Effects of dietary silkrose of Antheraea yamamai on gene expression profiling and disease resistance to Edwardsiella tarda in Japanese medaka (Oryzias latipes).

We previously identified a novel acidic polysaccharide, silkrose-AY, from the Japanese oak silkmoth (Antheraea yamamai), which can activate an innate immune response in mouse macrophage cells. However, innate immune responses stimulated by silkrose-AY in teleosts remain unclear. Here, we show the influence of dietary silkrose-AY in medaka (Oryzias latipes), a teleost model, in response to Edwardsiella tarda infection. Dietary silkrose-AY significantly improved the survival of fish and decreased the number of bacteria in their kidneys after the fish were artificially infected with E. tarda by immersion. We also performed a microarray analysis of the intestine, which serves as a primary barrier against microbial infection, to understand the profiles of differentially expressed genes (DEGs) evoked by silkrose-AY. The dietary silkrose-AY group showed differential expression of 2930 genes when compared with the control group prior to E. tarda infection. Gene ontology and pathway analysis of the DEGs highlighted several putative genes involved in pathogen attachment/recognition, the complement and coagulation cascade, antimicrobial peptides/enzymes, opsonization/phagocytosis, and epithelial junctional modification. Our findings thus provide fundamental information to help understand the molecular mechanism of bacterial protection offered by insect-derived immunostimulatory polysaccharides in teleosts.

Dietary Black Soldier Fly (Hermetia illucens)-Dipterose-BSF-Enhanced Zebrafish Innate Immunity Gene Expression and Resistance to Edwardsiella tarda Infection.

Dietary management using immunostimulants to protect fish health and prevent bacterial infection is widely practiced. Many insect species possess various bioactive substances that can improve animal health. We previously identified several bioactive polysaccharides derived from insects, including dipterose-BSF from black soldier fly (Hermetia illucens) larvae; this can stimulate innate immunity in mammalian macrophage RAW264.7 cells. However, the effect of dietary dipterose-BSF on the immune system of teleosts remains unclear. Here, we analyzed the immune status of zebrafish (Danio rerio) after 14 days of dietary inclusion of dipterose-BSF (0.01, 0.1, and 1 µg/g), followed by an immersion challenge using Edwardsiella tarda. To identify changes in the transcriptional profile induced by dipterose-BSF, we performed RNA-sequencing analyses of the liver and intestine. Differentially expressed genes were investigated, with both organs showing several upregulated genes, dominated by nuclear factor and tumor necrosis factor family genes. Gene Ontology analysis revealed several terms were significantly higher in the experimental group compared with the control group. Challenge tests suggested that dietary dipterose-BSF had some positive effects on disease resistance in fish, but these effects were not pronounced.

Growth of Yellowtail (Seriola quinqueradiata) Fed on a Diet Including Partially or Completely Defatted Black Soldier Fly (Hermetia illucens) Larvae Meal.

Against a background of increased demand for fish meal (FM), black soldier fly larva is a promising alternative feed source for sustainable aquaculture. Yellowtail, the most popular farmed fish in Japan, is a carnivorous fish; therefore, it requires a high proportion of FM in its diet. This study represents the first example of yellowtail fed on a diet including insect meal as a replacement for FM. Partially defatted black soldier fly meal (PDBM) comprised 49.0% crude protein and 23.2% crude fat, while completely defatted black soldier fly meal (CDBM) contained less than 10% crude fat, as the same level as FM was achieved with defatting PDBM using hexane. In feeding trials, growth of the fish was reduced in accordance with PDBM content: 10%, 20%, and 30% in their diet. Although a diet including 8% CDBM (with the same protein composition as 10% PDBM) also resulted in decreased fish growth, growth with a diet including 16% CDBM (with the same protein composition as 20% PDBM) was significantly higher than that of 20% PDBM, and equivalent to that of 10% PDBM. Therefore, even 10% of partially or completely black soldier fly larvae meal in diets inhibited growth in juvenile yellowtail, and we found that removal of the fat fraction could improve fish growth.

Serial Passage of Cryptococcus neoformans in Galleria mellonella Results in Increased Capsule and Intracellular Replication in Hemocytes, but Not Increased Resistance to Hydrogen Peroxide.

To gain insight into how pathogens adapt to new hosts, Cryptococcus neoformans (H99W) was serially passaged in Galleria mellonella. The phenotypic characteristics of the passaged strain (P15) and H99W were evaluated. P15 grew faster in hemolymph than H99W, in vitro and in vivo, suggesting that adaptation had occurred. However, P15 was more susceptible to hydrogen peroxide in vitro, killed fewer mouse macrophages, and had less fungal burden in human ex vivo macrophages than H99W. Analysis of gene expression changes during Galleria infection showed only a few different genes involved in the reactive oxygen species response. As P15 sheds more GXM than H99W, P15 may have adapted by downregulating hemocyte hydrogen peroxide production, possibly through increased capsular glucuronoxylomannan (GXM) shedding. Hemocytes infected with P15 produced less hydrogen peroxide, and hydrogen peroxide production in response to GXM-shedding mutants was correlated with shed GXM. Histopathological examination of infected larvae showed increased numbers and sizes of immune nodules for P15 compared to H99W, suggesting an enhanced, but functionally defective, response to P15. These results could explain why this infection model does not always correlate with murine models. Overall, C. neoformans' serial passage in G. mellonella resulted in a better understanding of how this yeast evolves under selection.

The Dipterose of Black Soldier Fly (Hermetia illucens) Induces Innate Immune Response through Toll-Like Receptor Pathway in Mouse Macrophage RAW264.7 Cells.

In our study, a novel bioactive polysaccharide was identified in the larvae of the black soldier fly (BSF) (Hermetia illucens) as a molecule that activates the mammalian innate immune response. We attempted to isolate this molecule, which was named dipterose-BSF, by gel-filtration and anion-exchange chromatography, followed by nitric oxide (NO) production in mouse RAW264.7 macrophage cells as a marker of immunomodulatory activity. Dipterose-BSF had an average molecular weight of 1.47 × 105 and consisted of ten monosaccharides. Furthermore, in vitro assays demonstrated that dipterose-BSF enhanced the expression of proinflammatory cytokines and interferon ß (IFNß) in RAW264.7 cells. The inhibition of Toll-like receptor 2 (TLR2) and 4 (TLR4) significantly attenuated NO production by dipterose-BSF, indicating that dipterose-BSF stimulates the induction of various cytokines in macrophages via the TLR signaling pathway. This observation was analogous with the activation of nuclear factor kappa B in RAW264.7 cells after exposure to dipterose-BSF. Our results suggest that dipterose-BSF has immunomodulatory potential through activating the host innate immune system, which allows it to be a novel immunomodulator for implementation as a functional food supplement in poultry, livestock, and farmed fish.

The silkrose of Bombyx mori effectively prevents vibriosis in penaeid prawns via the activation of innate immunity.

We previously identified novel bioactive polysaccharides from Bactrocera cucurbitae and Antheraea yamamai that activate innate immunity in RAW264 murine macrophages. However, in terms of potential applications in the cultivation of prawns, there were problems with the availability of these insects. However, we have now identified a polysaccharide from Bombyx mori that activates innate immunity in RAW264 cells and penaeid prawns. This purified polysaccharide, termed silkrose of B. mori (silkrose-BM), has a molecular weight of 1,150,000 and produces a single symmetrical peak on HPLC. Eight of nine constitutive monosaccharides of silkrose-BM are concomitant with dipterose of B. cucurbitae (dipterose-BC) and silkrose of A. yamamai (silkrose-AY). The major differences are found in the molar ratios of the monosaccharides. Silkrose-BM is approximately 500-fold less potent than silkrose-AY (EC50: 2.5 and 0.0043 µg/mL, respectively) in a nitrite oxide (NO) production assay using RAW264 cells. However, the maximum NO production for silkrose-BM and AY were comparable and higher than that of the lipopolysaccharide of Escherichia coli. The survival of penaeid prawns (Litopenaeus vannamei and Marsupenaeus japonicus) after infection with Vibrio penaecida was significantly improved by both dietary silkrose-BM and B. mori pupae. This suggests that silkrose-BM effectively prevents vibriosis in penaeid prawns via the activation of innate immunity.