Melanin and Melanogenesis at Different Life Stages in <Emphasis Type="Italic">Hermetia illucens</Emphasis>

This work has demonstrated that the life stages of the Hermetia illucens fly are characterized by varying melanin contents with the maximum melanin amount being detected in the pupa. It has been observed that active synthesis of the pigment takes place in the prepupa and coincides with the time when the population migrated for pupation. It has been noted that a high manganese content in the prepupa and pupa indicates the important role that this biogenic element plays in organogenesis and melanogenesis in H. illucens. It has been revealed that in the imago and pupa, the melanin demonstrates broad-range antimicrobial activity and contribute to the insect protection against bacterial and fungal infections.     

Fresh and Dry Mass Estimates of <Emphasis Type="Italic">Hermetia illucens</Emphasis> (Linnaeus, 1758) (Diptera: Stratiomyidae) Larvae Associated with Swine Decomposition in Urban Area of Central Amazonia

Information on biomass is substantial for calculating growth rates and may be employed in the medicolegal and economic importance of Hermetia illucens (Linnaeus, 1758). Although biomass is essential to understanding many ecological processes, it is not easily measured. Biomass may be determined by directly weighing or indirectly through regression models of fresh/dry mass versus body dimensions. In this study, we evaluated the association between morphometry and fresh/dry mass of immature H. illucens using linear, exponential, and power regression models. We measured width and length of the cephalic capsule, overall body length, and width of the largest abdominal segment of 280 larvae. Overall body length and width of the largest abdominal segment were the best predictors for biomass. Exponential models best fitted body dimensions and biomass (both fresh and dry), followed by power and linear models. In all models, fresh and dry biomass were strongly correlated (>75%). Values estimated by the models did not differ from observed ones, and prediction power varied from 27 to 79%. Accordingly, the correspondence between biomass and body dimensions should facilitate and motivate the development of applied studies involving H. illucens in the Amazon region.     

Taxonomy of Soldier Flies (Diptera: Stratiomyidae) of Sunderban Biosphere Reserve,India

The present communication deals with recording of nine species of soldier flies (Insecta: Diptera: Stratiomyidae) from Sunderban Biosphere Reserve of India, of which, Tinda indica (Walker, 1851), Hermetia illucens (Linnaeus, 1758), Odontomyia kashmirensis Brunetti, 1920, Odontomyia viridana (Wiedemann, 1824), Prosopochrysa vitripennis (Doleschall, 1856) are being reported for the first time from this unique and globally acclaimed mangrove ecosystem. Odontomyia kashmirensis Brunetti, 1920 is reported for the first time from the state of West Bengal. A key to identify these species is provided with taxonomic notes.     

Characterization of an inhibitor-resistant endo-1,4-β-mannanase from the gut microflora metagenome of <Emphasis Type="Italic">Hermetia illucens</Emphasis>

Objective

Hermetia illucens is a voracious insect scavenger that efficiently decomposes food waste. To exploit novel hydrolytic enzymes from this insect, we constructed a fosmid metagenome library using unculturable H. illucens intestinal microorganisms.

Results

Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone with a product displaying hydrolytic activity. Fosmid sequence analysis revealed a novel mannan-degrading gene (ManEM17) composed of 1371 base pairs, encoding 456 amino acids with a deduced 54 amino acid N-terminal signal peptide sequence. Conceptual translation and domain analysis revealed that sequence homology was highest (46%) with endo-1,4-β-mannosidase of Anaerophaga thermohalophila. Phylogenetic and domain analysis indicated that ManEM17 belongs to a novel β-mannanase containing a glycoside hydrolase family 26 domain. The recombinant protein (rManEM17) was expressed in Escherichia coli, exhibiting the highest activity at 55 °C and pH 6.5. The protein hydrolyzed substrates with β-1,4-glycosidic mannoses; maximum specific activity (5467 U mg^?1) occurred toward locust bean gum galactomannan. However, rManEM17 did not hydrolyze p-Nitrophenyl-β-pyranosides, demonstrating endo-form mannanase activity. Furthermore, rManEM17 was highly stable under stringent conditions, including polar organic solvents as well as chemical reducing and denaturing reagents.

Conclusions

ManEM17 is an attractive candidate for mannan degradation under the high-organic-solvent and protein-denaturing processes in food and feed industries.

     

Paramagnetics melanin and Mn<Superscript>2+</Superscript> in black soldier fly <Emphasis Type="Italic">Hermetia illucens</Emphasis>

Larva, prepupa (last instar larva), pupa, and an empty shell of pupa after hatching of the black soldier fly Hermetia illucens contain eumelanin, an active synthesis of which is observed at the prepupal stage, which is probably due to the release of prepupa from the feed substrate thickness to the open space for pupation. It was shown for the first time that prepupa contains high quantities of the magnetically active form of manganese Mn²⁺. This fact indicates that Mn²⁺ stimulates the copper-containing tyrosinase—the key enzyme in the synthesis of melanin in the period of migration and adaptation of the insect to the solar radiation.     

Susceptibility of shore fly <Emphasis Type="Italic">Scatella stagnalis</Emphasis> to five entomopathogenic nematode strains in bioassays

The shore fly, Scatella stagnalis (Fallén) (Diptera: Ephydridae) is an important insect pest of greenhouse crops. We evaluated two different Spanish isolates of entomopathogenic nematodes, Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) and Steinernema arenarium (Artyukhovsky) (Rhabditida: Steinernematidae), and two commercially available strains, Steinernema feltiae (Nemaplus^®) and Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae) (Nematop^®) against shore flies. In tests conducted in 24-well plate filter paper applied at 5, 11, 22, 44 and 88 nematodes per larva, all nematodes produced significant shore fly larval mortality. The lowest concentration tested was enough to obtain high larval mortality (65.2–87.0%). The nematodes Steinernema feltiae and Steinernema arenarium, which parasitized the shore fly larvae faster, also penetrated in higher number in the shore fly larva (4.6–8.8% penetration rate). In bioassays conducted in algae, Steinernema feltiae, applied at 50 nematodes/cm², caused highest (100%) and Steinernema arenarium lowest shore fly mortality (94%). Our results suggest that entomopathogenic nematodes appear feasible for controlling shore flies but further tests are needed to determine their efficacy in the field.     

Somatic organic content of the ctenophores <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis> (Ctenophora: Lobata) and <Emphasis Type="Italic">Beroe ovata</Emphasis> (Ctenophora: Beroida) in early ontogenetic stages

The organic matter content in the eggs and early larvae of the ctenophores Mnemiopsis leidyi and Beroe ovata from the Black Sea was determined using the adapted microtechnique of dichromate oxidation. The content of organic matter in the eggs of M. leidyi (0.25 μg/indiv.) was 5 times less than in B. ovata (1.28 ± 0.29 μg/indiv.). The somatic content of organic matter was 0.25 ± 0.09 μg/indiv. (25.1 ± 8.3 μg/mg, wet wt) for 2-day-old larvae of M. leidyi (0.2–0.3 mm in body length) and 1.37 ± 0.19 μg/indiv. (67.1 ± 5.7 μg/mg wet wt) for larvae of B. ovata (0.4 mm in body length). The specific organic content of larvae of both species steadily decreased with an increase in ctenophore body size and weight, approaching 3–4 μg/mg of wet weight for 2 mm specimens of M. leidyi and 3–5 μg/mg of wet weight for 6 mm B. ovata. The specific organic content of early larvae was 20–30 times higher than that of adult ctenophores. The results of this investigation could be useful in the evaluation of the energy budget for somatic growth and generative production in these species. Calculations indicate that with specific wet weight growth rates of 0.43/day for M. leidyi larvae and 0.29/day for B. ovata larvae, their true organic increases are respectively 30 and 38% less, i.e., no more than 0.31/day for the former and 0.18/day for the latter species.     

Biological Efficiency of the Prepupae <Emphasis Type="Italic">Hermetia illucens</Emphasis> in the Diet of the Young Mozambique Tilapia <Emphasis Type="Italic">Oreochromis mossambicus</Emphasis>

It has been noted that the introduction of small doses of dry prepupae of Hermetia illucens flies into the diet of the young Mozambique tilapia Oreochromis mossambicus (0.5 g/kg feed) had a positive effect on the growth and development of fish. It was found that the average daily gain in the experimental group was 0.7 g. It was established that the blood biochemical parameters were within the norm in the fish of all groups; however, in the experimental tilapia, the erythrocyte sedimentation rate was significantly lower and a tendency to increase in the amount of hemoglobin and glucose was noted. It was revealed that in the seminiferous tubules of fish in the experimental group, the number of mature sperm was 5.9 times higher compared with the control, indicating an increase in the rate of spermatogenesis. It is assumed that the observed effects are due to the high content of manganese in the prepupae present in a biologically active divalent form.     

Bacterial diversity obtained by culturable approaches in the gut of <Emphasis Type="Italic">Glossina pallidipes</Emphasis> population from a non sleeping sickness focus in Tanzania: preliminary results

Background

Glossina pallidipes is a haematophagous insect that serves as a cyclic transmitter of trypanosomes causing African Trypanosomiasis (AT). To fully assess the role of G. pallidipes in the epidemiology of AT, especially the human form of the disease (HAT), it is essential to know the microbial diversity inhabiting the gut of natural fly populations. This study aimed to examine the diversity of G. pallidipes fly gut bacteria by culture-dependent approaches.

Results

113 bacterial isolates were obtained from aerobic and anaerobic microorganisms originating from the gut of G. pallidipes. 16S rDNA of each isolate was PCR amplified and sequenced. The overall majority of identified bacteria belonged in descending order to the Firmicutes (86.6%), Actinobacteria (7.6%), Proteobacteria (5.5%)and Bacteroidetes (0.3%). Diversity of Firmicutes was found higher when enrichments and isolation were performed under anaerobic conditions than aerobic ones. Experiments conducted in the absence of oxygen (anaerobiosis) led to the isolation of bacteria pertaining to four phyla (83% Firmicutes, 15% Actinobacteria, 1% Proteobacteria and 0.5% Bacteroidetes, whereas those conducted in the presence of oxygen (aerobiosis) led to the isolation of bacteria affiliated to two phyla only (90% Firmicutes and 10% Proteobacteria). Phylogenetic analyses placed these isolates into 11 genera namely Bacillus, Acinetobacter, Mesorhizobium, Paracoccus, Microbacterium, Micrococcus, Arthrobacter, Corynobacterium, Curtobacterium, Vagococcus and Dietzia spp.which are known to be either facultative anaerobes, aerobes, or even microaerobes.

Conclusion

This study shows that G. pallidipes fly gut is an environmental reservoir for a vast number of bacterial species, which are likely to be important for ecological microbial well being of the fly and possibly on differing vectorial competence and refractoriness against AT epidemiology.

     

The combined effects of blue light and dilution rate on lipid class and fatty acid composition of <Emphasis Type="Italic">Tisochrysis lutea</Emphasis>

The lipid class and the fatty acid compositions of microalgae highly influence bivalve larval and post-larval development. Light is an essential environmental factor for microalgal culture, and quantity and quality of light may induce changes in the biochemical composition of the algae. The objective of this study was to investigate the effect of light spectrum (blue vs. white light) on lipid class and fatty acid compositions of Tisochrysis lutea cultured in a chemostat. Two different dilution rates (D) were assayed for each light spectrum: 0.2 and 0.7 day^?1. Triacylglycerol (TAG), sterol, and hydrocarbon (HC) content increased sharply at low D. The proportion of alkenones was significantly reduced under blue light. Polyunsaturated fatty acids (PUFA), and particularly n-3 PUFA, content in phospholipids (PL) increased under blue light compared to white light at low D. Thus, blue light raised 22:6(n-3) levels in total lipids of T. lutea at low D. The cultivation of T. lutea in a chemostat at low D under blue light may improve nutritional value as feed for bivalve larvae by modifying the PUFA profile, especially increasing 22:6(n-3).     

Walnut husk fly, <Emphasis Type="Italic">Rhagoletis completa</Emphasis> (Diptera: Tephritidae), invades Europe: invasion potential and control strategies

Rhagoletis completa Cresson (Diptera: Tephritidae) is native to North America and invaded Western Europe in the late 1980s, causing important damage to its principal host, walnut (Juglans spp.). In this review, we summarize the important elements of R. completa’s biology, phytosanitary status and methods used in Europe for its control, and then present the main conclusions associated with a completed risk analysis performed in 2014 to evaluate the dispersion and establishment potential of R. completa in Europe. The walnut husk fly was initially identified in Switzerland (1988) and Italy (1991), from where it spread to at least seven additional countries: France, Spain, Germany, Austria, Croatia, Slovenia and Hungary. R. completa has not reached the limits of its potential distribution. The main dissemination pathways within Europe include: (1) natural adult dissemination; (2) adult hitchhiker behaviour; and, to a lesser extent, (3) transportation of larvae in fresh fruits. R. completa host plants are widely distributed in Europe, either as isolated wild trees or in orchards, favouring the probability of fly establishment in currently fly-free areas. In addition, the European territories where Juglans species are present share biogeographic similarities. In orchards where R. completa is present and uncontrolled, 100% of walnut trees can be infested, causing losses in walnut yields of up to 80%. The negative effect is low (<10% yield loss) under phytosanitary control, although additional costs must also be considered to support specific monitoring for R. completa. The information presented here underlines a strong need for better walnut husk fly monitoring across European countries, as well as for increasing efforts to develop biological methods to control this emerging pest.     

Bacterial diversity of the outflows of a Polichnitos (Lesvos,Greece) hot spring,laboratory studies of a <Emphasis Type="Italic">Cyanobacterium</Emphasis> sp. strain and potential medical applications

The bacterial diversity of the outflows of Polichnitos (Lesvos, Greece) hot spring has been investigated. Cyanobacteria showing high sequence homologies with Phormidium sp. and Cyanobacterium aponinum were found. Members of the Alphaproteobacteria closely related to Rhodobium sp. Albidovulum sp., Rhodobacter sp., Microvigra sp., Nitratireductor sp. and Phaeobacter sp. Gammaproteobacteria, Betaproteobacteria and Firmicutes were represented by members of Idiomarina sp., Marinobacter sp., Shinella sp., Bacillus sp. and Clostridium sp. with sequence homologies ranging from 92% to 100%. Members of the Bacteroidetes and Planctomycetes were represented by sequences of novel phylogenetic linkages exhibiting 87–90% sequence homology with type strains. When the hot spring consortium was cultivated in bioreactor repeated batch culture under photo-autotrophic growth conditions at temperature < 30 °C, Cyanobacterium sp. dominated over Phormidium sp. Cyanobacterium sp. seems to have biotechnological potential since its extracellular broth exhibited a strong insecticidal activity against larvae of Aedes aegypti (a vector of important human diseases) and significant anti-cancer activity against the PC3 human prostate cancer cell line, while its toxicity against human endothelial cells was relatively low.     

Improving biocontrol of black vine weevil (<Emphasis Type="Italic">Otiorhynchus sulcatus</Emphasis>) with entomopathogenic fungi in growing media by incorporating spent mushroom compost

Amending a peat-based growing medium with 10% v/v spent mushroom compost, a source of fungal chitin and other nutrients, prolonged the persistence of entomopathogenic fungi (Metarhizium brunneum Petsch and Beauveria bassiana (Balsamo) Vuillemin; Hypocreales: Clavicipitaceae). This resulted in improved efficacy of M. brunneum against black vine weevil, Otiorhynchus sulcatus F. (Coleoptera: Curculionidae) larvae compared with using inoculum without spent mushroom compost. B. bassiana only controlled larvae when used in combination with spent mushroom compost (75?±?7% reduction in live larvae). Mixing entomopathogenic fungal inoculum with spent mushroom compost and growing medium was as effective in controlling black vine weevil larvae as using spent mushroom compost colonised with M. brunneum or B. bassiana in the growing medium (80?±?12% reduction in live larvae). The former method is preferable since it does not require production and storage of colonised spent mushroom compost, or registration of new substrate formulations of M. brunneum or B. bassiana.     

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.     

Isolation,characterization and virulence of bacteria from <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis> (Lepidoptera: Pyralidae)

Isolation, characterization and virulence of the culturable bacteria from entire tissues of larval Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) were studied to obtain new microbes for biological control. A total of 16 bacteria were isolated from living and dead larvae collected from different maize fields in the Eastern Black Sea Region of Turkey. The bacterial microbiota of O. nubilalis were identified as Pseudomonas aeruginosa (On1), Brevundimonas aurantiaca (On2), Chryseobacterium formosense (On3), Acinetobacter sp. (On4), Microbacterium thalassium (On5), Bacillus megaterium (On6), Serratia sp. (On7), Ochrobactrum sp. (On8), Variovorax paradoxus (On9), Corynebacterium glutamicum (On10), Paenibacillus sp. (On11), Alcaligenes faecalis (On12), Microbacterium testaceum (On13), Leucobacter sp. (On14), Leucobacter sp. (On15) and Serratia marcescens (On16) based on their morphological and biochemical characteristics. A partial sequence of the 16S rRNA gene was also determined to confirm strain identification. The highest insecticidal activities were obtained from P. aeruginosa On1 (80%), Serratia sp. On7 (60%), V. paradoxus On9 (50%) and S. marcescens On16 (50%) against larvae 14 days after treatment (p < 0.05). Also, the highest activity from previously isolated Bacillus species was observed from Bacillus thuringiensis subsp. tenebrionis Xd3 with 80% mortality within the same period (p < 0.05). Our results indicate that P. aeruginosa On1, Serratia sp. On7, V. paradoxus On9, S. marcescens On16 and B. thuringiensis subsp. tenebrionis Xd3 show potential for biocontrol of O. nubilalis.     

Optimization of <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacteria culturing for obtaining myristoleic acid

Plankton filament cyanobacteria Prochlorothrix hollandica is characterized by a very high content of C₁₄ and C₁₆ fatty acids (FA) in the lipid membranes. Depending on culturing conditions of the cyanobacteria, total concentrations of myristic and myristoleic acids can reach 35% and those of palmitic and palmitoleic acids can reach 60% of all esterified FA cells. In P. hollandica, a variety of monounsaturated FA is represented by myristoleic and palmitic acids, and by hexadecenoic (C_16:1) acid with olefin bond of cis-configuration, located in the Δ4 position. The process of intensive culturing for P. hollandica cells to yield a maximal biomass in order to isolate the pure drug of myristoleic acid derivative has been optimized. The use of a threestage purification gives 30 mg of chromatographically pure myristoleic acid methyl ester from 17 g of P. hollandica raw biomass (dry mass is 3 g), which is 1% of dry cell mass.     

Laboratory and simulated-field bioassays for assessing mixed cultures of <Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> against <Emphasis Type="Italic">Aedes aegypti</Emphasis> (Diptera: Culicidae) larvae resistant to temephos

Aedes aegypti (L.) is the main vector of tropical diseases such as dengue, chikungunya and Zika. Due to the overuse of insecticides, Ae. aegypti resistant populations have increased. Biological control with Lysinibacillus sphaericus (Ahmed) has been used against Culex sp. and Anopheles sp. Although Ae. aegypti is refractory to the binary toxin of L. sphaericus spores, vegetative cells have been shown to be effective against Ae. aegypti larvae. In this work, the effect of L. sphaericus vegetative cells on Ae. aegypti temephos-resistant larvae was assessed under lab and simulated field conditions. L. sphaericus caused about 90% mortality of insecticide-resistant Ae. aegypti larvae under simulated field conditions. Likewise, Ae. aegypti larvae were more sensitive to mixed cultures of L. sphaericus than to individual strains; then, the most effective mixed culture exhibited an LC₅₀ of 1.21 × 10⁵ CFU/mL with Rockefeller larvae and 8.04 × 10⁴ CFU/mL with field-collected larvae. Additionally, we found that mixed cultures composed of two L. sphaericus strains were more effective than a culture formed by the three strains. Our results suggest that mixed cultures comprising L. sphaericus vegetative cells could be useful for controlling temephos-resistant populations of Ae. aegypti, as evidenced by the effectiveness demonstrated under laboratory and simulated field conditions.     

Development and validation of KASP markers for the greenbug resistance gene <Emphasis Type="Italic">Gb7</Emphasis> and the Hessian fly resistance gene <Emphasis Type="Italic">H32</Emphasis> in wheat

Key message

Greenbug and Hessian fly are important pests that decrease wheat production worldwide. We developed and validated breeder-friendly KASP markers for marker-assisted breeding to increase selection efficiency.

Abstract

Greenbug (Schizaphis graminum Rondani) and Hessian fly [Mayetiola destructor (Say)] are two major destructive insect pests of wheat (Triticum aestivum L.) throughout wheat production regions in the USA and worldwide. Greenbug and Hessian fly infestation can significantly reduce grain yield and quality. Breeding for resistance to these two pests using marker-assisted selection (MAS) is the most economical strategy to minimize losses. In this study, doubled haploid lines from the Synthetic W7984 × Opata M85 wheat reference population were used to construct linkage maps for the greenbug resistance gene Gb7 and the Hessian fly resistance gene H32 with genotyping-by-sequencing (GBS) and 90K array-based single nucleotide polymorphism (SNP) marker data. Flanking markers were closely linked to Gb7 and H32 and were located on chromosome 7DL and 3DL, respectively. Gb7-linked markers (synopGBS773 and synopGBS1141) and H32-linked markers (synopGBS901 and IWB65911) were converted into Kompetitive Allele Specific PCR (KASP) assays for MAS in wheat breeding. In addition, comparative mapping identified syntenic regions in Brachypodium distachyon, rice (Oryza sativa), and sorghum (Sorghum bicolor) for Gb7 and H32 that can be used for fine mapping and map-based cloning of the genes. The KASP markers developed in this study are the first set of SNPs tightly linked to Gb7 and H32 and will be very useful for MAS in wheat breeding programs and future genetic studies of greenbug and Hessian fly resistance.