Fermented crop straws by Trichoderma viride and Saccharomyces cerevisiae enhanced the bioconversion rate of Musca domestica (Diptera: Muscidae).

Crop straw is an abundant renewable resource whose usage is limited due to its high cellulose, hemicellulose, and lignin contents. Here, Trichoderma viride, Saccharomyces cerevisiae, and Musca domestica were used to transform crop straws, and we investigated their impact on housefly rearing performance and optimized their utilization. The weights of cellulose, hemicellulose, and lignin in fermented crop straw diets significantly decreased after bioconversion by M. domestica larvae. The highest bioconversion rate was recorded in corn straw diet (16.19%), followed by wheat straw diet (10.31%) and wheat bran diet (8.97%). Similarly, high larval weight (yield) and pupation rate and fecundity and fertility rate were recorded in fermented crop straw diets composed of corn straw and wheat bran in 1:1 proportions. These results indicated that fermenting crop straw with T. viride and S. cerevisiae represented an efficient strategy that enhanced crop straw bioconversion and improved the rearing capacity of the housefly larvae. The resulting larvae could further be used as proteinaceous feed in poultry and aquaculture industries. Graphical abstract.

Efficacy of Origanum vulgare essential oil and carvacrol against the housefly, Musca domestica L. (Diptera: Muscidae).

The toxicity of Origanum vulgare essential oil to the housefly Musca domestica L. was evaluated. The major constituents of the O. vulgare essential oil by gas chromatographic mass spectrometry (GC-MS) analysis were carvacrol (58.13%), p-cymene (17.85%), thymol (8.15%), γ-terpinene (4.96%), and linalool (3.69%). Toxicity of O. vulgare essential oil against larvae and pupae was evaluated using fumigation and contact assays. The contact toxicity (LC50) of O. vulgare essential oil and carvacrol for larvae was 0.23 and 0.03 µL/cm2, respectively. The fumigation toxicity (LC50) of O. vulgare essential oil and carvacrol for larvae was 9.52 and 2.78 µL/L, respectively. Pupal toxicity was evaluated by percentage inhibition rate (PIR). PIR of O. vulgare essential oil at 0.25 µL/cm2 was 90.9% for the contact assay and 100% at 20 µL/L for the fumigation assay. PIR of carvacrol was 29.5% (0.025 µL/cm2) and 81.8% (1.25 µL/L) for the contact toxicity and fumigation assay, respectively. O. vulgare essential oil and carvacrol have significant toxicity to the housefly and are potential insecticides for housefly control.

Metabolites of stable fly reduce diarrhea in mice by modulating the immune system, antioxidants, and composition of gut microbiota.

Escherichia coli (E. coli) O1-induced diarrhea is associated with intestinal microbial imbalance, however, the results of using oral antibiotics still remain poor. To overcome such problem, our study investigates the role of metabolites from stable flies (MSF) in the occurrence of diarrhea. The amino acid composition and molecular weight analysis of MSF by RP-HPLC and GPC, respectively. Besides the normal control group, SPF mice in other group were inoculated with E. coli O1 received treatment as follows over a period of 7 days saline solution (E. coli control), ciprofloxacin (0.13 g/kg; positive control) and MSF (2, 4 and 8 mg/kg) dosage. Throughout the experiment, defecation and body weights were examined and recorded. On the eighth day, after administering anesthesia, blood, tissue of small intestine samples were obtained for immunological and anti-oxidant. Small intestinal tissues and cecum contents samples were used for histopathological and 16S rDNA sequencing analysis. Our showed that MSF was rich in isoleucine, and its molecular weight less than 400 Da is 60.03%. MSF (4 and 8 mg/kg) and ciprofloxacin, significantly decreased IL-6, IL-8 and TNF-α levels, whereas, increased IL-2, IL-4, IL-10, INF-γ, IgA and IgG levels in mice having diarrhea. These treatments also reversed intestinal flora imbalance as indicated by the increased in Firmicutes-to-Bacteroidetes ratio and Clostridium levels (P < 0.05) and improved 5-HT, CAT and SOD levels. MSF favored diarrhea management as compared to ciprofloxacin, suggesting that MSF can be used in the management of E. coli O1-induced diarrhea, in normal gut microbiota and normal intestinal antioxidant function.

A Natural Cattle Immune Response Against Horn Fly (Diptera: Muscidae) Salivary Antigens May Regulate Parasite Blood Intake.

The horn fly, Haematobia irritans (L.), is a blood-sucking ectoparasite that is responsible for sizeable economic losses in livestock. The salivary gland products facilitate blood intake. Taking advantage of the identification of novel H. irritans salivary antigens (Hematobin, HTB and Irritans 5, IT5), we investigated the parasite loads, H. irritans blood intake, and antibody response of naturally infected bovines during the fly season. Fly loads and fly hemoglobin content fluctuated during the trial. Each time horn fly loads exceeded 200 flies per cattle, a reduction in horn fly blood intake was observed three weeks later. All of the cattle elicited an antibody response against HTB and IT5 that declined once the fly season was over. Cattle anti-IT5 titers were positively correlated with parasite loads and negatively correlated with fly blood intake. These results suggest that the natural changes in the H. irritans blood intake observed in this study were associated with a natural host response against horn fly salivary antigens.

The functional interaction between abaecin and pore-forming peptides indicates a general mechanism of antibacterial potentiation.

Long-chain proline-rich antimicrobial peptides such as bumblebee abaecin show minimal activity against Gram-negative bacteria despite binding efficiently to specific intracellular targets. We recently reported that bumblebee abaecin interacts with Escherichia coli DnaK but shows negligible antibacterial activity unless it is combined with sublethal doses of the pore-forming peptide hymenoptaecin. These two bumblebee peptides are co-expressed in vivo in response to a bacterial challenge. Here we investigated whether abaecin interacts similarly with pore-forming peptides from other organisms by replacing hymenoptaecin with sublethal concentrations of cecropin A (0.3 µM) or stomoxyn (0.05 µM). We found that abaecin increased the membrane permeabilization effects of both peptides, confirming that it can reduce the minimal inhibitory concentrations of pore-forming peptides from other species. We also used atomic force microscopy to show that 20 µM abaecin combined with sublethal concentrations of cecropin A or stomoxyn causes profound structural changes to the bacterial cell surface. Our data indicate that the potentiating functional interaction between abaecin and pore-forming peptides is not restricted to specific co-expressed peptides from the same species but is likely to be a general mechanism. Combination therapies based on diverse insect-derived peptides could therefore be used to tackle bacteria that are recalcitrant to current antibiotics.

Molecular identification of necrophagous muscidae and sarcophagidae fly species collected in Korea by mitochondrial cytochrome C oxidase subunit I nucleotide sequences.

Identification of insect species is an important task in forensic entomology. For more convenient species identification, the nucleotide sequences of cytochrome c oxidase subunit I (COI) gene have been widely utilized. We analyzed full-length COI nucleotide sequences of 10 Muscidae and 6 Sarcophagidae fly species collected in Korea. After DNA extraction from collected flies, PCR amplification and automatic sequencing of the whole COI sequence were performed. Obtained sequences were analyzed for a phylogenetic tree and a distance matrix. Our data showed very low intraspecific sequence distances and species-level monophylies. However, sequence comparison with previously reported sequences revealed a few inconsistencies or paraphylies requiring further investigation. To the best of our knowledge, this study is the first report of COI nucleotide sequences from Hydrotaea occulta, Muscina angustifrons, Muscina pascuorum, Ophyra leucostoma, Sarcophaga haemorrhoidalis, Sarcophaga harpax, and Phaonia aureola.

GC-MS analysis of cuticular lipids in recent and older scavenger insect puparia. An approach to estimate the postmortem interval (PMI).

An analytical method was developed to characterize puparia cuticular lipids (hydrocarbons, waxes) and to compare the molecular distribution patterns in the extracts from either recent or older puparia. Acid-catalyzed transesterification and solvent extraction and purification, followed by combined gas chromatography coupled to mass spectrometry, were optimized for the determination of hydrocarbons and fatty acid ethyl esters from transesterified waxes, extracted from a single species of a fly scavenger (Hydrotaea aenescens Wiedemann, 1830). Comparison between recent (2012) or older (1997) puparia contents has highlighted significant composition differences, in particular, a general decrease of the chain length in the n-alkane distribution pattern and, on the contrary, an increase of the ester chain length. Both extracts contain traces of three hopane hydrocarbon congeners. Preliminary results evidence the change in puparia lipid composition over time, thus potentially providing new indices for estimating postmortem interval.

Behavioural and electroantennogram responses of the stable fly (Stomoxys calcitrans L.) to plant essential oils and their mixtures with attractants.

BACKGROUND: Insect olfactory organs possess many olfactory receptor neurons, which detect many different sets of odorants in nature. In order to feed on blood meals, stable flies locate host animals and humans using chemical cues such as 1-octen-3-ol and butyric acid. In the present study, behavioural and electroantennogram (EAG) response patterns to repellent volatiles from essential oils (EOs) of Zanthoxylum piperitum and Z. armatum in combination with the attractants were investigated. RESULTS: Components of the EOs such as cuminaldehyde, citronellal, neral, linalool, linalool oxide, terpinen-4-ol, 1,8-cineole, and piperitone induced remarkable repellent behaviours in the stable fly. EAG responses in the fly antenna to these chemicals showed a dose-dependent manner. The patterns of behavioural and EAG responses were significantly altered depending on the ratios of 1-octen-3-ol or butyric acid to the EOs or compounds in the air mixtures. CONCLUSION: The present study demonstrated that the Zanthoxylum EOs decreased the levels of response of flight behaviours of the stable fly towards host volatile compounds. The combinations of odorant mixtures of the attractants with the EOs and their components affect the representation of behavioural and EAG responses of the flies. The summation and integration patterns of olfactory responses measured by the EAG indicated that the peripheral olfactory networks in antennae could process the odorant complexity of different odorant mixtures between attractants and repellents.

Host blood meal identification by multiplex polymerase chain reaction for dispersal evidence of stable flies (Diptera:Muscidae) between livestock facilities.

A species-specific multiplex polymerase chain reaction targeting the cytochrome b gene of cattle, horses, humans, and dogs was developed to determine the blood meal sources of stable flies, Stomoxys calcitrans (L.), collected from Florida equine facilities. Of 595 presumptive blood-fed stable flies analyzed, successful host amplification was obtained in 350, for a field host-detection efficiency of 58.8%. The majority of analyzed stable flies had fed on cattle (64.6%), followed by horses (24.3%), humans (9.5%), and dogs (1.6%). A survey of animal-enclosed pastures occurring within 3 km of stable fly collection sites revealed that the nearest cattle were between 0.8 and 1.5 km from the four horse farm sampling sites. Cattle-feeding frequencies were greater on farms where cattle were located at distances of 0.8 km, suggesting that between farm differences in host-feeding frequency is related to the number of and distance from a particular host type. Time course evaluations of previously laboratory-fed stable flies demonstrated that host-detection efficiency with this system was 100, 50, and 0% when flies were evaluated at 16, 24, and 48 h postblood feeding, respectively. The results of this study suggest short-term stable fly dispersal of up to 1.5 km in a 48-h time period. The implications of these findings are discussed.

An insight into the transcriptome and proteome of the salivary gland of the stable fly, Stomoxys calcitrans.

Adult stable flies are blood feeders, a nuisance, and mechanical vectors of veterinary diseases. To enable efficient feeding, blood sucking insects have evolved a sophisticated array of salivary compounds to disarm their host's hemostasis and inflammatory reaction. While the sialomes of several blood sucking Nematocera flies have been described, no thorough description has been made so far of any Brachycera, except for a detailed proteome analysis of a tabanid (Xu et al., 2008). In this work we provide an insight into the sialome of the muscid Stomoxys calcitrans, revealing a complex mixture of serine proteases, endonucleases, Kazal-containing peptides, anti-thrombins, antigen 5 related proteins, antimicrobial peptides, and the usual finding of mysterious secreted peptides that have no known partners, and may reflect the very fast evolution of salivary proteins due to the vertebrate host immune pressure. Supplemental Tables S1 and S2 can be downloaded from http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T1/Sc-tb1-web.xls and http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T2/Sc-tb2-web.xls.

Identification of PVK/CAP2b neuropeptides from single neurohemal organs of the stable fly and horn fly via MALDI-TOF/TOF tandem mass spectrometry.

MALDI-TOF/TOF tandem mass spectrometry has been applied to determine the complete sequences of the PVK/CAP2b neuropeptides in the stable fly Stomoxys calcitrans and horn fly Haematobia irritans, insect pests of livestock. This peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The sequences are as follows: Stoca-PVK/CAP2b-1, AGGASGLYAFPRVa; Stoca-PVK/CAP2b-2, NAKLYPVPRVa; and Haeir-PVK/CAP2b-1, AGGASGLYAFPRVa; Haeir-PVK/CAP2b-1, NAKLYPMPRVa. Both Stoca-PVK/CAP2b-1 and -2 stimulate Malpighian tubule fluid secretion in the stable fly, with EC50 values between 3 and 11 nM. The identification of these novel neuropeptides adds to our knowledge of the peptidomes of flies, and can aid in the development of neuropeptide-based control strategies of these insect pests.

Solution structures of stomoxyn and spinigerin, two insect antimicrobial peptides with an alpha-helical conformation.

Stomoxyn and spinigerin belong to the class of linear cysteine-free insect antimicrobial peptides that kill a range of microorganisms, parasites, and some viruses but without any lytic activity against mammalian erythrocytes. Stomoxyn is localized in the gut epithelium of the nonvector stable fly that is sympatric with the trypanosome vector tsetse fly. Spinigerin is stored and secreted by hemocytes from the fungus-growing termite. The structure of synthetic stomoxyn and spinigerin in aqueous solution and in TFE/water mixtures was analyzed by CD and NMR spectroscopy combined with molecular modeling calculations. Stomoxyn and spinigerin adopt a flexible random coil structure in water while both assume a stable helical structure in the presence of TFE. In 50% TFE, the structure of stomoxyn is typical of cecropins, including an amphipathic helix at the N-terminus and a hydrophobic C-terminus with helical features that probably fold in a helical conformation at higher TFE concentration. In contrast to stomoxyn, spinigerin acquires very rapidly a helical conformation. In 10% TFE the helix is highly bent and the structure is poorly defined. In 50% TFE, the helical structure is well defined all along its sequence, and the slightly bent alpha-helix displays an amphiphilic character, as observed for magainin 2. The structural similarities between stomoxyn and cecropin A from Hyalophora cecropia and between spinigerin and magainin 2 suggest a similar mode of action on the bacterial membranes of both pairs of peptides. Our results also confirm that TFE induces helix formation and propagation for amino acids showing helical propensity in water but also enhances the helix propagation propensity of nonpolar beta-branched residues.

Cuticular hydrocarbons of buffalo fly, Haematobia exigua, and chemotaxonomic differentiation from horn fly, H. irritans.

We determined the quantity and chemical composition of cuticular hydrocarbons of different strains, sexes, and ages of buffalo flies, Haematobia exigua. The quantity of cuticular hydrocarbons increased from less than 1 microg/fly for newly emerged flies to over 11 microg/fly in 13-d-old flies. The hydrocarbon chain length varied from C(21) to C(29), with unbranched alkanes and monounsaturated alkenes the major components. Newly emerged flies contained almost exclusively C(27) hydrocarbons. Increasing age was accompanied by the appearance of hydrocarbons with shorter carbon chains and an increase in the proportion of alkenes. 11-Tricosene and 7-tricosene were the most abundant hydrocarbons in mature H. exigua. Cuticular hydrocarbons of H. exigua are distinctly different from those of horn flies, Haematobia irritans. The most noticeable differences were in the C(23) alkenes, with the major isomers 11- and 7-tricosene in H. exigua and (Z)-9- and (Z)-5-tricosene in H. irritans, respectively. Cuticular hydrocarbon analysis provides a reliable method to differentiate the two species, which are morphologically difficult to separate. The differences in cuticular hydrocarbons also support their recognition as separate species, H. exigua and H. irritans, rather than as subspecies.

The role of HiTI, a serine protease inhibitor from Haematobia irritans irritans (Diptera: Muscidae) in the control of fly and bacterial proteases.

Blood-sucking arthropods are vectors responsible for the transmission of several pathogens and parasites to vertebrate animals. The horn fly Haematobia irritans irritans (Diptera: Muscidae) and the tick Boophilus microplus are important hematophagous ectoparasites that cause losses in cattle production. A serine protease inhibitor from a thorax extract of the fly H. irritans irritans (HiTI) was previously isolated, characterized and cloned. In the present study we described the expression, purification, and characterization of the recombinant HiTI (rHiTI) and its possible role in the control of different endogenous and bacterial proteases. rHiTI was successfully expressed using the pPIC9 expression vector with a yield of 4.2 mg/L of active rHiTI. The recombinant HiTI purified by affinity chromatography on trypsin-Sepharose had a molecular mass of 6.53 kDa as determined by LS-ESI mass spectrometry and inhibition constants (Kis) similar to those of native HiTI for bovine trypsin and human neutrophil elastase of 0.4 and 1.0 nM, respectively. Purified rHiTI also showed inhibitory activity against the trypsin-like enzyme of H. i. irritans using its possible natural substrates, fibrinogen and hemoglobin; and also inhibited the OmpT endoprotease of Escherichia coli using fluorogenic substrates. The present results confirm that HiTI may play a role in the control of fly endogenous proteases but also suggest a role in the inhibition of pathogen proteases.

Purification, characterization, and cloning of a serine proteinase inhibitor from the ectoparasite Haematobia irritans irritans (Diptera: Muscidae).

The fly Haematobia irritans irritans is one of the most important ectoparasites in cattle production, due to its ability to suck large amounts of blood. This report describes the purification and characterization of a serine proteinase inhibitor (HiTI) present in H. i. irritans head and thorax extracts. The HiTI purified by affinity chromatography on trypsin-Sepharose has a molecular mass of 7029Da by MALDI-TOF mass spectrometry. HiTI inhibited bovine trypsin, human neutrophil elastase, and a trypsin-like enzyme purified from H. i. irritans abdomen with dissociation constants of 0.57, 1.30, and 0.20nM, respectively. The HiTI partial amino acid sequence allowed its classification into the BPTI-Kunitz-type family. An HiTI cDNA fragment was cloned in the pGEMT vector using RT-PCR. The translated amino acid sequence of HiTI cDNA confirmed a unique Kunitz-type-domain protein. Our results suggest that HiTI could control some endogenous enzyme, e.g., the H. i. irritans trypsin-like protein.

Distinctive hydrocarbons of the black dump fly, Hydrotaea aenescens (Diptera: Muscidae).

Hydrotaea aenescens (Wiedemann), the black dump fly, is a potential biological control agent originally from the western hemisphere, now found in many parts of the Palearctic region except for the United Kingdom, where it cannot be imported for any reason. A complication of classical biological control is the problem of strain identification, as one must be able to somehow mark or follow a particular strain that has been introduced into the field or is contemplated for release. Gas chromatographic analysis of the surface hydrocarbons of pooled and individual dump fly adults resulted in reproducible hydrocarbon patterns that differentiated widely distributed strains of H. aenescens and showed similarities between strains that were related. Sexual dimorphism was observed in the surface hydrocarbons. Conspecific similarities included identities of the hydrocarbons found in colony material collected worldwide, with differences being found in the quantities of compounds present.

Localization of myosuppressinlike peptides in the hypocerebral ganglion of two blood-feeding flies: horn fly and stable fly (Diptera:Muscidae).

The insect peptides leucomyosuppressin (pEDVDHVFLRFamide) and dromyosuppressin (TDVDHVFLRFamide) have identical chemical sequences with the exception of the N-terminal amino acid; both inhibit spontaneous contraction of insect visceral muscles. Neurons in the hypocerebral ganglion of horn fly, Hematobia irritans (L.), and stable fly, Stomoxys calcitrans (L.), were found to contain material immunoreactive to antiserum produced against the C-terminal of leucomyosuppressin, but not to the N-terminal of dromyossuppressin. Two large lateral clusters containing 8 cells, linked dorsally and ventrally by 2 chains of 6 cells, encircled the anterior surface of the proventriculus and were immunoreactive of leucomyosuppressin and FMRFamide antisera. Axons from these cells were traced to the wall of the aorta and over the surface of the proventriculus. Ultrastructural analysis revealed these cells contained a singular type of elementary secretory granule that contained material of relatively low electron density, both in the cell body and at the axon terminals.

Pterin quantity and gonotrophic stage as indicators of age in Musca autumnalis (Diptera: Muscidae).

Spectrofluorimetric methods were used to measure pterin quantities in the head capsules of adult face flies, Musca autumnalis De Geer. Quantities at eclosion were greater in males than in females, and were proportional to the head capsule width in each sex. Pterin accumulated as a linear function of time at constant temperature, at rates of 3.8 relative fluorescence units per degree-day in males and 1.68 units in females, both above a common base of 9.8 degrees C. A mark-release-recapture experiment revealed that pterin accumulated at a rate of 6.40 units per degree-day among males, twice as fast as in the laboratory. In contrast, accumulation among females was 1.35 units per degree-day, approximating their laboratory rate. Gonotrophic age grading of recaptured females indicated median development rates corresponded with expectations based on earlier laboratory studies, but substantial variation among females led to imprecise estimates of chronological age. Calibration curves for each sex are presented to estimate degree-day age from head capsule width and pterin content. The pterin age-grading method allows the field study of longevity of male face flies, and complements gonotrophic methods for the study of longevity and reproductive success of females.

Age structure and reproductive composition of summer Musca autumnalis (Diptera: Muscidae) populations estimated by pterin concentrations.

Pterins in face flies, Musca autumnalis DeGeer, were measured spectrofluorimetrically to estimate the chronological ages of adults in natural summer populations. The age distributions showed a systematic undersampling of young males. Exponential, Gompertz, and Weibull models were fitted to the age distributions. A 1-parameter exponential model suggested mean daily survival rates of 0.83 for female face flies, and a mean life expectation of 5.5 d. But the Weibull model best fit the age distributions, providing mean life expectations of 10.1 and 11 d for males and females, respectively. Ovarian dynamics were examined with respect to chronological age and delays were detected in vitellogenesis and oviposition. Face fly populations seemed to reproduce at less than the maximum rate allowed by prevailing temperatures, at which it was estimated that an average female would undergo 2.2 ovarian cycles and have a lifetime fecundity of 28.5 female progeny.

Isolation and characterization of a diuretic peptide common to the house fly and stable fly.

An identical CRF-related diuretic peptide (Musca-DP) was isolated and characterized from whole-body extracts of the house fly, Musca domestica, and stable fly, Stomoxys calcitrans. The peptide stimulates cyclic AMP production in Manduca sexta Malpighian tubules and increases the rate of fluid secretion by isolated Musca domestica tubules. The 44-residue peptide, with a mol.wt. of 5180, is amidated, and has the primary structure: NKPSLSIVNPLDVLRQRLLLEIARRQMKENTRQVELNRAILKNV-NH2. Musca-DP has a high percentage of sequence identity with other characterized CRF-related insect diuretic peptides.