicted. The relative expression of six selected miRNAs was further investigated, including miRNAs that are related to reproduction and neural processes in other insects. Mature miRNAs were also characterized across an evolutionary time scale, suggesting that the majority of them have been conserved since the emergence of the Arthropoda [540 million years ago (Ma)], Hexapoda (488 Ma) and Brachycera (195 Ma) lineages. This study is the first report of miRNAs for screwworm flies. We also performed a comparative analysis with the hereby predicted miRNAs from the sheep blowfly, Lucilia cuprina. The results presented may advance our understanding of parasitic habits within Calliphoridae and assist further functional studies in blowflies.