Exploitation of Tenebrio molitor larvae as biological factories for human probiotics, an exploratory study

The exploitation of yellow mealworm (Tenebrio molitor) larvae for the bioaugmentation of probiotic Bacillus clausii strains was evaluated during a 7-day rearing period. qPCR was applied to evaluate the persistence and growth of B. clausii in the rearing substrate and larvae (washed and non-washed). Moreover, the effect of freeze-drying of larvae on B. clausii viability was evaluated. The results demonstrated the suitability of yellow mealworm as biological factories for the multiplication of B. clausii through a simple and inexpensive procedure, in view of the further application of larvae as foods and food ingredients. In more detail, an increase in the load of B. clausii was observed during the 7-day rearing of larvae fed wheat middlings spiked with 1 Log cells g−1. Further research is needed to evaluate the most suitable technologies and processing parameters for obtaining yellow mealworm-based ingredients with a stable and active population of probiotic B. clausii

Microbial dynamics in rearing trials of Hermetia illucens larvae fed coffee silverskin and microalgae

In the present study, Hermetia illucens larvae were reared on a main rearing substrate composed of a coffee roasting byproduct (coffee silverskin, Cs) enriched with microalgae (Schizochytrium limacinum or Isochrysis galbana) at various substitution levels. The microbial diversity of the rearing substrates, larvae, and frass (excrement from the larvae mixed with the substrate residue) were studied by the combination of microbial culturing on various growth media and metataxonomic analysis (Illumina sequencing). High counts of total mesophilic aerobes, bacterial spores, presumptive lactic acid bacteria, coagulase-positive cocci, and eumycetes were detected. Enterobacteriaceae counts were low in the rearing diets, whereas higher counts of this microbial family were observed in the larvae and frass. The microbiota of the rearing substrates was characterized by the presence of lactic acid bacteria, including the genera Lactobacillus, Leuconostoc and Weissella. The microbiota of the H. illucens larvae fed Cs was characterized by the dominance of Paenibacillus. H. illucens fed diets containing I. galbana were characterized by the presence of Enterococcus, Lysinibacillus, Morganella, and Paenibacillus, depending on the algae inclusion level, while H. illucens fed diets containing S. limacinum were characterized by high relative abundances of Brevundimonas, Enterococcus, Paracoccus, and Paenibacillus, depending on the algae inclusion level. Brevundimonas and Alcaligenes dominated in the frass from larvae fed I. galbana; the predominance of Brevundimonas was also observed in the frass from larvae fed Schyzochitrium-enriched diets. Based on the results of the present study, an effect of algae nutrient bioactive substances (e.g. polysaccharides, high-unsaturated fatty acids, taurine, carotenoids) on the relative abundance of some of the bacterial taxa detected in larvae may be hypothesized, thus opening new intriguing perspectives for the control of the entomopathogenic species and foodborne human pathogens potentially occurring in edible insects. Further studies are needed to support this hypothesis. Finally, new information on the microbial diversity occurring in insect frass was also obtained

Distribution of Transferable Antibiotic Resistance Genes in Laboratory-Reared Edible Mealworms (Tenebrio molitor L.)

In the present study, the distribution of antibiotic resistance genes in laboratory-reared fresh mealworm larvae (Tenebrio molitor L.), their feeding substrates (carrots and wheatmeal), and frass was assessed. Microbial counts on selective media added with antibiotics highlighted the presence of lactic acid bacteria resistant to ampicillin and vancomycin and, more specifically, enterococci resistant to the latter antibiotic. Moreover, staphylococci resistant to gentamicin, erythromycin, tetracycline, and vancomycin were detected. Enterobacteriaceae resistant to ampicillin and gentamicin were also found, together with Pseudomonadaceae resistant to gentamicin. Some of the genes coding for resistance to macrolide-lincosamide-streptogramin B (MLSB) [erm(A), erm(C)], vancomycin [vanA, vanB], tetracycline [tet(O)], and β-lactams [mecA and blaZ] were absent in all of the samples. For the feeding substrates, organic wheatmeal was positive for tet(S) and tet(K), whereas no AR genes were detected in organic carrots. The genes tet(M), tet(K), and tet(S) were detected in both mealworms and frass, whereas gene aac-aph, coding for resistance to amynoglicosides was exclusively detected in frass. No residues for any of the 64 antibiotics belonging to 10 different drug classes were found in either the organic wheatmeal or carrots. Based on the overall results, the contribution of feed to the occurrence of antibiotic resistance (AR) genes and/or antibiotic-resistant microorganisms in mealworm larvae was hypothesized together with vertical transmission via insect egg smearing

Transmission and scanning electron microscopic observations on antennal apical pegs in the wasp species Pimplinae (Insecta: Hymenoptera)

Pimplinae are parasitoids belonging to the family Ichneumonidae that attack and develops inside hidden host; female wasps evolved a peculiar host recognition strategy, that involves the use of self-produced vibrations which are transmitted through the antennae on the substrate and perceived back as an echo using the leg subgenual organ. In this study we investigated, using both scanning and transmission electron microscopy, the antennal tips of a few Pimplinae. In all the investigated species, the antennal tips present peculiar apical pegs with different shape and number, often defining a flattened sole devoid of other antennal structures, such as sensilla. These pegs are present in both sexes with different number and development, are inserted on the antennal wall through an inflexible socket and present a cuticular shaft with cuticle of different thickness. We never found the presence of sensory neurons or glandular epithelium associated with these pegs. Because of their peculiar morphological features, we hypothesize for the antennal apical pegs a role in the context of host searching behavior (in the case of the female through the vibrational sounding strategy), as well as during mating behavior

Morphology and distribution of antennal multiporous gustatory sensilla related to host recognition in some Trichogramma spp.

Trichogramma spp. (Hymenoptera Trichogrammatidae) are endoparasitic wasps that attack the eggs of mainly Lepidoptera species. The morphology of the antennal multiporous gustatory sensilla of five Trichogramma species is studied and compared: Trichogramma cacoeciae Marchal, Trichogramma chilonis Ishii, Trichogramma dendrolimi Matsumura, Trichogramma turkestanica Meyer, and Trichogramma sibericum Sorokina. Scanning electron microscopy of the female antennae shows the presence of numerous peculiar sensilla on the ventral apical side of the club, for which a gustatory function is hypothesized. These appear as multiporous sensilla, although the pores are not distributed all over the surface of the hair, as they are only on the ventral margin. Also, these sensilla are located exclusively on antennal areas that come into contact with the substrate. Variations in the numbers and distributions of the multiporous gustatory sensilla were found between the species examined, as well as within individuals belonging to the same species. Functional aspects and variations in the distributions of these sensilla are discussed

Fine structure of antennal sensilla of the spittlebug Philaenus spumarius L. (Insecta: Hemiptera: Aphrophoridae). I. Chemoreceptors and thermo-/hygroreceptors

The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Cercopoidea: Aphrophoridae), is a polyphagous species that transmits Xylella fastidiosa, a bacterium associated with “Olive Quick Decline Syndrome” in Southern Italy. In this study, the morphology and the ultrastructure of the antennal sensilla of P. spumarius were investigated. The antennae consist of three segments: a basal scape, a pedicel and a flagellum composed of a basal enlargement (ampulla) and a long segment (filament). The pedicel bears a single campaniform sensillum while the ampulla houses twelve coeloconic sensilla and three large basiconic sensilla. These latter sensilla show a smooth multiporous external cuticular wall and a total number of 27 sensory neurons per sensillum. The coeloconic sensilla belong to two morphologically distinct types: double-walled and single-walled sensilla. The sensory peg of the double-walled sensilla is smooth at the base and distally has a grooved cuticular surface with pores organized in spoke channels between each ridge. Three sensory neurons enter the lumen while at the basal level, before entering the peg, a fourth sensory neuron is found. The single-walled sensilla show an aporous thick cuticular wall and two sensory neurons entering the sensillar lumen, with a third neuron ending at the sensillum base

Sensory Receptors Associated with the Labial Tip and Precibarium of Philaenus spumarius L. (Hemiptera: Aphrophoride)

The meadow spittlebug, Philaenus spumarius (Linnaeus) (Hemiptera: Aphrophoridae), is an important vector for the xylem-limited bacterium Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner), which is associated with olive quick decline syndrome in southern Italy. The mouthparts of Hemiptera have important roles in host plant selection, feeding behavior and for vectoring pathogens that cause plant diseases. In this study, the functional morphology of the sensory structures located on the labium tip and precibarium of P. spumarius was investigated using scanning and transmission electron microscopy. The labium tip is composed of two symmetrical sensory complexes, each with five different types of sensilla: aporous sensilla trichodea type 1 and 2; uniporous sensilla chaetica type 1 and 2; and multiporous sensilla basiconica. The precibarium of P. spumarius has two kinds of sensory structures: bulbous sensilla and papillae sensilla. In particular, two groups of sensilla are located on the epipharynx: a distal group that consists of ten papillae sensilla and a proximal group composed of six papillae sensilla and two bulbous sensilla, while the hypopharynx has only two papillae sensilla. The involvement of these sensory structures in the context of feeding behavior and pathogen transmission is discussed

Female sex pheromone gland of the boxwood leafminer, Monarthropalpus buxi (Diptera: Cecidomyiidae): Morphological and behavioural evidence

Morphological studies were carried out to determine whether females of the boxwood leafminer, Monarthropalpus buxi (Laboulbène) (Diptera: Cecidomyiidae) have a pheromone gland, its position and ultra-structure. In addition, the reproductive behaviour of this gall midge was studied in the laboratory in a wind-tunnel. The source of the sex pheromone was identified as the epidermis of the inter-segmental membrane between the 8th and 9th abdominal segments of females. The epidermis there was hypertrophied and consisted of a unicellular layer of secretory cells with ultra-structural features that include a well developed smooth endoplasmic reticulum and Golgi complexes that are typical of pheromone-producing glands. At adult emergence the sex ratio was 1 : 1. The virgin females extruded their ovipositor and moved it slowly (visible using a video-camera at high magnification), which was followed by mating. The females were monogamous, while the males usually mated several times with different females. Wind-tunnel bioassays using virgin females, ovipositor-ectomised virgins and mated females provided further evidence that virgin females produce a sex pheromone