Larvae Of Strain Research Articles

Larvicidal activity evaluation of the chemical compounds isolated from n-hexane extract of Derris elliptica root against the Temephos-susceptible strain of Aedes aegypti larvae

Abstract. Sayono S, Anwar R, Sumanto D. 2021. Larvicidal activity evaluation of the chemical compounds isolated from n-hexane extract of Derris elliptica root against the Temephos-susceptible strain of Aedes aegypti larvae. Biodiversitas 23: 757-764. Aedes aegypti is the main vector of Dengue, Chikungunya, and Zika diseases. Temephos resistance of this species has hampered vector control efforts worldwide. Studies proved that Derris elliptica extracts effectively controlled Aedes larvae, so the active compound needed to be isolated. This study evaluated the larvicidal activity of the chemical compounds isolated from n-hexane fractions of Tuba roots against the temephos-susceptible Ae. aegypti larvae. Six isolates were obtained from three of the seven n-hexane fractions. Three levels of concentration of each isolate were formulated for a preliminary bioassay test, and resulted in the two most active compounds, isolates 3 and 6. The final bioassay test results indicated that isolate 3 was more active than isolate 6 with LC50 and LC90 after 24 hours of exposure were 1.607 and 7.399 ppm, and after 48 hours of exposure were 0.926 and 3.206 ppm respectively. Isolate 6 also had high larvicidal activity after 48 hours of exposure, with LC50 and LC90 were 1.056 and 4.647 ppm. Further studies need to be carried out to determine the chemical structures and toxicity mechanisms of the bioactive compounds.

A novel multiplex PCR assay to detect and distinguish between different types of Paenibacillus larvae and Melissococcus plutonius, and a survey of foulbrood pathogen contamination in Japanese honey.

Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.

Parasitic fitness of a strain of Diachasmimorpha longicaudata (Hymenoptera, Braconidae) reared on irradiated larvae of a Vienna-8 strain of Ceratitis capitata (Diptera, Tephritidae)

Parasitic fitness of a strain of Diachasmimorpha longicaudata (Hymenoptera, Braconidae) reared on irradiated larvae of a Vienna-8 strain of Ceratitis capitata (Diptera, Tephritidae)

ANTIBACTERIAL EFFECTS OF ANATOLIAN PROPOLIS ON PAENIBACILLUS LARVAE

Paenibacillus larvae (P. larvae) is a pathogenic bacterium causing American Foulbrood Disease (AFB) in honeybee larvae. It is necessary to develop alternative strategies for the control of this disease due to the serious honeybee colonies loses and the use of antibiotics. Recent studies are aimed at the investigating natural fighting agents against P. larvae. In our study, it was aimed to demonstrate potential antibacterial efficacy of ethanol extract of Anatolian Propolis (EAP) against P. larvae strains PB35 and SV35 which were isolated in Turkey. The total phenolic content (TPC) and flavonoid content (TFC) of EEAP were determined as 181.73±5.20 mg Gallic Acid Equivalents (GAE)/g, and 42.33±1.40 mg Quercetin Equivalents (QE)/g, respectively. It was found that EAP contains different amounts of ferulic, caffeic, coumaric acids, rutin, and caffeic acid phenethyl ester (CAPE). The antibacterial activity of the EAP was determined by using agar-well diffusion, microdilution, and Bioscreen C techniques. The Minimal Inhibition Concentration (MIC) values of the EAP were determined as 74.87 μg/ml against strain PB35 and SV35 using both microdilution and Bioscreen C teqnique. In both techniques, Minimal Bactericidal Concentration (MBC) values of the EAP were evaluated as 149 and 598.4 μg/ml against strain PB35 and SV35, respectively. The fact that EAP shows low concentrations of bacteriostatic (MIC) and bactericide (MBC) activity values against P. larvae strains, spore-forming bacilli, which are agents of AFB disease, suggests that it may be a potential source in AFB disease control.

Dry and Rainy Seasons Significantly Alter the Gut Microbiome Composition and Reveal a Key Enterococcus sp. (Lactobacillales: Enterococcaceae) Core Component in Spodoptera frugiperda (Lepidoptera: Noctuidae) Corn Strain From Northwestern Colombia.

Spodoptera frugiperda is a polyphagous pest of several crops of economic importance. Nowadays, the insect is broadly distributed in America and, recently, in Africa, Asia, and Australia. The species has diverged into corn and rice strains. The role of the gut microbiota in insect physiology is relevant due to its participation in crucial functions. However, knowledge of seasonal variations that alter the gut microbiome in pests is limited. Gut microbiome composition between the dry and rainy seasons was analyzed with cultured and uncultured approaches in S. frugiperda corn strain larvae collected at Northwest Colombia, as seasonal microbiome changes might fluctuate due to environmental changes. On the basis of culture-dependent methods, results show well-defined microbiota with bacterial isolates belonging to Enterococcus, Klebsiella (Enterobacteriales: Enterobacteriaceae), Enterobacter (Enterobacterales: Enterobacteriaceae), and Bacillus (Bacillales: Bacillaceae) genera. The community composition displayed a low bacterial diversity across all samples. The core community detected with uncultured methods was composed of Enterococcus, Erysipelatoclostridium (Erysipelotrichales: Erysipelotrichaceae), Rasltonia (Burkholderiales: Burkholderiaceae), and Rhizobium (Hyphomicrobiales: Rhizobiaceae) genera, and Enterobacteriaceae family members. Significant differences in microbiome diversity were observed between the two seasons. The relative abundance of Erysipelatoclostridium was high in the dry season, while in the phylotype ZOR0006 (Erysipelotrichales: Erysipelotrichaceae) and Tyzzerella (Lachnospirales: Lachnospiraceae) genus, the relative abundance was high in the rainy season. The overall low gut bacterial diversity observed in the S. frugiperda corn strain suggests a strong presence of antagonist activity as a selection factor possibly arising from the host, the dominant bacterial types, or the material ingested. Targeting the stability and predominance of this core microbiome could be an additional alternative to pest control strategies, particularly in this moth.

Short-term metabolic resistance inductive effect of different agrochemical groups on Anopheles gambiae mosquitoes

Background: In order to assess the impact of the different groups of agricultural pesticides used in Côte d'Ivoire on the increase of mosquitoes resistance to insecticides, the expression profiles of 7 P450 cytochromes and one GSTE2 of Anopheles gambiae involved in mosquito resistance to insecticides were studied. The goal of this study was to determine the effect of short exposure of mosquito larvae to different groups of agricultural pesticides on mosquito resistance. Methods: Three groups of pesticides were selected: (i) agricultural insecticide solutions, (ii) non-insecticide pesticide solutions (a mixture of herbicides and fungicides), and (iii) a mixture of the first two. A fourth non-pesticide solution was used as a control. Four groups of each stage 2 larvae (strain Kisumu, male and female) were exposed to 20% concentrated solution for 24 hours. Susceptibility tests for dichlorodiphenyltrichloroethane (DDT) and Deltamethrin were carried out on adults aged 2-5 days. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to quantify the expression of eight metabolic genes involved in mosquito resistance to insecticides. Results: Susceptibility to DDT showed a similar increase in the time required to knock down 50% of mosquitoes (kdt50) in l colonies exposed to insecticides and non-insecticides compared to the control colony. As for deltamethrin, kdt50 was higher in the colonies exposed to insecticides and the pesticide mixture compared to the colony exposed to non-insecticides. Of all the genes studied in all colonies, except for CYP6P1 induced only in the colony consisting of the pesticide mixture, no genes were induced. Conclusions: This study confirmed that induction is influenced by the duration, the concentration of the solution and the type of xenobiotic used as an inducer. The overexpression of CYP6P1 confirmed the inductive effect that a short exposure of mosquito larvae to agricultural pesticides could have.

Fatty acids and their derivatives from Chlorella vulgaris extracts exhibit in vitro antimicrobial activity against the honey bee pathogen Paenibacillus larvae

The green microalga Chlorella vulgaris Beijerinck (Chlorellaceae) is widely used as a food supplement for humans and animals. In beekeeping practice, Chlorella vulgaris has potential as a pollen supplement. We studied whether Chlorella extracts display antimicrobial properties against Paenibacillus larvae, the causative agent of the honey bee bacterial disease American foulbrood. We identified components responsible for antimicrobial activity and evaluated the added values of Chlorella as a food supplement for honey bees. Different extracts (water, acetone, methanol) were prepared from Chlorella biomass (phototrophically and heterotrophically cultivated) and screened for antimicrobial activity against ERIC I and ERIC II genotypes of P. larvae. Active acetone extracts of phototrophically cultivated Chlorella vulgaris biomass were fractioned via preparative reverse-phase chromatography. Antimicrobial activity was detected for 9 of the resulting 33 fractions. Further analysis revealed the chemical composition of the active fractions. C. vulgaris extracts showed a significant antimicrobial effect against vegetative cells and spores of P. larvae strains of ERIC I and ERIC II genotypes. The lowest MIC of the most active acetone extract was 6.3 µg/mL for both tested genotypes. In the majority of the active fractions, monolinolenin, fatty acid linoleic acid, and methyl esters of linoleic and/or palmitic acid were identified via high-performance liquid chromatography coupled with high-resolution mass spectrometry analysis. Based on our results, we concluded that algal C. vulgaris food supplements not only contain nutritional but also potential prophylactic properties for honey bee health.

Isolation, characterization, and comparative genomic analysis of vB_PlaP_SV21, new bacteriophage of Paenibacillus larvae

Paenibacillus larvae cause an American foulbrood disease (AFB) that is responsible for the extinction of honeybee colonies and is a honeybee bacterial disease that has to be obligatory notified worldwide. Recently, bacteriophage studies targeting Paenibacillus larvae have emerged as a promising alternative treatment method. The inability of bacteria to create resistance against bacteriophages makes this method advantageous. As a consequence, this study was conducted to describe the genome and biological characteristics of a novel phage capable of lysing Paenibacillus larvae samples isolated from honeybee larva samples in Turkey. The Paenibacillus phage SV21 (vB_PlaP_SV21) was isolated by inducing Paenibacillus larvae strain SV21 with Mitomycin-C. Whole-genome sequencing, comparative genomics, and phylogenetic analysis of vB_PlaP_SV21 were performed. Transmission electron microscopy images showed that vB_PlaP_SV21 phage was a Podovirus morphology. The vB_PlaP_SV21 phage specific for Paenibacillus larvae was determined to belong to the Podoviridae family. Host range and specificity, burst size, lytic activity, and morphological characteristics of the phage were determined. Bioinformatic analysis of the Paenibacillus phage SV21 showed 77 coding sequences in its linear 44,949 bp dsDNA genome with a GC content of 39.33%. In this study, we analysed the genomes of all of the currently sequenced P. larvae phage genomes and classified them into five clusters and a singleton. According to molecular, morphological, and bioinformatics results, ıt was observed that API480 (podovirus), which was reported as a singleton in previous studies and public databases, and Paenibacillus phage SV21 phage could form a new cluster together

Effect of Feeding Rate on Survival, Zootechnical Performance of Tilapia Oreochromis niloticus (Linnaeus, 1758) Brazil Strain Larvae Fed on 17-α-methyltestosterone Treated Feed

This study was conducted to evaluate the feeding levels on survival, growth performance and feed utilization in the Brazil strain of Nile tilapia Oreochromis niloticus. Four (4) batches of larvae with mean initial weight 0.012 ± 0.005 g and mean initial total length 9 ± 0.003 mm were formed in triplicate, three per feeding rate. The rationing rates according to fry biomass applied in this trial were: batch 1: 50, 40, 30 and 25%, batch 2: 40, 30, 25 and 20%, batch 3: 30, 25, 20 and 18% and batch 4: 25, 20, 18 and 15% of biomass. The different feeding rates were tested during the 1st, 2nd, 3rd and 4th week of larval rearing respectively. The fish were all fed a commercial feed (48% protein) distributed manually at a frequency of 5 meals per day. After 28 days of rearing, the results show that apart from the survival rate, the other parameters such as final average weight, daily growth and food consumption index were affected by the different rationing rates tested. The larvae of batch 2 rationed at 40, 30, 25 and 20 % of their biomass during the 1st, 2nd, 3rd and 4th week of rearing respectively recorded significantly higher growth parameters (MWF: 1.092±0.027 g and DG: 0.038±0.000 g) and a more interesting food converion ratio (0.88 ) than the other batches of fry. From this study, it is concluded that an optimal feeding rate of 40, 30, 25 and 20% of the larval biomass during the 1st, 2nd, 3rd and 4th week of rearing, respectively, should be applied in order to avoid either wastage or underfeeding of the subjects during hormone treatment with 17 α-methyltestosterone.

Study on emergency metabolic changes of Anopheles sinensis larvae following exposure to deltamethrin

To analyze the changes of small molecular metabolites in the larvae of a deltamethrin-sensitive strain of Anopheles sinensis following exposure to deltamethrin, so as to provide the scientific basis for investigating the metabolic pathway and screening metabolic markers of deltamethrin in An. sinensis. The 50% and 75% lethal concentrations (LC50 and LC75) of deltamethrin against the larvae of a deltamethrin-sensitive strain of An. sinensis were calculated in laboratory. The type and content of An. sinensis larvae metabolites were detected using high performance liquid chromatography and mass spectrometry (LC-MS/MS) following exposure to deltamethrin at LC50 and LC75 for 30 min and 24 h, and the changes of metabolites were analyzed. The LC50 and LC75 values of deltamethrin were 4.36 × 10-3 µg/mL and 1.12 × 10-2 µg/mL against thelarvae of a deltamethrin-sensitive strain of An. sinensis. Following exposure of the larvae of a deltamethrin-sensitive strain of An. sinensis to deltamethrin at LC50 and LC75 for 30 min, the differential metabolites mainly included organic oxygen compounds, carboxylic acid and its derivatives, fatty acyl and pyrimidine nucleotides, with reduced glucose levels. Following exposure for 24 h, the differential metabolites mainly included organic oxygen compounds, carboxylic acid and its derivatives, aliphatic acyl and purine nucleotides, with increased glucose level detected. Carbohydrate, carboxylic acid and its derivatives, fatty acyls, amino acids and their derivatives may play important roles in deltamethrin metabolism in the larvae of a deltamethrin-sensitive strain of An. sinensis.

American Foulbrood in the Czech Republic: ERIC II Genotype of Paenibacillus Larvae Is Prevalent.

American foulbrood (AFB) is a dangerous disease of honeybees (Apis mellifera) caused by the spore-forming bacterium Paenibacillus larvae. According to the ERIC (enterobacterial repetitive intergenic consensus) classification, five genotypes are distinguished, i.e., I, II, III, IV, and V, which differ in their virulence and prevalence in colonies. In the Czech Republic, AFB prevalence is monitored by the State Veterinary Administration; however, the occurrence of specific P. larvae genotypes within the country remains unknown. In this study, our aim was to genotype field P. larvae strains collected in the Czech Republic according to the ERIC classification. In total, 102 field isolates from colonies with AFB clinical symptoms were collected from various locations in the Czech Republic, and the PCR genotypization was performed using ERIC primers. We confirmed the presence of both ERIC I and II genotypes, while ERIC III, IV, and V were not detected. The majority of samples (n = 82, 80.4%) were identified as ERIC II, while the ERIC I genotype was confirmed only in 20 samples (19.6%). In contrast to other European countries, the ERIC II genotype is predominant in Czech honeybee colonies. The ERIC I genotype was mostly detected in border regions close to Poland, Slovakia, and Austria.

Developmental plasticity among strains of the yellow mealworm,Tenebrio molitor L., under dry conditions

Edible insect production relies, among others, on the use of high-quality strains with good productivity characteristics. However, studies on the evaluation of the growth performance of different insect strains are limited. In this framework, the objective of the present study was to evaluate the growth and development under dry conditions of larvae of sixTenebrio molitor strains of different geographical origins, i.e. Greece, Italy, Turkey, Spain and USA, in terms of larval survival and development, as well as feed utilisation efficiency. In light of our results, we report the variable growth performance of differentT. molitor strains in the absence of a moisture source. Although no significant differences were shown among the strains tested with regard to the final larval weights and survival rates, there were significant differences in the development time and the growth rate, which may affect the overall efficiency ofT. molitor larvae production. Our study aims to highlight the significance of strain for the growth performance and consequently the productivity ofT. molitor.

Short communication: A potential role of tannins in the control of American foulbrood

Aim of study: To evaluate the use of tannins extracts in the control of the American foulbrood pathology and to investigate if these extracts present levels of toxicity on Apis mellifera.Area of study: Paenibacillus larvae strains C1 and C2 were from Balcarce, province of Buenos Aires, strain C6 from Rio Cuarto, in Cordoba province and strain C9 from Concordia in Entre Rios province. Bees larvae used for toxicological assays were collected in Santa Paula experimental apiary, Mar del Plata (belonging to the Centro de Investigación en Abejas Sociales (CIAS-IIPROSAM) from UNMdP.Material and methods: The minimal inhibitory concentration (MIC) of five different tannin extracts were obtained by agar diffusion method on four P. larvae strains; using the MIC value, the toxicity test on A. mellifera larvae was performed afterwards.Main results: The MIC value was in the range of 6.9 to 898.6 µg/mL. Three tannin extracts did not show toxicity against bee larvae; however, those that were fed with the latter showed a significant increase in weight.Research highlights: Three tanninS extracts showed a good antimicrobial activity against P. larvae and they did not show toxicity against bee larvae.

Integrated miRNA and transcriptome profiling to explore the molecular determinism of convergent adaptation to corn in two lepidopteran pests of agriculture

BackgroundThe degree to which adaptation to same environment is determined by similar molecular mechanisms, is a topic of broad interest in evolutionary biology, as an indicator of evolutionary predictability. We wished to address if adaptation to the same host plant in phytophagous insects involved related gene expression patterns. We compared sRNA-Seq and RNA-Seq data between two pairs of taxa of Ostrinia and Spodoptera frugiperda sharing maize as host-plant. For the latter, we had previously carried out a reciprocal transplant experiment by feeding of the larvae of the Corn strain (Sf-C) and the Rice strain (Sf-R) on corn versus rice and characterized the mRNA and miRNA responses.ResultsFirst, we predicted the genes encoding miRNA in Ostrinia nubilalis (On) and O. scapulalis (Os). Respectively 67 and 65 known miRNA genes, as well as 196 and 190 novel ones were predicted with Os genome using sncRNAs extracted from whole larvae feeding on corn or mugwort. In On, a read counts analysis showed that 37 (55.22%) known miRNAs and 19 (9.84%) novel miRNAs were differentially expressed (DE) on mugwort compared to corn (in Os, 25 known miRs (38.46%) and 8 novel ones (4.34%)). Between species on corn, 8 (12.5%) known miRNAs and 8 (6.83%) novel ones were DE while only one novel miRNA showed expression variation between species on mugwort. Gene target prediction led to the identification of 2953 unique target genes in On and 2719 in Os, among which 11.6% (344) were DE when comparing species on corn. 1.8% (54) of On miR targets showed expression variation upon a change of host-plant.We found molecular changes matching convergent phenotype, i.e., a set of nine miRNAs that are regulated either according to the host-plant both in On and Sf-C or between them on the same plant, corn. Among DE miR target genes between taxa, 13.7% shared exactly the same annotation between the two pairs of taxa and had function related to insect host-plant interaction.ConclusionThere is some similarity in underlying genetic mechanisms of convergent evolution of two distant Lepidopteran species having adopted corn in their host range, highlighting possible adaptation genes.

Divergence of Cultivable Bacteria Associated with Larvae and Adult Bactrocera dorsalis (Diptera: Tephritidae) Laboratory-Reared Strains

Insects entertain intricate and mutualistic relationship with an array of microorganisms, which significantly influence their fitness, ecology and evolution. In recent decades, there has been increasing interest toward studying the effects of microbiome on many host insects (Dipterans, Lepidopterans, and Coleopterans). Studies so far realized indicate that gut microbiome contribute to host nutritional ecology, defense, immunity and lifespan.
 Bactrocera dorsalis (Tephritidae: Diptera) is a polyphagous fruit fly which attacks a huge variety of fruits and vegetables worldwide and has been placed as a quarantine species by many countries. To investigate the specific functions of the gut endosymbionts, it is a prerequisite to know the composition of gut bacterial communities whose manipulation will help to decipher their ecological relevance. Here, we used the culture-dependent technique to isolate and identify gut bacteria from B. dorsalis at different developmental stages. The results revealed 11 bacterial species from the third instar larvae, 18 and 12 from female and male populations, respectively. These bacteria were assigned to six families, namely, Enterobacteriaceae, Enterococcaceae, Staphylococcaceae, Streptococcaceae, Micrococcaceae and Bacillaceae. Bacterial species from these families were differentially represented in various samples, except Klebsiella oxytoca , Enterobacter cloacae, Pantoea dispers and Enterococcus faecalis that were detected at all developmental stages. Overall, Enterobacteriaceae was the most dominant family in females and third instar larvae accounting for 57.89% and 26.32%, respectively, while Enterococcaceae was dominant in males with 75% of the total bacterial taxa. These results suggest that B. dorsalis possesses a huge variety of cultivable bacteria that could be used to explore their specific functions on host physiology and fitness.

Short-term metabolic resistance inductive effect of different agrochemical groups on Anopheles gambiae mosquitoes

Background: In order to assess the impact of the different groups of agricultural pesticides used in Côte d'Ivoire on the increase of mosquitoes resistance to insecticides, the expression profiles of 7 P450 cytochromes and one GSTE2 of Anopheles gambiae involved in mosquito resistance to insecticides were studied. The goal of this study was to determine the effect of short exposure of mosquito larvae to different groups of agricultural pesticides on mosquito resistance. Methods: Three groups of pesticides were selected: (i) agricultural insecticide solutions, (ii) none-insecticide pesticide solutions (a mixture of herbicides and fungicides), and (iii) a mixture of the first two. A fourth non-pesticide solution was used as a control. Four groups of each stage 2 larvae (strain Kisumu, male and female) were exposed to 20% concentrated solution for 24 hours. Susceptibility tests for dichlorodiphenyltrichloroethane (DDT) and Deltamethrin were carried out on adults aged 2-5 days. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to quantify the expression of eight metabolic genes involved in mosquito resistance to insecticides. Results: Susceptibility to DDT showed a similar increase in the time required to knock down 50% of mosquitoes (kdt50) in l colonies exposed to insecticides and none-insecticides compared to the control colony. As for deltamethrin, kdt50 was higher in the colonies exposed to insecticides and the pesticide mixture compared to the colony exposed to none-insecticides. Of all the genes studied in all colonies, except for CYP6P1 induced only in the colony consisting of the pesticide mixture, no genes were induced. Conclusions: This study confirmed that induction is influenced by the duration, the concentration of the solution and the type of xenobiotic used as an inducer. The overexpression of CYP6P1 confirmed the inductive effect that a short exposure of mosquito larvae to agricultural pesticides could have.

Immune-related gene expression in honey bee larva (Apis mellifera) exposed to plant extracts from Humulus lupulus with antimicrobial activity against Paenibacillus larvae

American foulbrood caused by Paenibacillus larvae brings with it several problems on beekeepers for the use of synthetic antibiotics, likewise led to the appearance of resistant strains and the presence of chemical residues in the commercial products of the hive. In recent years, the use of natural compounds for the control of P. larvae has increased. Herein, we propose to determine the value of the Minimal Inhibitory Concentration (MIC) of a hydroalcoholic extract of Humulus lupulus (hop) leaves of Victoria variety on P. larvae strain, and to evaluate its toxicity and effect on the expression of three genes of the immune system on honey bee larvae. For the extract, the MIC value was 83.79 μg/mL. For larvae, the extract did not show toxicity, on the other hand presented a down-regulation in the gene expression for the three genes evaluated. The results obtained here are a first report on the evaluation in the effect of natural extracts on the expression of genes related to the immune system in bee larvae.

Transcriptome analysis reveals a complex response to the RGNNV/SJNNV reassortant Nervous Necrosis Virus strain in sea bream larvae

The gilthead sea bream (Sparus aurata) is a marine fish of great importance for Mediterranean aquaculture. This species has long been considered resistant to Nervous Necrosis Virus (NNV), an RNA virus that causes massive mortalities in several farmed fish animals. However, the recent appearance of RGNNV/SJNNV reassortant strains started to pose a serious threat to sea bream hatcheries, as it is able to infect larvae and juveniles of this species. While host response to NNV has been extensively studied in adult fish, little attention has been devoted to early life history stages, which are generally the most sensitive ones. Here we report for the first time a time-course RNA-seq analysis on 21-day old fish gilthead sea bream larvae experimentally infected with a RGNNV/SJNNV strain. NNV-infected and mock-infected samples were collected at four time points (6 h, 12 h, 24 h, and 48 h post infection). Four biological replicates, each consisting of five pooled larvae, were analysed for each time point and group. A large set of genes were found to be significantly regulated, especially at early time points (6 h and 12 h), with several heat shock protein encoding transcripts being up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at T12). A gene set enrichment analysis (GSEA) identified several altered pathways/processes. For instance, the formation of peroxisomes, which are important anti-viral components as well as essential for nervous system homeostasis, and the autophagy pathway were down-regulated at 6 h and 24 h post infection (hpi). Finally, two custom “reactomes” (i.e. significant gene sets observed in other studies) were defined and used. The first reactome integrated the transcriptomic response to NNV in different fish species, while the second one included all genes found to be stimulated either by interferon (IFN) or by IFN and Chikungunya virus in zebrafish. Genes in both reactomes showed predominant up-regulation at 6hpi and 12hpi and a general down-regulation at 24hpi. Such evidence suggest a certain degree of similarity between the response of sea bream and that of other fish species to NNV, while the observed down-regulation of IFN- and viral-stimulated pathways argues for a possible interference of NNV against the host response.

Characterization of CRISPR Spacer and Protospacer Sequences in Paenibacillus larvae and Its Bacteriophages.

The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage–host systems.

The evaluation of culture performance and mantle coloration of two boring giant clam (Tridacna crocea) strains

The boring giant clam (Tridacna crocea) is the smallest species of the Tridacna genus, but they strongly express mantle color polymorphism. These color morphs represent potential genetic resources for aquaculture to supply the aquarium market, and the evaluation of their genetic and production characteristics is important. Wild yellow-green clams were consistently larger than blue clams in both the Huangyan Island and Zhaoshu Island populations in the Southern China Sea. To determine the relationship between mantle color and phenotypic traits, blue, yellow-green and control strains (various colors) were constructed and cultured for 1.5 years at an experimental station in Sanya, Hainan Island Province. All three strains had a high fertilization rate, but the survival to D larvae of the blue strain was significantly lower than the other two strains. Larval survival rate was over 85 % for all three strains, but the metamorphosis rate of the control strain was significantly lower than that of the other two strains. During the grow-out stage, the survival rate of the control strain from day 120 to day 480 was highest and that of the blue strain was lowest of the three groups. After metamorphosis, growth divergence of the three strains varied until day 120. Consistent growth trends appeared from day 120 to day 180, with the yellow-green strain largest, followed by the control strain. The process of mantle coloration was divided into three stages: the non-coloring period (day 0–60), the coloring period (day 60–180), and the stable expression period (day 180 onward). The yellow-green and blue mantle colors are genetically stable. This study provides a base for analysis of mantle color heredity and the genetic breeding of giant clams, and has great potential applications in the aquaculture industry, aquarium market and the coral reef tourism.