Minor impact of probiotic bacteria and egg white on Tenebrio molitor growth, microbial composition, and pathogen infection

The industrial rearing of the yellow mealworm (Tenebrio molitor) for feed and food purposes on agricultural by-products may expose larvae and adults to entomopathogens used as biocontrol agents in crop production. Bacterial spores/toxins or fungal conidia from species such as Bacillus thuringiensis or Metarhizium brunneum could affect the survival and growth of insects. Therefore, the aim of this study was to investigate the potential benefits of a wheat bran diet supplemented with probiotic bacteria and dried egg white on larval development and survival and its effects on the gut microbiome composition. Two probiotic bacterial species, Pediococcus pentosaceus KVL B19-01 and Lactiplantibacillus plantarum WJB, were added to wheat bran feed with and without dried egg white, as an additional protein source, directly from neonate larval hatching until reaching a body mass of 20 mg. Subsequently, larvae from the various diets were exposed for 72 h to B. thuringiensis, M. brunneum, or their combination. Larval survival and growth were recorded for 14 days, and the bacterial microbiota composition was analyzed using 16S rDNA sequencing prior to pathogen exposure and on days 3 and 11 after inoculation with the pathogens. The results showed increased survival for T. molitor larvae reared on feed supplemented with P. pentosaceus in the case of co-infection. Larval growth was also impacted in the co-infection treatment. No significant impact of egg white or of P. pentosaceus on larval growth was recorded, while the addition of Lb. plantarum resulted in a minor increase in individual mass gain compared with infected larvae without the latter probiotic. On day 14, B. thuringiensis was no longer detected and the overall bacterial community composition of the larvae was similar in all treatments. On the other hand, the relative operational taxonomic unit (OTU) abundance was dependent on day, diet, and probiotic. Interestingly, P. pentosaceus was present throughout the experiments, while Lb. plantarum was not found at a detectable level, although its transient presence slightly improved larval performance. Overall, this study confirms the potential benefits of some probiotics during the development of T. molitor while underlining the complexity of the relationship between the host and its microbiome

Impact and Persistence of Serratia marcescens in Tenebrio molitor Larvae and Feed under Optimal and Stressed Mass Rearing Conditions

International audienceIndustrial insect mass rearing aims to produce quality insects under safe sanitary conditions which can be compromised by pathogens and abiotic stressors. Therefore, knowledge on pathogen persistence, virulence and means of detection is of importance. This study focuses on the opportunistic pathogen Serratia marcescens (Sm) as a possible candidate to reveal sanitary issues in Tenebrio molitor (Tm) breeding. A screening test was performed to assess the impact of abiotic stressors (starvation, density and sieving) in presence and absence of Sm. Two Sm detection methods were conducted, and the kinetics of Sm persistence were investigated. Our results show that (i) the presence of Sm had a low but significant effect on Tm mortality, (ii) a short temporary starvation period had a negative impact on larval growth, (iii) the detection of Sm by q-PCR was sensitive but less convenient than a specific Sm growth media, (iv) the kinetics of persistence showed that Sm declined but survived for nine days in the feed and in the feces for three weeks. Both the relatively low virulence and the persistence in the environment suggest that Sm could be used as an indicator for the sanitary status of mealworm production

Genetics of resistance to transgenic Bacillus thuringiensis poplars in the poplar leaf beetle, Chrysomela tremulae F. (Coleoptera: Chrysomelidae)

International audienceThe area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B.thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F-2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6,400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D-LC = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D-ML = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations

The CodY-dependent <i>clhAB2</i> operon is involved in cell shape, chaining and autolysis in <i>Bacillus cereus</i> ATCC 14579

<div><p>The Gram-positive pathogen <i>Bacillus cereus</i> is able to grow in chains of rod-shaped cells, but the regulation of chaining remains largely unknown. Here, we observe that glucose-grown cells of <i>B</i>. <i>cereus</i> ATCC 14579 form longer chains than those grown in the absence of glucose during the late exponential and transition growth phases, and identify that the <i>clhAB</i>_<i>2</i> operon is required for this chain lengthening phenotype. The <i>clhAB</i>_<i>2</i> operon is specific to the <i>B</i>. <i>cereus</i> group (i.e., <i>B</i>. <i>thuringiensis</i>, <i>B</i>. <i>anthracis</i> and <i>B</i>. <i>cereus</i>) and encodes two membrane proteins of unknown function, which are homologous to the <i>Staphylococcus aureus</i> CidA and CidB proteins involved in cell death control within glucose-grown cells. A deletion mutant (Δ<i>clhAB</i>_<i>2</i>) was constructed and our quantitative image analyses show that Δ<i>clhAB</i>_<i>2</i> cells formed abnormal short chains regardless of the presence of glucose. We also found that glucose-grown cells of Δ<i>clhAB</i>_<i>2</i> were significantly wider than wild-type cells (1.47 μm ±CI_95% 0.04 vs 1.19 μm ±CI_95% 0.03, respectively), suggesting an alteration of the bacterial cell wall. Remarkably, Δ<i>clhAB</i>_<i>2</i> cells showed accelerated autolysis under autolysis-inducing conditions, compared to wild-type cells. Overall, our data suggest that the <i>B</i>. <i>cereus clhAB</i>_<i>2</i> operon modulates peptidoglycan hydrolase activity, which is required for proper cell shape and chain length during cell growth, and down-regulates autolysin activity. Lastly, we studied the transcription of <i>clhAB</i>_<i>2</i> using a <i>lacZ</i> transcriptional reporter in wild-type, <i>ccpA</i> and <i>codY</i> deletion-mutant strains. We found that the global transcriptional regulatory protein CodY is required for the basal level of <i>clhAB</i>_<i>2</i> expression under all conditions tested, including the transition growth phase while CcpA, the major global carbon regulator, is needed for the high-level expression of <i>clhAB</i>_<i>2</i> in glucose-grown cells.</p></div

Two models for <i>B</i>. <i>cereus clhAB</i>_<i>2</i> regulation.

<p>Left: regulation of <i>clhAB</i>_<i>2</i> expression in an amino acid- rich medium. In a nutrient-rich medium (such as LB medium [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref013" target="_blank">13</a>]), ILV uptake is sufficient to maintain the endogenous pool of ILV. The CodY global regulatory protein displays enhanced affinity for its DNA target when bound to ILV[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref027" target="_blank">27</a>]. ILV-bound CodY binds to the CodY binding sequence upstream of <i>clhAB</i>_<i>2</i> and assists RNA polymerase with transcribing the <i>clhAB</i>_<i>2</i> operon (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g004" target="_blank">4</a>). The expression level of <i>clhAB</i>_<i>2</i> is constant and moderate (i.e. basal level) during the late exponential and transition growth phases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g001" target="_blank">Fig 1</a>). Right: regulation of <i>clhAB</i>_<i>2</i> expression in an amino acid- glucose- rich medium. In LB medium with 0.35% glucose, ILV-CodY binds to the CodY motif and activates <i>clhAB</i>_<i>2</i> expression, but the expression profile is different (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g001" target="_blank">Fig 1</a>). CcpA plays a positive role by indirectly regulating the transcription of <i>clhAB</i>_<i>2</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g002" target="_blank">Fig 2</a>) and this regulatory pathway remains to be characterized. The role of known or hypothetical effector molecules is depicted with a dashed arrow. In the left part, the ILV effector [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref027" target="_blank">27</a>] is depicted with a green dashed arrow; in right part, the Fru-6-P [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref025" target="_blank">25</a>] and unknown glycolytic intermediate effectors are depicted with blue dashed arrows. Moderate and constant <i>clhAB</i>_<i>2</i> expression is depicted by three identical gray arrows. High and gradual <i>clhAB</i>_<i>2</i> expression is depicted by three non-identical gray arrows. The intracellular ILV pool is depicted as a green box. The CodY motif sequence is depicted as a red box. The unknown DNA-binding motif is depicted as a blue box.?, unknown CcpA-dependent signaling pathway; ?, unknown transcriptional regulator. Fru-6-P, fructose-6-phosphate, Glc-6-P, glucose-6-phosphate. ILV, isoleucine, leucine, valine. CodY, CodY transcriptional regulator. CcpA, CcpA global carbon regulator; PTS, phosphotransferase system. T, unknown transporter of ILV.</p

Phenotypic analyses of Δ<i>clhAB</i>_<i>2</i> isogenic mutant.

<p>(A) Box plot of growth rates in the absence (LB) or presence of 0.35% glucose (LBG). Growth rates were determined using output files of OD₆₁₀ values from the microplate reader (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials & Methods</a>). NS—no statistical significance. (B, C) Stationary phase survival of <i>Bc</i> and Δ<i>clhAB</i>_<i>2</i> isogenic mutant in LB and in LBG. Cell viability tests were performed at 0, 4, 24, 48, and 72h. Significance is based on Mann & Whitney test with a <i>P</i> <0.05*.</p

Expression of <i>clhAB</i>_<i>2</i> in the presence or presence of glucose.

<p>(A) Genetic organization of <i>clhAB</i>_<i>2</i> locus in the <i>Bc</i> genome and schematic representation of the <i>clhAB</i>_<i>2</i>’Z transcriptional fusion construct. (B) <i>Bc</i> strain <i>clhAB</i>_<i>2</i>’Z was grown in LB in the presence (open circles) or absence (closed circles) of 0.35% glucose. Optical densities (OD₆₀₀, in blue circles) of cell cultures and β-galactosidase specific activities (U/mg protein, in black losanges) in the presence (open losanges) or absence (closed losanges) of 0.35% glucose are shown. The levels of <i>lacZ</i> expression of pHT304-18’Z (background level) were around 15 U/mg protein. (C) Filtered supernatants were measured for glucose concentration (Glucose Assay Kit, Sigma, in blue squares) and for pH determination (black triangles). The start of the transition growth phase is indicated as <i>t</i>₀ for time zero. The glucose concentration of LB medium (closed squares) was below 0.01% and pH (closed triangles) was 7 ± 0.2. The data presented are representative of four independent experiments.</p

Inter-constriction cell arrangements and cell width measures in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB2ΩclhAB</i>_<i>2</i> populations in the presence or absence of glucose.

<p>(A) Close view of <i>Bc</i>, Δ<i>clhAB2</i>, and Δ<i>clhAB2</i>,<i>ΩclhAB2</i> chains at <i>t</i>₀ using fluorescence microscopy. Cells were grown in LB and LB with 0.35% glucose (LBG). Division septa and cytoplasmic membranes were imaged using the FM4-64 lipophilic dye. Chains exhibited constrictions that occurred at septa spaced 4 cells apart in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB</i>_<i>2</i><i>ΩclhAB</i>_<i>2</i> in LB medium and in Δ<i>clhAB</i>_<i>2</i> in LBG medium. They also exhibited constrictions that occurred at septa spaced 8 cells apart in <i>Bc</i> and Δ<i>clhAB</i>_<i>2</i>,Ω<i>clhAB</i>_<i>2</i> in LBG medium. (B) Distributions of “short” (≤4) and “long” (>4) inter-constriction cell types in the <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and complemented mutant populations (N>250 cell arrangements). Two inter-constriction arrangement types in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB</i>_<i>2</i>Ω<i>clhAB</i>_<i>2</i> populations were defined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>). The first type, containing cell arrangements with two to four cells ("short") and the second type, including cell arrangements with six to eight cells ("long"). The significant effects of glucose (dashed line) and <i>clhAB</i>_<i>2</i> mutation (solid line) are based on a Binomial analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>) with <i>P</i> <0.01** and <0.05*. (C) Cell width measures in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i> and Δ<i>clhAB</i>_<i>2</i>Ω<i>clhAB</i>_<i>2</i> populations (N>250 bacilli) in LB and LBG media. The significant effect of <i>clhAB</i>_<i>2</i> mutation in LBG medium is based on a Student’s t test and ANOVA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>) with <i>P</i> <0.001***. A <i>P</i> value close to the cutoff 0.05 was considered as non significant (NS). Mean ± CI _95% is depicted.</p

CodY-dependent regulation of the <i>clhAB</i>_<i>2</i> operon in the presence or absence of glucose.

<p>Effect of <i>codY</i> mutation on the expression of <i>clhAB</i>_<i>2</i>. Cells of <i>Bc</i> and isogenic mutant strains (Δ<i>codY</i>, <i>codY</i>-complemented mutant), which all harbored the transcriptional P_{<i>clhAB2</i>}’-<i>lacZ</i> fusion construct, were grown in LB (closed symbols) or in LBG (open symbols) media. See legend of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g002" target="_blank">Fig 2</a> for additional informations.</p