Sarcophaga Bullata Research Articles

Insecticidal efficacy of the drug «Phytocreolin», which affects the number of zoophilic flies

This article examines the insectoacaricidal effectiveness of the drug «Phytocreolin» on the number of zoophilic flies in open and closed livestock premises. For the first time, the authors conducted studies of the insectoacaricidal efficacy of the drug «Phytocreolin» based on pine tar, how it differs from creolin, and having a repellent effect against insects and, thereby, contributing to a decrease in the number of zoophilic flies in livestock premises. Studies conducted to establish the background number of imago flies in open and closed types, followed by an entomological assessment of zoophilic flies, showed that the dominant biological species was the gray meat fly (Sarcophaga bullata), which accounted for about 80% of the total number of flies. Such a large number of this species is due to the active summer season, as well as the time of research. The housefly (Musca domestica) was also found in the insects, the number of which amounted to 10% of the total number of zoophilic flies. In addition, the autumn stable fly (Stomoxys calcitrans), which accounted for about 7%, and the common green carrion (Lucilia caeza), 3% of the total number of zoophilic flies, were also found in livestock facilities. It was found that the effectiveness of the insecticide drug in the disinfection of livestock premises of a closed type for the entire period of experience, when disinfecting with a 25% solution of the drug in reducing the number of insects was 15,3%, when treated with a 50% solution, the effectiveness was the best and amounted to 29,8% and when disinfecting with a 100% solution, the decrease in the number of zoophilic flies was about 21,7%. Similar results of the effectiveness of the use of an insecticidal drug were obtained in open-type livestock facilities. So, over the entire period of the experiment, when disinfecting with a 25% solution of the drug, the decrease in the number of insects was 9,6%, when treated with a 50% solution – 23% and when disinfecting with a 100% solution, the insecticidal efficiency was the best and amounted to 26%.

Insecticidal activity of brown seaweed (Sargassum latifolium) extract as potential chitin synthase inhibitors: Toxicokinetic and molecular docking approaches

In this study, Sargassum latifolium (S. latifolium) n-hexane extract as a novel source for insecticidal bioactive compounds was investigated. The composition of the extract was identified by using GC-MS analysis. We performed the study on four types of insects and larvae sp (Sarcophaga bullata, Tribolium castaneum, Musca domestica, and Solenopsis invicta) which cause a deleterious impact on the crops and human health. We used scanning electron microscopy to show the destructive effects of S. latifolium extract on the exoskeleton. The treatment exhibited severe morphological changes and a high mortality rate. Also, different bioinformatic approaches were utilized to investigate the possible insecticidal mechanism of the extract. Molecular docking showed the potential of the compounds to be chitin synthase inhibitors. Besides, the toxicokinetic parameters of the most promising compounds were evaluated to assess the impact of using the extract as an insecticidal agent on human health. S. latifolium extract exhibited promising insecticidal activity and acceptable toxicokinetic properties as compared to other known pesticide groups.

Silver Ion High-Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry: A Tool for Analyzing Cuticular Hydrocarbons.

Aliphatic hydrocarbons (HCs) are usually analyzed by gas chromatography (GC) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, analyzing long-chain HCs by GC is difficult because of their low volatility and the risk of decomposition at high temperatures. MALDI cannot distinguish between isomeric HCs. An alternative approach based on silver ion high-performance liquid chromatography (Ag-HPLC) is shown here. The separation of HC standards and cuticular HCs was accomplished using two ChromSpher Lipids columns connected in series. A gradient elution of the analytes was optimized using mobile phases prepared from hexane (or isooctane) and acetonitrile, 2-propanol, or toluene. HCs were detected by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Good separation of the analytes according to the number of double bonds, cis/trans geometry, and position of double bonds was achieved. The retention times increased with the number of double bonds, and trans isomers eluted ahead of cis isomers. The mobile phase significantly affected the mass spectra of HCs. Depending on the mobile phase composition, deprotonated molecules, molecular ions, protonated molecules, and various solvent-related adducts of HCs were observed. The optimized Ag-HPLC/APCI-MS was applied for characterizing cuticular HCs from a flesh fly, Neobellieria bullata, and cockroach, Periplaneta americana. The method made it possible to detect a significantly higher number of HCs than previously reported for GC or MALDI-MS. Unsaturated HCs were frequently detected as isomers differing by double-bond position(s). Minor HCs with trans double bonds were found beside the prevailing cis isomers. Ag-HPLC/APCI-MS has great potential to become a new tool in chemical ecology for studying cuticular HCs.

Chiral analysis of β-alanyl-d,l-tyrosine and its derivatives and estimation of binding constants of their complexes with 2-hydroxypropyl-β-cyclodextrin by capillary electrophoresis.

Chiral CE methods were developed for the elucidation of l- or d-configuration of tyrosine residue in antimicrobial dipeptide β-alanyl-tyrosine (β-Ala-Tyr) isolated from the hemolymph of larvae of fleshfly Neobellieria bullata and for the evaluation of enantiopurity of its synthetic isomers (β-Ala-d-Tyr and β-Ala-l-Tyr), and enantiomers of their amidated and acetylated derivatives, β-Ala-d,l-Tyr-NH2 and N-Ac-β-Ala-d,l-Tyr, respectively. Baseline separations were achieved for all three pairs of enantiomers: (i) for β-Ala-d,l-Tyr in acidic background electrolyte composed of 32/50mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 2.5, and 20mg/mL 2-hydroxypropyl-β-cyclodextrin as chiral selector; (ii) for β-Ala-d,l-Tyr-NH2 enantiomers in acidic background electrolyte consisting of 48/50mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 3.5, and 30mg/mL 2-hydroxypropyl-β-cyclodextrin; and (iii) for enantiomers of N-Ac-β-Ala-d,l-Tyr in alkaline background electrolyte composed of 50/49mM Na2 B4 O7 /NaOH, pH 10.5, and 60mg/mL 2-hydroxypropyl-β-cyclodextrin. From CE analyses of mixed samples of isolated β-Ala-Tyr and synthetic standards β-Ala-l-Tyr and β-Ala-d-Tyr, it turned out that isolated β-Ala-Tyr was pure l-enantiomer. In addition, the average apparent binding constants, Kb , and average actual ionic mobilities of the complexes of β-Ala-d,l-Tyr and its above derivatives with 2-hydroxypropyl-β-cyclodextrin were determined. These complexes were weak, with Kb values ranging from 11.2 to 79.1 L/mol. Their cationic mobilities were equal to (5.6-9.2)×10-9 m2 /V/s, and anionic mobilities to (-1.3-1.6)×10-9 m2 /V/s.

Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial β-Alanyl-Tyrosine from Hemolymph of Fleshfly Neobellieria bullata.

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide β-alanyl-tyrosine (β-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of β-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of β-Ala-Tyr and its admixtures have been converted into preparative purification of β-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the β-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

New thinking for filth fly control: residual, non-chemical wall spray from volcanic glass.

Filth flies are of medical and veterinary importance because of the transfer of disease organisms to animals and humans. The traditional control methods include the use of chemical insecticides. A novel mechanical insecticide made from volcanic glass and originally developed to control mosquitoes (Imergard™ WP; ImG) was investigated for control of adult grey flesh flies, Sarcophaga bullata (Parker), secondary screwworms, Cochliomyia macellaria (F.), and house flies, Musca domestica L. In a modified WHO cone test device, the time to 50% mortality (LT50 ) when applied at 5 g/m2 (tested at 30 °C and 50% relative humidity (rH)) was 7.1, 4.3 and 3.2 h, respectively. When knockdown was included, the LT50 s were 5.5, 1.5 and 2.8 h, respectively. Application rates of 1.25 and greater g/m2 had the shortest LT50 s. The time to the LT50 increased for M. domestica as rH increased, but ImG was still active at the highest rH tested of 70%. Scanning electron micrographs showed ImG was present on all body parts, unlike that for mosquitoes where it was found mostly on the lower legs. These first studies on the use of Imergard WP against flies suggest this could be an alternative method for filth fly control.

Carbon Nanotube Fibers for Neural Recording and Stimulation.

Recordings and stimulations of neuronal electrical activity are topics of great interest in neuroscience. Many recording techniques, and even treatment of neurological disorders, can benefit from a microelectrode that is flexible, chemically inert, and electrically conducting and preferentially transfers electrons via capacitive charge injection. Commercial electrodes that currently exist and other electrodes that are being tested with the purpose of facilitating and improving the electron transport between solid materials and biological tissues still have some limitations. This paper discusses carbon nanotube (CNT)-based microelectrodes to record and stimulate neurons and compares their electron transport capabilities to noble metals such as Au and Ag. The recording ability of electrodes is tested through electroretinography on Sarcophaga bullata fly eyes by using Au and Ag wires and CNT fibers as electrodes. Stimulation is demonstrated through the implantation of Au wire and CNT fibers into the antennas of the Madagascar hissing cockroach (Gromphadorhina portentosa) to control their locomotion. Our results demonstrate that a particular property of the CNT fiber is its high rate of electron transfer, leading to an order of magnitude lower impedance compared to Au and Ag and an impressive 15.09 charge injection capacity. We also established that this carbon nanomaterial assembly performs well for in vivo electrophysiology, rendering it a promising prospect for neurophysiological applications.

Is rapid cold-hardening an aerobic process? Characterization of changes in metabolic activity during its induction and effects of anoxia in flesh fly

Rapid cold-hardening (RCH) is a type of phenotypic plasticity that promotes a swift improvement of cold tolerance in insects. A brief exposure to mild cold dramatically increases insect survival to a subsequent cold exposure that would be lethal otherwise. In adult male flesh fly, Sarcophaga bullata, as little as 15 min at 5 °C significantly improved organismal survival at −7°C from 0 to 66.7 ± 11.1%. In this study, we investigated whether this RCH response is an aerobic process in S. bullata by characterizing changes in metabolic activity during its induction. At the level of whole organism, CO2 production continued at a level above our detection limit, and a relatively greater rate was observed during the early phase before it stabilized after ~1 h of the RCH induction. Similarly, in isolated flight muscle tissues, those maintained at 5 °C for 10 min exhibited significantly greater rates of oxygen consumption, compared to those maintained at 5 °C for 1 h (2.82 ± 0.29 vs. 1.36 ± 0.22 μl O2 mg−1 DM h−1). When these tissues were exposed to LaCl3, a treatment that should inhibit RCH ex vivo, oxygen consumption rates of the muscles were reduced significantly to a level similar to those that had been maintained at 5 °C for 1 h. Interestingly, however, the RCH response was still evident among individuals exposed to chilling under anoxia. Compared to those exposed to anoxia for 30 min only at 25 °C, flies exposed to 5 °C for 2 h under anoxia following the initial exposure exhibited a significantly greater level of cold tolerance at −7.5 °C (41.7 ± 7.1 vs. 91.8 ± 3.9%). Our results suggest that while relatively greater rates of metabolic activity are associated with the early phase of the RCH induction, it can proceed under the anoxic condition, thereby suggesting its independence to aerobic respiration.

Detection of fly artifacts from four species of necrophagous flies on household materials using immunoassays.

An immunoassay was previously developed as a technique to improve methods for detection and analysis of fly artifacts found at crime scenes. The dot blot assay utilized a polyclonal antiserum (anti-md3) based on a unique digestive cathepsin D found in cyclorrhaphous Diptera. In this study, artifacts produced by adults of Calliphora vicina, Cynomya cadaverina, Sarcophaga bullata, and Protophormia terraenovae were examined using the immunoassay to determine if insect-derived stains could be distinguished from a range of human body fluid stains. A lift technique was developed which permitted transfer of fly artifacts from test materials to filter paper for dot blot analyses. All species readily deposited artifacts on all test household materials regardless of diet consumed. Despite differences in texture and porosity of the household materials, artifacts of all species transferred to the filter paper. With all fly species, anti-md3 serum bound to artifacts produced after feeding on semen, blood, feces, urine, and saliva. By contrast, anti-md3 serum did not react with any of the human fluids tested, nor with any of the lifts from household materials not exposed to flies. There was no evidence of false positives with any of the fly species tested, regardless of diet consumed. There was also no indication of false negatives with any of the dot blot assays. These observations suggest that immunoassays using anti-md3 serum performed on a simple lift of suspected fly artifacts can be used effectively as a confirmatory assay to distinguish fly regurgitate and fecal stains from human body fluids.

Control of Filth Flies, Cochliomyia macellaria (Diptera: Calliphoridae), Musca domestica (Diptera: Muscidae), and Sarcophaga bullata (Diptera: Sarcophagidae), Using Novel Plant-Derived Methyl Ketones.

Filth flies live in close proximity to humans and livestock and transmit pathogens. Current control relies on chemical insecticides, and flies can develop resistance to these insecticides. The public is also interested in natural and safer insecticides. Therefore, alternative pesticides compatible with the synanthropic nature of flies are needed. Four plant aliphatic methyl ketones were evaluated for control of adult house flies, Musca domestica L., blow flies, Cochliomyia macellaria (F.), and gray flesh flies, Sarcophaga bullata (Parker). In sealed petri dish assays, 2-heptanone, 2-octanone, 2-nonanone, and 2-undecanone exhibited fumigant activity against house flies with 24-h LC50s of 6.9, 7.5, 8.0, and 9.2 µg/cm3, respectively. Further research focused on undecanone (a U.S. EPA-registered biopesticide). When tested in larger enclosures at 1.7, 2.3, and 2.8 µg/cm3, undecanone provided 60.4, 82.2, and 94.4% house fly mortality; 56.9, 75.6, and 92.5% flesh fly mortality; and 62.1, 84.5, and 97.9% blow fly mortality, respectively, after a 2-h exposure. In a two-choice behavioral assay with 194.6 µg/cm2 of the test compound on the treatment versus an untreated surface of the same area, the overall mean repellencies for blow flies were 84.7% for undecanone versus 87.6% for N,N-diethyl-meta-toluamide (DEET). For house flies, mean repellencies were 80.7% for undecanone and 84.9% for DEET. The house fly topical LD50 for undecanone was 58.1 µg per fly. Undecanone was far less expensive for filth fly control than the gold standard for insect fumigation, methyl bromide.

Genome and Ontogenetic-Based Transcriptomic Analyses of the Flesh Fly,Sarcophaga bullata

The flesh fly, Sarcophaga bullata, is a widely-used model for examining the physiology of insect diapause, development, stress tolerance, neurobiology, and host-parasitoid interactions. Flies in this taxon are implicated in myiasis (larval infection of vertebrates) and feed on carrion, aspects that are important in forensic studies. Here we present the genome of S. bullata, along with developmental- and reproduction-based RNA-Seq analyses. We predict 15,768 protein coding genes, identify orthology in relation to closely related flies, and establish sex and developmental-specific gene sets based on our RNA-Seq analyses. Genomic sequences, predicted genes, and sequencing data sets have been deposited at the National Center for Biotechnology Information. Our results provide groundwork for genomic studies that will expand the flesh fly’s utility as a model system.

Disruptive Effectiveness of Methoprene (A Juvenile Hormone Analogue) on Adult Performance and Natality of the Grey Flesh Fly Parasarcophaga argyrostoma (Diptera: Sarcophagidae)

The grey flesh fly Parasarcophaga argyrostoma plays a role in human cutaneous wounds and eye myiasis as well as it acts as a parasitoid of various animals. The present study aimed to investigate the efficacy of methoprene on the adult performance and natality of this fly. Five dose levels (10.0, 5.0, 1.0, 0.1 and 0.01 μg/larva) of methoprene was topically applied onto the early last instar larvae and prepupae. The adult emergence of flies was completely blocked at the highest dose, regardless of the time of treatment. At other dose levels, the adult eclosion was partially blocked. Survival of the adult flies was adversely affected by the toxic potency of methoprene. Only at doses of 5.0 and 1.0 μg/larva, some morphologically deformed flies were observed. The total longevity of adult females was remarkably shortened in a dose-dependent course. Topical application of methoprene onto either last instar larvae or prepupae resulted in a considerably prolonged pre-larviposition period, but significantly shortened larviposition period. Topical treatment of either last instar larvae or prepupae with methoprene led to dramatic reduction in natality, in a dose-dependent course.

Evaluating the evolution and function of the dynamic Venom Y protein in ectoparasitoid wasps.

Venom of the parasitoid wasp Nasonia vitripennis changes the metabolism and gene expression in its fly host Sarcophaga bullata to induce developmental arrest, suppression of the immune response and various other venom effects. Yet, the venom of ectoparasitoid wasps has not been fully characterized. A major component of N.vitripennis venom is an uncharacterized, high-expressing protein referred to as Venom Y. Here we describe the evolutionary history and possible functions of this venom protein. We found that Venom Y is a relatively young gene that has duplicated to form two distinct paralogue groups. A copy of Venom Y has been recruited as a venom protein in at least five wasp species. Functional analysis found that Venom Y affects detoxification and immunity genes in envenomated fly hosts. Many of these genes are fat-body specific, suggesting that Venom Y may have a targeted effect on fat body tissue. We also show that Venom Y may mitigate negative effects of other venom proteins. Finally, protein sequencing indicates that Venom Y is post-translationally modified. This study contributes to elucidating parasitoid venom by using RNA interference knockdown to investigate venom protein function in the context of the whole venom cocktail.

Two isoforms of Pepck in Sarcophaga bullata and their distinct expression profiles through development, diapause, and in response to stresses of cold and starvation

Pepck is a metabolic enzyme that participates in gluconeogenesis through the conversion of oxaloacetate into phosphoenol pyruvate. Numerous transcriptomic studies have identified Pepck as a potential key player during diapause and various stresses responses. Here, we describe expression patterns of both cytosolic and mitochondrial isoforms of Pepck throughout development, during diapause, and in response to starvation and cold shock in the flesh fly, Sarcophaga bullata. We cloned full-length transcripts for both Pepck isoforms and observed that expression of both genes varied throughout development. Diapausing pupae have the highest relative expression of both isoforms, suggesting participation in the anticipatory production of sugars and sugar alcohols that occurs during this overwintering stage. In response to acute stress, the cytosolic isoform was upregulated whereas the mitochondrial variant remained unchanged. Cytosolic Pepck was strongly upregulated after 2 h recovery from cold shock and returned to baseline levels within 8 h. In response to 24 h of starvation, the cytosolic isoform was similarly upregulated and returned to control levels after 24 h of recovery. Acute stress is known to incur a metabolic cost, and Pepck could be a key player in this response. Although it remains unclear why there is such a dramatic divergence in the expression of the two isoforms, the distinction suggests specific roles for the two isoforms that depend on the developmental status of the fly and the stress conditions to which it is exposed.

Toxicity And Physiological Activity of Methoprene, A Juvenile Hormone Analog, Against Development and Metamorphosis of The Grey Flesh Fly, Parasarcophaga argyrostoma (Robineau- Desvoidy)(Diptera: Sarcophagidae).

The grey flesh fly Parasarcophaga argyrostoma plays a role in human cutaneous wounds and eye myiasis and it is also known as parasitoid of various animals. The present study aimed to investigate the efficacy of Methoprene on survival, development and metamorphosis of this fly species. Five dose levels (10.0, 5.0, 1.0, 0.1 and 0.01µg/larva) of Methoprene was topically applied onto the early last instar larvae and prepupae. Methoprene exhibited larvicidal, pupicidal and adulticidal activities against P. argyrostoma. LD50 values were found 0.155 and 0.258 µg/insect after topical treatment of early last instar larvae and prepupae, respectively. The maximal body weight of treated larvae was considerably decreased. The duration of treated larvae was prolonged. The coefficient of growth of treated larvae was depressed. The pupal duration was remarkably prolonged. Some larval-pupal intermediates had been produced, only at the higher two doses. Topical treatment of prepupae only with the lower two doses induced a state of 'permanent prepupae'. The treated last instar larvae pupated in regressed rate. The pupation rate was slightly regressed after topical treatment of prepupae only with the lower two doses. The adult emergence of flies was completely blocked after topical treatment of either the early last instar larvae or prepupae with the highest dose of Methoprene. At other dose levels, the adult eclosion of flies was partially blocked. Different percentages of deformed pupaeand adults were recorded.

Semiochemical Exploitation of Host-Associated Cues by Seven Melittobia Parasitoid Species: Behavioral and Phylogenetic Implications

Chemical compounds (infochemicals or semiochemicals) play an important role both in intra-specific and inter-specific communication. For example, chemical cues appear to play a key role in the host selection process adopted by insect parasitoids. In recent years significant advances have been made in order to understand the chemical ecology of insect parasitoids. However, little information is available about the evolution of semiochemical use in the host location process of insect parasitoids. Here we investigated the strategy adopted by seven closely related parasitoid species in the genus Melittobia when foraging for four different suitable hosts. By using an integrated approach that combined olfactometer bioassays and phylogenetic investigations, we found that: 1) exploitation of host-derived semiochemicals is widespread in the Melittobia genus; 2) there is specificity of attraction towards the different host species tested; in particular, the early-branching species in the Melittobia genus are attracted to odors associated with leaf cutting bees (Megachile rotundata) whereas the most-diverged species are attracted to odors associated with solitary mud dauber wasps (Trypoxyilon politum). Regardless of the phylogenetic relationships, no Melittobia species exhibited attraction towards odors of factitious laboratory hosts (i.e. the flesh fly Sarcophaga bullata). Interestingly, five Melittobia species are also attracted by odors associated with honeybees hosts which indicate that these parasitoids could be potential pests of honeybees. Our study shed light on the host location within the Melittobia genus and represents a first attempt to understand semiochemical use in an evolutionary perspective in the context of parasitoids’ foraging behavior.

Copper(II) complexes of the Neb-colloostatin analogues containing histidine residue structure stability biological activity

Neb-colloostatin was discovered during the isolation of Neb-TMOF from the ovary of the grey fleshfly Neobellieria bullata. Its amino acid sequence was determined as SIVP4LGLP8VP10IGP13IVVGP18R. The analogues with point mutation P4H and P8H were synthesized and their copper(II) complexes were studied by potentiometry, UV–Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation 1:1 and 2:1 metal-to-ligand molar ratios for both peptides were studied. At physiological pH (7.4), both peptides form mononuclear the CuH-1L complex with 3N{NH2, N−, NIm} coordination mode. For the P4H and P8H peptides at high pH (∼11) the binding sites of copper(II) ions are quite different. For the CuH-4L complex of the P4H peptide the 4N{4N−} coordination mode is dominante, while for the P8H peptide the 4N{NIm, 3N−} binding site towards N-termini is formed with (6,5,5) chelate ring. At high pH the P8H peptide cannot form the 4N {NH2, 3N−} complex because of the presence of the Pro4 residue in the amino acid sequence (a break point in the metal coordination). At pH 7.4 dinuclear the Cu2H-4L complexes dominate with suggested the 3N{NH2, 2N−}3N{NIm, 2N−}binding sites where the 3N{NIm, 2N−} coordination mode for the P4H peptide towards C-termini, and for P8H peptide towards N-termini are formed. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The systems studied have lower apoptotic properties compared to those of Neb-colloostatin and its copper(II) complex at pH 7.4.

Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9

The revolutionary RNA-guided endonuclease CRISPR/Cas9 system has proven to be a powerful tool for gene editing in a plethora of organisms. Here, utilizing this system we developed an efficient protocol for the generation of heritable germline mutations in the parasitoid jewel wasp, Nasonia vitripennis, a rising insect model organism for the study of evolution, development of axis pattern formation, venom production, haplo-diploid sex determination, and host–symbiont interactions. To establish CRISPR-directed gene editing in N. vitripennis, we targeted a conserved eye pigmentation gene cinnabar, generating several independent heritable germline mutations in this gene. Briefly, to generate these mutants, we developed a protocol to efficiently collect N. vitripennis eggs from a parasitized flesh fly pupa, Sarcophaga bullata, inject these eggs with Cas9/guide RNA mixtures, and transfer injected eggs back into the host to continue development. We also describe a flow for screening mutants and establishing stable mutant strains through genetic crosses. Overall, our results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in N. vitripennis, with strong potential for expansion to target critical genes, thus allowing for the investigation of several important biological phenomena in this organism.

Changes in microRNA abundance may regulate diapause in the flesh fly, Sarcophaga bullata

Diapause, an alternative developmental pathway characterized by changes in developmental timing and metabolism, is coordinated by molecular mechanisms that are not completely understood. MicroRNA (miRNA) mediated gene silencing is emerging as a key component of animal development and may have a significant role in initiating, maintaining, and terminating insect diapause. In the present study, we test this possibility by using high-throughput sequencing and qRT-PCR to discover diapause-related shifts in miRNA abundance in the flesh fly, Sarcophaga bullata. We identified ten evolutionarily conserved miRNAs that were differentially expressed in diapausing pupae compared to their nondiapausing counterparts. miR-289-5p and miR-1-3p were overexpressed in diapausing pupae and may be responsible for silencing expression of candidate genes during diapause. miR-9c-5p, miR-13b-3p, miR-31a-5p, miR-92b-3p, miR-275-3p, miR-276a-3p, miR-277-3p, and miR-305-5p were underexpressed in diapausing pupae and may contribute to increased expression of heat shock proteins and other factors necessary for the enhanced environmental stress-response that is a feature of diapause. In S. bullata, a maternal effect blocks the programming of diapause in progeny of females that have experienced pupal diapause, and in this study we report that several miRNAs, including miR-263a-5p, miR-100-5p, miR-125-5p, and let-7-5p were significantly overexpressed in such nondiapausing flies and may prevent entry into diapause. Together these miRNAs appear to be integral to the molecular processes that mediate entry into diapause.

Desiccation enhances rapid cold-hardening in the flesh fly Sarcophaga bullata: evidence for cross tolerance between rapid physiological responses.

Many insects use rapid cold-hardening (RCH), a physiological response to sub-lethal exposure to stressors, such as chilling and desiccation, to enhance their cold tolerance within minutes. Recently, drought-induced RCH, triggered by brief, mild desiccation, was described in larvae of the freeze-tolerant gall fly (Eurosta solidaginis). However, its prevalence and ecological significance in other insects is not known. Consequently, we used a freeze-intolerant model, the flesh fly, Sarcophaga bullata, to investigate the effects and mechanisms of drought-induced RCH. In addition, we investigated how drought- and cold-induced RCH interact by exposing flies to both desiccation and chilling. Desiccation for 3h increased larval pupariation after cold shock from 28 to 40%-the first example of drought-induced RCH in both a freeze-intolerant insect and in a non-overwintering life stage. We also found that desiccation and chilling together enhanced the cold hardiness of larvae and adults more than either did separately, suggesting that drought and cold trigger distinct physiological mechanisms that interact to afford greater cold tolerance. These results suggest that drought-induced RCH is a highly conserved response used by insects with diverse life history strategies. Furthermore, the protective interaction between drought- and cold-induced RCH suggests that, in nature, insects use multiple cues and physiological mechanisms to fine-tune their response to changing ambient conditions.