Diatoms in maggots; a potential tool for drowning diagnosis - A preliminary study.

Advanced putrefaction causes extensive loss of soft tissue, rendering it difficult to use the diatom test as a reliable diagnostic tool for drowning investigations. A positive diatom test in carrion insect larvae may provide significant assistance in overcoming the challenge of decomposition. The studies determined the utility of diatom test in carrion larvae on severely decomposed bodies. A modified acid digestion method involving nitric acid, K2Cr2O7 and HCl, was used to digest the blowfly larvae feeding on piglet carrion previously drowned in freshwater and sea water, respectively. Extracted diatom frustules were analysed and characterised using light microscopy coupled to a digital camera. Diatoms recovered from maggots on sea-drowned piglets were similar to diatoms from sea water (drowning medium). Centric diatoms recovered in maggots were higher (200 ± 60 diatoms/ mL) than pennate diatoms (80 ± 20 diatoms/mL). Isolated diatoms common to both maggots and water samples included Coscinodiscus sp. and Navicular spp. Albeit, there were no diatoms recovered from maggots on freshwater-drowned piglets. The findings of this study suggest that the diatom test is still a reliable diagnostic tool to determine if drowning was involved in the death of a fully decomposed body. This is the first study that isolated diatoms from maggots feeding on drowned bodies. It serves as the basis for further research into the utility of maggots for drowning investigations.

Effects of lighted incubation and foraging enrichment during rearing on individual fear behavior, corticosterone, and neuroplasticity in laying hen pullets.

Environmental conditions during incubation and rearing can influence stress responsivity of laying hen pullets throughout their lifespan, and therefore have important implications for their welfare. In this study, a 12:12h green LED light-dark cycle during incubation and larvae provisioning as enrichment during rearing were tested as strategies to optimize early-life conditions and thereby decrease stress responsivity in ISA Brown laying hens. A combination of parameters was measured to indicate neuronal, physiological, and behavioral changes that may affect fear and stress. The proteins calbindin D28k (calbindin1), doublecortin (DCX), and neuronal nuclein protein (NeuN) were quantified after hatch as a proxy for brain plasticity. Plasma and feather corticosterone levels were measured after hatch and at the end of the rearing phase, and fearfulness was investigated through a series of behavioral tests (i.e., voluntary approach, open field, tonic immobility, and manual restraint tests). No effects of light during incubation were found on calbindin1, DCX, or NeuN. Neither of the treatments affected corticosterone levels in blood plasma and feathers. Light-incubated pullets showed less fearfulness towards humans in the voluntary approach test, but not in the other behavioral tests reported in this study. Larvae provisioning had no effect on behavior. Our study showed minor effects of light during incubation and no effects of enrichment during rearing on stress responsivity of laying hen pullets. The small effects may be explained by the enriched rearing conditions for all birds in this experiment (low stocking density, natural daylight, and 24/7 classical music). Given the promising results of lighted incubation in other studies, which were mostly performed in broiler chickens, and evidence regarding the positive effects of enrichment during rearing, the potential of these strategies to improve laying hen welfare needs to be explored further.

Relationship of black soldier fly larvae (BSFL) gut microbiota and bioconversion efficiency with properties of substrates.

Treating food waste using black soldier fly larvae (BSFL) is widely regarded as a promising nature-based measure. This study explored the influence of food waste particle sizes on substrate properties and its subsequent effects on bioconversion efficiency and gut microbiota. The results indicated that particle sizes mainly ranging from 4 mm to 10 mm (T1) significantly increased the weight loss rate of food waste by 35 % and larval biomass by 38 % compared to those in T4 (particle sizes mostly less than 2 mm) and promoted the bioconversion of carbon and nitrogen into larvae and gases. Investigation of substrates properties indicated that the final pH value of T1 was 7.79 ± 0.10, with Anaerococcus as the predominant substrate microorganism (relative abundance: 57.4 %), while T4 exhibited a final pH value of 5.71 ± 0.24, with Lactobacillus as the dominant microorganism (relative abundance: 95.2 %). Correlation analysis between substrate chemical properties and microbial community structure unveiled a strong relationship between substrate pH and the relative abundance of Anaerococcus and Lactobacillus. Furthermore, beneficial microorganisms such as Lactobacillus and Enterococcus colonized the BSFL gut of T1, while pathogenic bacterium Morganella, detrimental to BSFL gut function, was enriched in T4 (relative abundance: 60.9 %). Nevertheless, PCA analysis indicated that alterations in the gut microbial community structure may not be attributed to the substrate microorganisms. This study establishes particle size as a crucial parameter for BSFL bioconversion and advances understanding of the relationship between gut microbiota and substrate microbiota.

Near-infrared Absorption and Emission Probes with Optimal Connection Bridges for Live Monitoring of NAD(P)H Dynamics in Living Systems.

Two NAD(P)H-biosensing probes consisting of 1,3,3-trimethyl-3H-indolium and 3-quinolinium acceptors, linked by thiophene, A, and 3,4-ethylenedioxythiophene, B, bridges are detailed. We synthesized probes C and D, replacing the thiophene connection in probe A with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe E was prepared by substituting probe A's 3-quinolinium unit with a 1-methylquinoxalin-1-ium unit. Solutions are non-fluorescent but in the presence of NADH, exhibit near-infrared fluorescence at 742.1 nm and 727.2 nm for probes A and B, respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes C and D displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe E exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe A led to a significant increase in fluorescence intensity. Additionally, subjecting probe A to lactate and pyruvate molecules resulted in opposite changes in NAD(P)H levels, with lactate causing a substantial increase in fluorescence intensity, conversely, pyruvate resulted in a sharp decrease. Treatment of A549 cells with varying concentrations of the drugs cisplatin, gemcitabine, and camptothecin (5, 10, and 20 µM) led to a concentration-dependent increase in intracellular fluorescence intensity, signifying a rise in NAD(P)H levels. Finally, fruit fly larvae were treated with different concentrations of NADH and cisplatin illustrating applicability to live organisms. The results demonstrated a direct correlation between fluorescence intensity and the concentration of NADH and cisplatin, respectively, further confirming the efficacy of probe A in sensing changes in NAD(P)H levels within a whole organism.

Exploiting Agri-Food Waste as Feed for Tenebrio molitor Larvae Rearing: A Review.

The agri-food industry generates substantial amounts of waste, including by-products and residues. The increasing demand for sustainable and eco-friendly practices in the agri-food sector has sparked an interest in finding alternative uses for such waste materials. One promising approach is the utilization of waste from the agri-food industry as feed for the rearing of mealworms (Tenebrio molitor). Since agri-food waste is rich in proteins, carbohydrates, lipids, and vitamins, as well as other bioactive compounds, all of which are essential for insect growth and development, incorporating such waste into the diet of mealworms promotes sustainable insect production, reducing the economic and environmental problems associated with waste disposal. This practice can also be beneficial for the rearing of mealworms since their nutritional value can also be enhanced. To this end, various waste materials, such as fruit and vegetable peels, spent grains, and food processing residues, have been investigated as potential feed sources, leading to increased mass production, lower cost, and enhanced nutritional value. This review aims to highlight the potential of agri-food waste as a feed source for mealworms, as well as their potential to enhance their nutritional value. Furthermore, the potential applications of mealworms reared on agri-food waste are highlighted, including their potential as a sustainable protein source for human consumption and as feed ingredients in the livestock and aquaculture sectors.

Potential of Thuja occidentalis L. Essential Oil and Water Extracts against Field Crop Pests.

Thuja occidentalis L. essential oil (EOTO) and its compounds, such as terpinyl acetate, bornyl acetate, and ß-thujone, are claimed to be highly effective against some storage pests, sanitary insects, or pests of fruit trees, while data about its use in protecting field crops are very scarce. There is also a lack of information in the literature about the insecticidal value of water extracts from T. occidentalis (WETOs). Both essential oils (EOs) and water extracts (WEs) from various plants have advantages and disadvantages in terms of their use as insecticides. EOs are generally more effective, but their preparation is more complicated and quite expensive. In turn, WEs are simple to prepare and cheap, but they often have limited effectiveness. Moreover, significant differences in responses exist depending on the species of the donor plant, the method of preparing the extract, its concentration, the species of the pest being controlled, the developmental stage, and even the gender of the pest. The goals of the research were to assess the effect of EOTO and WETOs prepared from dry and fresh matter on the mortality, feeding, and body mass changes of important crop pests, i.e., the black bean aphid, pea leaf weevil, and Colorado potato beetle (CPB), respectively, as well as on the mortality and voracity of non-target organism Asian lady beetle young larvae. EOTO showed significant aphicidal activity with LC50 = 0.8267% and 0.2453% after 42 h of the experiment for nymphs and wingless females of black bean aphid, respectively. Adults of CPB were more resistant to EOTO than aphids, with LC50 values for females equal to 1.5327% and 1.3113% after 48 h and after 72 h of the experiment. There was no significant effect of EOTO on CPB foraging. Calculated LC50 values for pea leaf weevil adults were lower than those for CPB (0.9638% and 0.8573% for males after 12 h and 24 h, respectively). In the case of this pest, a clear reduction in foraging was obtained, with higher concentrations of EOTO resulting in more pronounced reductions in foraging behavior. Concentrations of EOTO above 0.5%, which showed efficacy against the aphid, were lethal to 3-day-old larvae of the Asian lady beetle. WETOs, in turn, showed significant potential in inhibiting adult pea leaf weevil feeding, with very low or no effectiveness in reducing A. fabae and CPB, respectively.

The incorporation of strontium and barium into the otoliths of the flounder Paralichthys olivaceus at early life stages demonstrates resilience to ocean acidification.

Ocean acidification could modify the bioavailability and chemical properties of trace elements in seawater, which could affect their incorporation into the calcareous structures of marine organisms. Fish otoliths, biomineralized ear stones made by aragonite, are suspended within the endolymph fluid of teleosts, indicating that the elemental incorporation of otoliths might also be susceptible to ocean acidification. In this study, we evaluated the combined effects of CO2-induced ocean acidification (pH 8.10, 7.70, and 7.30, corresponding to ocean acidification scenarios under the representative concentration pathway 8.5 model as projected by the Intergovernmental Panel on Climate Change) and water elemental concentrations of strontium (Sr) and barium (Ba; low, medium, and high) on elemental incorporation into otoliths of the flounder Paralichthys olivaceus at early life stages. Our results revealed that the elemental incorporation of Sr and Ba into otoliths was principally dependent on the corresponding water elemental concentrations rather than on ocean acidification. Moreover, the partition coefficients (DMe) of Sr and Ba may stabilize after dynamic equilibrium is reached as the water elemental concentration increases, but are not affected by ocean acidification. Therefore, the incorporation of Sr and Ba into otoliths of the flounder at early life stages may not serve as an effective indicator of ocean acidification. In other words, the findings suggest that ocean acidification does not impact the incorporation of Sr and Ba incorporation into otoliths when tracing the temperature or salinity experiences of the flounder. Our findings will provide new knowledge for understanding the potential ecological effects of ocean acidification on the recruitment dynamics of fish species.

Unraveling the interplay between environmental microplastics and salinity stress on Mytilus galloprovincialis larval development: A holistic exploration.

The rise of plastic production has triggered a surge in plastic waste, overwhelming marine ecosystems with microplastics. The effects of climate change, notably changing salinity, have shaped the dynamics of coastal lagoons. Thus, understanding the combined impact of these phenomena on marine organisms becomes increasingly crucial. To address these knowledge gaps, we investigated for the first time the interactive effects of environmental microplastics (EMPs) and increased salinity on the early development of Mytilus galloprovincialis larvae. Morphological assessments using the larval embryotoxicity test revealed larval anomalies and developmental arrests induced by EMPs and increased salinity. Transcriptomic analyses targeting 12 genes involved in oxidative stress, apoptosis, DNA repair, shell formation, and stress proteins were conducted on D-larvae uncovered the potential effects of EMPs on shell biomineralization, highlighting the role of Histidine Rich Glycoproteine (HRG) and tubulin as crucial adaptive mechanisms in Mytilus sp. in response to environmental shifts. Furthermore, we explored oxidative stress and neurotoxicity using biochemical assays. Our findings revealed a potential interaction between EMPs and increased salinity, impacting multiple physiological processes in mussel larvae. Our data contribute to understanding the cumulative effects of emerging anthropogenic pollutants and environmental stressors, emphasizing the need for a holistic approach to assessing their impact on marine ecosystems.

Biodegradation of aged polyethylene (PE) and polystyrene (PS) microplastics by yellow mealworms (Tenebrio molitor larvae).

Globally, over 287 million tons of plastic are disposed in landfills, rivers, and oceans or are burned every year. The results are devastating to our ecosystems, wildlife and human health. One promising remedy is the yellow mealworm (Tenebrio molitor larvae), which has proved capable of degrading microplastics (MPs). This paper presents a new investigation into the biodegradation of aged polyethylene (PE) film and polystyrene (PS) foam by the Tenebrio molitor larvae. After a 35 - day feeding period, both pristine and aged MPs can be consumed by larvae. Even with some inhibitions in larvae growth due to the limited nutrient supply of aged MPs, when compared with pristine MPs, the aged MPs were depolymerized more efficiently in gut microbiota based on gel permeation chromatography (GPC) and Fourier transform infrared spectroscopy (FTIR) analysis. With the change in surface chemical properties, the metabolic intermediates of aged MPs contained more oxygen-containing functional groups and shortened long-chain alkane, which was confirmed by gas chromatography and mass spectrometry (GC-MS). High-throughput sequencing revealed that the richness and diversity of gut microbes were restricted in the MPs-fed group. Although MPs had a negative effect on the relative abundance of the two dominant bacteria Enterococcaceae and Lactobacillaceae, the aged MPs may promote the relative abundance of Enterobacteriaceae and Streptococcaceae. Redundancy analysis (RDA) further verified that the aged MPs are effectively biodegraded by yellow mealworm. This work provides new insights into insect-mediated mechanisms of aged MP degradation and promising strategies for MP sustainable and efficient solutions.

Extracellular traps development in canine neutrophils induced by infective stage Toxocara canis larvae.

Neutrophils, a crucial element of the host defense system, develop extracellular traps against helminth parasites. Neutrophils accumulate around the larvae of Toxocara canis (T. canis) in the tissues of the organism. This study aimed to determine the reaction in canine neutrophils after incubation with infective stage T. canis larvae (L3) in vitro. Most L3 were still active and moved between the extracellular traps (NETs) after 60-min incubation. NETs were not disintegrated by L3 movement. The L3 was only immobilized by NETs, entrapped larvae were still motile between the traps at the 24 h incubation. NETs were observed not only to accumulate around the mouth, excretory pole or anus but also the entire body of live L3. The extracellular DNA amount released from the canine neutrophils after being induced with phorbol 12-myristate 13-acetate was not affected by T. canis excretory/secretory products obtained from 250 L3. To the Authors'knowledge, the extracellular trap structures was firstly observed in canine neutrophils against T. canis L3 in vitro. NETs decorated with myeloperoxidase, neutrophil elastase and histone (H3) were observed under fluorescence microscope. There were not significant differences in the amount of extracellular DNA (P > 0.05), but the morphological structure of NETs was different in the live and head-inactivated T. canis larvae.

Expression of Heat Shock Protein 70 (HSP70) and Astacin Genes of Strongyloides stercoralis as well as HSP70 and HSP17.1 Genes of S. ratti in Adult and Larval Stages of S. stercoralis.

Background: Uncovering the roles and characteristics of pathogenesis-related molecules can help us develop novel management methods in parasitology. In this study, we studied the expression levels of Strongyloides stercoralis heat shock protein70 (HSP70) (Sst-hsp-70) and astacin (Sst-ast) as pathogenesis-related genes as well as the expression of S. ratti HSP70 and HSP17.1 (Sra-hsp-70, Sra-hsp-17.1) in the larvae and adult stages of S. stercoralis. Methods: A hyperinfection isolate of S. stercoralis from Gilan Province, northern Iran was cultivated on nutrient agar. After a couple of days, parasites in different stages of life were collected, and total RNA was extracted. The expression levels of astacin and HSP genes were compared by real-time PCR. Results: Statistically higher expression levels of Sst-ast, Sst-hsp-70, and Sra-hsp-70 genes in L3 larvae than in adults were observed. However, the expression level of Sra-hsp-17.1 was non-significantly lower in the larval stage than in adult worms. Conclusion: Higher expression levels of Sst-ast, Sst-hsp-70, and Sra-hsp-70 genes in the larval stages of S. stercoralis suggest the potential role of these enzymes in parasite cutaneous invasion and pathogenesis. However, higher expression of Srahsp-17.1 in adult forms is probably involved in resistance and survival mechanisms. The similarity in gene expression between S. stercoralis and S. ratti can provide helpful hints to better understand strongyloidiasis from various perspectives, including pathogenesis, proper diagnosis, and targeted treatment.

Ecotoxicological strategies employing biochemical markers and organisms to monitor the efficacy of Malathion photolysis treatment.

The objective of this study was to assess the photolysis-mediated degradation of malathion in standard and commercial formulations, and to determine the toxicity of these degraded formulations. Degradation tests were carried out with 500 µg L-1 of malathion and repeated three times. The initial and residual toxicity was assessed by using Lactuca sativa seeds for phytotoxicity, Stegomyia aegypti larvae for acute toxicity, and Stegomyia aegypti mosquitoes (cultivated from the larval stage until emergence as mosquitoes) to evaluate the biochemical markers of sublethal concentrations. For the standard formulations the photolytic process efficiently reduced the initial concentration of malathion to levels below the regulatory limits however, the formation of byproducts was revealed by chromatography, which allowed for a more complete proposal of photolytic-mediated malathion degradation route. The degraded formulations inhibited the growth of L. sativa seeds, while only the untreated formulations showed larvicidal activity and mortality. Both formulations slightly inhibited acetylcholinesterase activity in S. aegypti mosquitoes , while the standard formulation decreased and the commercial formulation increased glutathione S-transferase activity. However, there were no significant differences for superoxide dismutase, esterase-α, esterase-ß and lipid peroxidation. These findings indicate that in the absence of the target compound, the presence of byproducts can alter the enzymatic activity. In general, photolysis effectively degrade malathion lower than the legislation values; however, longer treatment times must be evaluated for the commercial formulation.

Immunomodulatory effects of a probiotic combination treatment to improve the survival of Pacific oyster (Crassostrea gigas) larvae against infection by Vibrio coralliilyticus.

Introduction: The culture of Pacific oysters (Crassostrea gigas) is of significant socio-economic importance in the U.S. Pacific Northwest and other temperate regions worldwide, with disease outbreaks acting as significant bottlenecks to the successful production of healthy seed larvae. Therefore, the current study aims to describe the mechanisms of a probiotic combination in improving the survival of C. gigas larvae. Specifically, we investigate changes in C. gigas larval gene expression in response to V. coralliilyticus infection with or without a pre-treatment of a novel probiotic combination. Methods: Treatment groups consisted of replicates of Pacific oyster larvae exposed to a) a combination of four probiotic bacteria at a total concentration of 3.0 x 105 CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 103 CFU/mL at 48 hpf, and c) the probiotic combination at 18 hpf and V. coralliilyticus RE22 at 48 hpf. RNA was extracted from washed larvae after 72 hpf, and transcriptome sequencing was used to identify significant differentially expressed genes (DEGs) within each treatment. Results: Larvae challenged with V. coralliilyticus showed enhanced expression of genes responsible for inhibiting immune signaling (i.e., TNFAIP3, PSMD10) and inducing apoptosis (i.e., CDIP53). However, when pre-treated with the probiotic combination, these genes were no longer differentially expressed relative to untreated control larvae. Additionally, pre-treatment with the probiotic combination increased expression of immune signaling proteins and immune effectors (i.e., IL-17, MyD88). Apparent immunomodulation in response to probiotic treatment corresponds to an increase in the survival of C. gigas larvae infected with V. coralliilyticus by up to 82%. Discussion: These results indicate that infection with V. coralliilyticus can suppress the larval immune response while also prompting cell death. Furthermore, the results suggest that the probiotic combination treatment negates the deleterious effects of V. coralliilyticus on larval gene expression while stimulating the expression of genes involved in infection defense mechanisms.

Development of a multi-entry identification key for economically important fruit fly larvae (Diptera, Tephritidae, Dacinae).

Identification of fruit fly larvae is difficult due to the limited morphological characteristics present. However, this is the stage at which fruit flies are intercepted at ports of entry through horticultural imports. Molecular tools are useful but are time-consuming and expensive compared to morphological identifications. This project aims to use available information from the literature and our own research to build a multi-entry identification key for thirteen tephritid species and species groups that are of economic concern for the European Union. Third-instar larvae were obtained from different regions and hosts. Thirteen species or representatives of species groups were obtained, including Ceratitis, Dacus, Bactrocera and Zeugodacus spp. The cephalopharyngeal skeletons were dissected out, cleared in a 10% NaOH solution, dehydrated and mounted in Euparal on glass slides. Images of at least 20 larvae/species were captured using a compound microscope fitted with a camera. Measurements were taken of the mounted mandibles and the number of tubules and their position in the anterior spiracles in relation to the cephalic skeleton were noted. Differences between morphometric parameters were tested via ANOVA and verified using discriminant function analysis. A matrix was compiled including nine characters for which significant inter-specific differentiation was preliminarily detected. The key was converted into a mobile application by LucID.

Cutaneous myiasis caused by Dermatobia hominis (Diptera: Oestridae) in a Polish traveller to South America - a case report.

Myiasis, an infestation caused by dipteran larvae, commonly known as maggots, is one of the most common parasitic skin disorders in the tropical regions. Authors report a case of cutaneous myiasis caused by Dermatobia hominis (Diptera: Oestridae) in a Polish traveller returning from a self-organized trip to South America. Species biology, prophylaxis, and medical implications of this tropical parasitosis are discussed.

Optimization of inclusion level of lipid in larval diet of Labeo rohita (Hamilton, 1822).

Formulation and preparation of larval feed according to the requirement of Indian major carp is a prerequisite for improving the survival (%) and growth during early developmental stages. A feeding trial of 50 days in a replicate of five was conducted to determine the optimal inclusion levels of fish oil (lipid) in the larval diet of Labeo rohita. Four isonitrogenous (50% CP) nanoparticulate diets with four lipid inclusion levels, L5 (5%), L7 (7%), L9 (9%), and L11 (11%) were prepared and fed to four groups of rohu (Labeo rohita) larvae. At the end of feeding trial, survival (%), growth performance, digestive enzyme activity, gut morphology, and expression of growth and feed intake genes were evaluated. All pairwise comparisons among groups indicated higher growth performance (weight gain, specific growth rate, and daily weight gain), survival (%), and IGF-1 gene expression of the L9 group followed by the L7 while the L11 showed poor performance even less than L5. All studied intestinal enzymes except amylase showed a similar trend. Amylase showed comparable results among L7, L9, and L5, while L11 showed the lowest value. The intestinal villi length also showed higher values in L9 followed by L7, and lowest in the L11 group. Feed intake regulating genes, leptin showed lipid inclusion level upregulation, while ghrelin showed the highest expression in the L9 group. Based on growth performance, gut morphology, intestinal enzyme activity, and gene expression analysis, 9% dietary lipid could be recommended to ensure the optimum growth and survival of L. rohita larvae.

On Ypsolopha micromoths (Lepidoptera, Ypsolophidae) associated with Adesmia shrubs (Fabaceae) in the arid western slope of the central Andes.

Ypsolopha Latreille, 1796 (Lepidoptera, Ypsolophidae) is a genus comprised mostly of Holarctic micromoth species with a fairly broad range of larval hosts (e.g. Aceraceae, Rosaceae, and Fagaceae). The only previous record of herbivory on a representative of the South American genus Adesmia DC. (Fabaceae) was based on the discovery of Ypsolophamoltenii Vargas, 2018 larvae feeding on Adesmiaverrucosa Meyen in the Andes of northern Chile. Further surveys revealed Adesmiaatacamensis Phil. as another host for Y.moltenii, and Adesmiaspinosissima Meyen as the single host of Ypsolopha sp. The genetic distance between DNA barcodes of the two micromoth species was 7.9-8.1% (K2P). These results suggest narrow host ranges for Adesmia-feeding Ypsolopha and highlight the need to further explore the taxonomic diversity of these micromoths in other South American environments.

Quantitative tyramine analysis method for Apis mellifera larvae infected with Melissococcus plutonius, the causative agent of European foulbrood.

Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus plutonius that causes European foulbrood in honey bee brood. In the present study, we developed a method for quantitative analysis of tyramine in culture medium and honey bee larvae with a limit of quantitation of 3 ng/mL and a recovery rate of >97% using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry and deuterium-labeled tyramine, demonstrating for the first time that a highly virulent M. plutonius strain actually produces tyramine in infected larvae. This method will be an indispensable tool to elucidate the role of tyramine in European foulbrood pathogenesis in combination with exposure bioassays using artificially reared bee larvae.

Isolation of a Novel Pythium Species, P. thermoculicivorax, and Trichoderma sp. from Natural Enzootic Mosquito Larval Infections.

Only a handful of microbial mosquito larval pathogens have been described to date. Sampling several natural enzootic infections of mosquito larvae in southwestern Florida indicated the presence of microbial pathogens capable of extensive larval mortality. A microscopic analysis of one sample site revealed extensive apparent growth of a Pythium-like microbe on mosquito larvae, with the highest degree of infection observed in the siphon and head regions. Structures consistent with sporangia were seen on infected insects after lactophenol blue staining, and higher-resolution scanning electron microscopy (SEM) micrographs showed sporangia and encysted zoospores targeting the head and siphon regions. The isolate was single-colony purified, and molecular identification targeting the ITS and COX1 loci coupled to phylogenetic reconstruction indicated that the isolate belonged to the Pythium genus but was distinct from its closest characterized species, P. inflatum. Morphological features were characterized, with the isolate showing rapid growth on all mycological media tested and relatively high thermotolerance, capable of robust growth at 37 °C; hence, it was designated P. thermoculicivorax. Sampling from a second series of natural infections of mosquito larvae resulted in the molecular identification of three Trichoderma isolates, one with high similarity to T. strigosum and the other two clustering closely with T. asperellum. These data highlight the occurrence of natural enzootic infections of mosquito larvae, potentially as a resource for the identification of new mosquito pathogens.

The Effect of Dietary Protein Hydrolysate from Black Soldier Fly Larvae and Schizochytrium on Palatability, Nutrient Metabolites and Health Status in Beagle Dogs.

Protein hydrolysate from black soldier fly larvae (BSFP) has garnered great attention with its lower allergenicity, high amount of essential amino acids, and small bioactive peptides. Schizochytrium is a promising alternative source of n-3 FUFA because it has enriched docosahexaenoic acid (DHA, C22: 6). The aim of this study was to assess palatability, the presence of diarrhea, plasma biochemistry panels, anti-oxidative and anti-inflammatory effects, and immune function in beagle dogs when supplementing a mixture of protein hydrolysate from black soldier fly larvae and schizochytrium (BSFPs) into their diets. Experiment I: 24 young beagle dogs (16 males and 8 females; 4-5 months; BW: 6.40 ± 0.15 kg) were randomly divided into four groups: (1) control (CON), (2) 5% BSFPs, (3) 10% BSFPs, (4) 15% BSFPs. Their body weights and fecal scores were recorded, and blood samples were collected for analysis. Experiment II: three diets containing 5%, 10%, and 15% BSFPs were evaluated by comparing them with a basal diet (CON) to evaluate palatability. These results suggested that a lower presence of diarrhea existed in the BSFP diet than the CON diet (p < 0.05). Three treatment groups remarkably increased their total protein (TP) and albumin (ALB) contents and decreased their concentrations of triglyceride (TG) and total cholesterol (TC) in plasma (p < 0.05). Moreover, the 5% and 15% BSFPs groups had a higher calcium (CA) content in plasma, and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and contents of creatinine (CREA) and urea nitrogen (BUN) were significantly reduced by supplementing BSFP in their diets (p < 0.05). Their anti-oxidative enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were dramatically enhanced, and their malondialdehyde (MDA) concentrations were remarkably reduced (p < 0.05). Immunoglobulin A and G (IgA and IgG) concentrations in the plasma in the 10% and 15% BSFPs groups were significantly increased (p < 0.05). Furthermore, lower interleukin-8 (IL-8) contents were shown in the BSFP diets than the CON diet (p < 0.05). Similarly, the diets supplemented with BSFPs exhibited a positive effect on palatability (p < 0.05). To sum up, the diets supplemented with BSFPs significantly enhanced palatability, immune function, and anti-oxidative and anti-inflammatory capacity to alleviate diarrhea and improve the general health of the beagle dogs.