Effect of different doses of the catecholamine epinephrine on antioxidant responses of larvae of the flesh fly Sarcophaga dux.

The production and use of pharmaceutical products have increased over the past decades, and several are considered potential or proved hazardous wastes. When contaminating the environment, they can severely impact biodiversity. The catecholamine epinephrine (adrenaline) is no exception. Epinephrine can be administered as growth promoter in cattle, and is used for anaphylaxis treatment in human. While a range of studies has examined the effects of this catecholamine on vertebrate tissues, and evidenced that it can disrupt the oxidative stress status, the effects epinephrine could have on insects have remained poorly considered. Here, we examined the physiological effects of different concentrations (0, 25, 50, and 100 µg/mL) of epinephrine on larvae of the flesh fly Sarcophaga dux. Following experimental treatments, levels of H2O2, GSH, CAT, GPx, and CEH were measured from the fat body, cuticle, gut, and hemolymph of 3rd instars. Significant differences are reported for these physiological endpoints among the considered body compartments, and epinephrine concentrations. Epinephrine treatments did not increase reactive oxygen species production (H2O2 amounts), except for gut tissues. Increased levels of GSH suggest that epinephrine may have enhanced glucose metabolism and flux towards the pentose phosphate pathway, while reducing glutamine oxidation. CAT activity was slightly increased when the concentration of epinephrine was higher. The decreased GPx activity in the fat body was consistent with GSH variations. In sum, the injection of epinephrine seemed to elicit the antioxidant response in S. dux larvae, in turn attenuating ROS production.

Effect of Terbufos (Organophosphate) on the Cadaveric Colonization Process: Implications for Postmortem Interval Calculation.

The determination of necrophagous fly specie's development time is considered an accurate method for estimating postmortem interval (PMI). However, pesticides and other chemicals can alter the flies' life cycle, inducing errors in PMI estimation. Thus, this work aimed to evaluate the effect of different doses of Terbufos (Organophosphates) on the temporal dispersion pattern and development of immature dipterans associated with decaying rat carcasses. For this, 150 g female Wistar rats received, via gavage, 200 µl of Terbufos (5 or 10 mg/kg) or distilled water (control) and, after 30 min of administration, the animals were euthanized and distributed in suspended traps to decompose under environmental conditions. The dispersing immatures were collected daily, and their development time was monitored until the emergence of adult flies. After data analysis, it was observed that Terbufos altered 1) the temporal pattern of larval dispersion; 2) the composition and structure of the colonizing assemblage (emerged adults); 3) species' development time, accelerating or delaying their cycle, depending on the dose used; and 4) the califorids and sarcophagids emergence rate, increasing the mortality of pupae from intoxicated carcasses. Thus, this work demonstrates experimentally that Terbufos directly influences the development of flies with forensic potential and discusses the implications for PMI estimation, which can assist in future investigative processes with suspected poisoning by this organophosphate.

A formulation of neem and hypericum oily extract for the treatment of the wound myiasis by Wohlfahrtia magnifica in domestic animals.

Untreated, traumatic, or wound myiases can cause severe consequences to animal health and welfare as well as economic losses to livestock productions. For healing myiasis-caused wounds, disinfectant such as creolin is wrongly but currently used in association with insecticides. Though effective, creolin is highly toxic to the patients, is inadequate with respect to the repellent effect, and may delay the healing of treated wounds. In this uncontrolled study, the efficacy of the patented plant-derived formulation 1 Primary Wound Dressing© (1-PWD), composed of neem oil (Azadirachta indica A. Juss.) and the oily extract of Hypericum perforatum (L.) flowers, has been investigated. Forty-four domestic animals of different species suffering from wound myiasis lasting for up to 25 days, at different parts of the body, were enrolled in the study. No systemic or local antibiotic or disinfectants' treatment was administered. Larvae recovered on open wounds and adults reared from mature larvae were identified as Wohlfahrtia magnifica. All the treated wounds healed in a range of 10 to 32 days without further infestation. None of the recruited animals presented bacterial complications. Data herein presented indicate that the tested natural plant-derived formulation is able to manage the infestation caused by W. magnifica larvae and the healing process of traumatic infested wounds in several domestic animal species, without any side effect on the living tissue and without the need to use local or systemic chemical or other products.

Immunosuppressive effects of the limonoid azadirachtin, insights on a nongenotoxic stress botanical, in flesh flies.

The tetranortriterpenoid azadirachtin (Aza) is a well-known insect growth disruptor of plant origin. Although its actions on insects have been extensively studied; fragmentary reports are available from the immunological point of view. Therefore, in the present study, total (THC) and differential hemocyte counts (DHC), nodulation, phenoloxidase (PO) activity, immune-reactive lysozymes and inducible nitric oxide (NO) were assessed, as measures of immune responses, in Sarcophaga argyrostoma 3rd instars challenged individually with M. luteus or Aza, or in combination with both compared to the control larvae. THC was significantly declined after 12 h and 24 h of treatment with Aza. DHC varied considerably; in particular, plasmatocytes were significantly decreased after 36 h and 48 h of treatment with Aza; whereas granulocytes were significantly increased. Nodulation was significantly increased with the increase of time after all treatments. Challenging with M. luteus significantly increased the activity of PO in hemocytes and plasma; whereas such activity was significantly decreased after treatment with Aza or combined Aza and M. luteus. Treatment with Aza or M. luteus alone or in couple significantly increased lysozyme activity of fat body, hemocytes and plasma. However, challenging with M. luteus significantly increased NO concentration in the same tissues. A hypothetical model of Aza as a potential mutagen is presented. However, no genotoxic effect was observed through tracking apoptosis-associated changes in Aza-treated hemocytes via flow cytometry-based apoptosis detection. Our study suggests that the integration of Aza, as an eco-friendly pesticide, with bacterial biopesticides may be a successful approach for controlling insect pests.

Insecticidal activity of Stemona collinsiae root extract against Parasarcophaga ruficornis (Diptera: Sarcophagidae).

In Thai indigenous knowledge, Stemona plant has traditionally been used as insecticide on plantations. Recently, S. collinsiae extract was showed to be an anti-feedant and growth inhibitor and to exert strong insecticidal activity. Here, the insecticidal activity of S. collinsiae root extract against Parasarcophaga ruficornis is studied. The larvicidal and pupicidal activities of the ethanolic root extract of S. collinsiae were tested using contact toxicity tests, and adulticidal activity was tested using the topical contact toxicity and sugar bait methods The ethanolic extract at concentration ranging of 0.3-320mg/larva for the direct contact toxicity test and from 0.3 to 3.2mg/cm2/larva for the secondary contact toxicity test showed 3.0-51.0 and 1.0-94.0% corrected mortality, respectively. Against third-instar larvae, the LD50 concentrations of the ethanolic extract were 31.7±0.0mg/larva and 1.4±0.0mg/cm2/larva for direct and secondary contact toxicity tests, respectively. Pupae were not eliminated at all concentrations of the ethanolic extract. Against adult flies, which were killed via oral administration, the LD50 concentration of the ethanolic extract was 0.145±0.070g extract/g glucose. Thus, the ethanolic extract of S. collinsiae was capable of eliminating P. ruficornis in larval and adult stages via topical and ingestion administration, respectively.

THE EFFECT OF THE TRAMADOL ACCUMULATED IN RAT LIVER ON THE DEVELOPMENT OF THE IMMATURE STAGES OF THE FLESH FLY SARCOPHAGA ARGYROSTOMA (ROBINEAU-DESVOIDY, 1830) (DIPTERA: SARCOPHAGIDAE).

The present study investigated the effects of the accumulation of tramadol on the development rates of the larvae and pupae. Larvae of the flesh fly Sarcophaga argyrostoma (Robineau-Desvoidy, 1830) were reared on three groups of Wister albino rat livers. One group was administered the recommended or normal dose (Dl) and the second group was injected with higher over or double dose (D2) of tramadol. Tramadol was administered by stomach tube once a day, for 3 months. The third group was injected with normal saline solution as the control. Using GC-Ms analysis, Rat livers contained 0.72 and 1.62mg/g of tramadol, in case of Dl and D2, respectively. Larvae of S. argyrostoma fed on DI and D2 rat livers contained (0.11 & 0.1 8mg/g), respectively. The corresponding tramadol concentrations persisted in the produced pupae of S. argyrostoma were (0.07 & 0.09mg/g), respectively. The accumulation of tramadol in tissues of S. argyrostoma larvae reduced the durations of larval stadia from (7.0408 1.0198 days) in case of the control to (6.6383±0.4857 days) when fed on DI liver and (6.3438±0.4826 days) when fed on D2 livers. The pupal durations was altered to (14.3750±0.4919 days) for D2 and (14.9574±0.6580 days) for DI, compared to (13.9167±1.0071 days), in case of the control. The average weight of the 3rd day larva of S. argyrostoma decreased from 18.1807±1.4949mg, in case of the control to 14.7279±1.3366mg and 14.9560±3.8210mg, when fed on Dl and D2 tramadol treated livers. In contrast, corresponding weight produced pupae increased from 14.1750±0.4667mg (control). to 15.2449±0.302mg & 15.9062±0.2888mg, in case of D1 and D2, respectively.

Lachesana tarabaevi, an expert in membrane-active toxins.

In the present study, we show that venom of the ant spider Lachesana tarabaevi is unique in terms of molecular composition and toxicity. Whereas venom of most spiders studied is rich in disulfide-containing neurotoxic peptides, L. tarabaevi relies on the production of linear (no disulfide bridges) cytolytic polypeptides. We performed full-scale peptidomic examination of L. tarabaevi venom supported by cDNA library analysis. As a result, we identified several dozen components, and a majority (∼80% of total venom protein) exhibited membrane-active properties. In total, 33 membrane-interacting polypeptides (length of 18-79 amino acid residues) comprise five major groups: repetitive polypeptide elements (Rpe), latarcins (Ltc), met-lysines (MLys), cyto-insectotoxins (CIT) and latartoxins (LtTx). Rpe are short (18 residues) amphiphilic molecules that are encoded by the same genes as antimicrobial peptides Ltc 4a and 4b. Isolation of Rpe confirms the validity of the iPQM (inverted processing quadruplet motif) proposed to mark the cleavage sites in spider toxin precursors that are processed into several mature chains. MLys (51 residues) present 'idealized' amphiphilicity when modelled in a helical wheel projection with sharply demarcated sectors of hydrophobic, cationic and anionic residues. Four families of CIT (61-79 residues) are the primary weapon of the spider, accounting for its venom toxicity. Toxins from the CIT 1 and 2 families have a modular structure consisting of two shorter Ltc-like peptides. We demonstrate that in CIT 1a, these two parts act in synergy when they are covalently linked. This finding supports the assumption that CIT have evolved through the joining of two shorter membrane-active peptides into one larger molecule.

Effects of juvenoid Pyriproxyfen on reproduction and F1 progeny in myiasis causing flesh fly Sarcophaga ruficornis L. (Sarcophagidae: Diptera).

Freshly emerged virgin female Sarcophaga ruficornis were topically treated with different doses of pyriproxyfen to test the efficacy on reproduction and subsequent F1 progeny. The results included mortality of the treated adults, significant reduction in fecundity, more than 90% inhibition in larvae production, mortality in F1 generation during larval instars, reduction in pupariation, and adult emergence and production of deformed adults. There was a dose-dependent response showing high degree of correlation in the doses administered and deformities observed. The effects in F1 generation show that an intraovarial transfer of pyriproxyfen was responsible for the various deformations observed, showing the potency of juvenoid pyriproxyfen for longer durations across generations. This is the first study that clearly demonstrates the efficacy of pyriproxyfen against reproduction in S. ruficornis and its potential for the management of this notorious pest of medico-veterinary importance.

Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species.

The imidazole derivative KK-42 is a synthetic insect growth regulator known previously to be capable of averting embryonic diapause in several Lepidoptera, but whether it also affects diapauses occurring in other developmental stages remains unknown. In the present study, we examined the effect of KK-42 on pupal diapause in two species of Lepidoptera, the Chinese oak silkworm Antheraea pernyi and the corn earworm Helicoverpa zea, and in one species of Diptera, the flesh fly Sarcophaga crassipalpis. In A. pernyi, KK-42 delayed pupal diapause termination under the long day conditions that normally break diapause in this species. Likewise, in H. zea, KK-42 delayed termination of pupal diapause, a diapause that, in this species, is normally broken by high temperature. KK-42-treated pupae of these two species eventually terminated diapause and successfully emerged as adults, but the timing of diapause termination was significantly delayed. KK-42 also significantly increased the incidence of pupal diapause in H. zea and S. crassipalpis when administered to larvae that were environmentally programmed for diapause, but it was not capable of inducing pupal diapause in H. zea if larvae were reared under environmental conditions that do not normally evoke the diapause response. Experiments with H. zea showed that the effect of KK-42 on pupal diapause was dose- and stage-dependent, but not temperature-dependent. Results presented here are consistent with a link between KK-42 and the ecdysteroid signaling pathway that regulates pupal diapause.

Inhibitory effects of extracellular self-DNA: a general biological process?

Self-inhibition of growth has been observed in different organisms, but an underlying common mechanism has not been proposed so far. Recently, extracellular DNA (exDNA) has been reported as species-specific growth inhibitor in plants and proposed as an explanation of negative plant-soil feedback. In this work the effect of exDNA was tested on different species to assess the occurrence of such inhibition in organisms other than plants. Bioassays were performed on six species of different taxonomic groups, including bacteria, fungi, algae, plants, protozoa and insects. Treatments consisted in the addition to the growth substrate of conspecific and heterologous DNA at different concentration levels. Results showed that treatments with conspecific DNA always produced a concentration dependent growth inhibition, which instead was not observed in the case of heterologous DNA. Reported evidence suggests the generality of the observed phenomenon which opens new perspectives in the context of self-inhibition processes. Moreover, the existence of a general species-specific biological effect of exDNA raises interesting questions on its possible involvement in self-recognition mechanisms. Further investigation at molecular level will be required to unravel the specific functioning of the observed inhibitory effects.

Host diapause status and host diets augmented with cryoprotectants enhance cold hardiness in the parasitoid Nasonia vitripennis.

Boosting cold hardiness in parasitoids is a goal that is particularly attractive for increasing shelf life and shipment of biological control agents. In the experiments reported here we use the parasitoid Nasonia vitripennis as a model to evaluate manipulations that may be capable of enhancing the wasp's cold tolerance. We altered the parasitoid's cold tolerance by manipulating the wasp's diapause status, the diapause status of the host fly (Sarcophaga crassipalpis), and the diet of the host. Larval diapause in N. vitripennis dramatically increased cold tolerance and the diapause status of the host also exerted a positive, although less dramatic, effect. Augmenting the host fly's diet with supplements of putative cryoprotectants (alanine, proline and glycerol) enhanced cold tolerance in parasitoids that fed on the flies, thus indicating a tri-trophic effect on parasitoid cold tolerance. The most pronounced improvement in cold tolerance was noted in parasitoids fed on fly hosts that had received a diet augmented with proline. These results suggest mechanisms that could be exploited for enhancement of cold tolerance in parasitoids of commercial interest.

Spider toxins comprising disulfide-rich and linear amphipathic domains: a new class of molecules identified in the lynx spider Oxyopes takobius.

In addition to the conventional neurotoxins and cytotoxins, venom of the lynx spider Oxyopes takobius was found to contain two-domain modular toxins named spiderines: OtTx1a, 1b, 2a and 2b. These toxins show both insecticidal activity (a median lethal dose against flesh fly larvae of 75 µg·g(-1)) and potent antimicrobial effects (minimal inhibitory concentrations in the range 0.1-10 µm). Full sequences of the purified spiderines were established by a combination of Edman degradation, mass spectrometry and cDNA cloning. They are relatively large molecules (~ 110 residues, 12.0-12.5 kDa) and consist of two distinct modules separated by a short linker. The N-terminal part (~ 40 residues) contains no cysteine residues, is highly cationic, forms amphipathic α-helical structures in a membrane-mimicking environment, and shows potent cytolytic effects on cells of various origins. The C-terminal part (~ 60 residues) is disulfide-rich (five S-S bonds), and contains the inhibitor cystine knot (ICK/knottin) signature. The N-terminal part of spiderines is very similar to linear cytotoxic peptides found in various organisms, whereas the C-terminal part corresponds to the usual spider neurotoxins. We synthesized the modules of OtTx1a and compared their activity to that of full-length mature toxin produced recombinantly, highlighting the importance of the N-terminal part, which retained full-length toxin activity in both insecticidal and antimicrobial assays. The unique structure of spiderines completes the range of two-domain spider toxins.