Tenebrio molitor larvae meal inclusion affects hepatic proteome and apoptosis and/or autophagy of three farmed fish species.

Herein, the effect of dietary inclusion of insect (Tenebrio molitor) meal on hepatic pathways of apoptosis and autophagy in three farmed fish species, gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss), fed diets at 25%, 50% and 60% insect meal inclusion levels respectively, was investigated. Hepatic proteome was examined by liver protein profiles from the three fish species, obtained by two-dimensional gel electrophoresis. Although cellular stress was evident in the three teleost species following insect meal, inclusion by T. molitor, D. labrax and O. mykiss suppressed apoptosis through induction of hepatic autophagy, while in S. aurata both cellular procedures were activated. Protein abundance showed that a total of 30, 81 and 74 spots were altered significantly in seabream, European seabass and rainbow trout, respectively. Insect meal inclusion resulted in individual protein abundance changes, with less number of proteins altered in gilthead seabream compared to European seabass and rainbow trout. This is the first study demonstrating that insect meal in fish diets is causing changes in liver protein abundances. However, a species-specific response both in the above mentioned bioindicators, indicates the need to strategically manage fish meal replacement in fish diets per species.

Solanum Nigrum Fruit Extract Increases Toxicity of Fenitrothion-A Synthetic Insecticide, in the Mealworm Beetle Tenebrio Molitor Larvae.

Synthetic insecticides are widely used for crop protection both in the fields and in the food stored facilities. Due to their toxicity, and assumptions of Integrated Pest Management, we conducted two independent experiments, where we studied the influence of Solanum nigrum unripe fruit extract on the toxicity of an organophosphorus insecticide fenitrothion. In the first variant of the experiment, Tenebrio molitor larvae were fed with blended fenitrothion (LC50) and the extract in four concentrations (0.01, 0.1, 1 and 10%) in ratio 1:1 for 3 days. In the second variant, a two-day application of fenitrothion (LC40) was preceded by a one-day extract treatment. The first variant did not show any increase in lethality compared to fenitrothion; however, ultrastructure observations exhibited swollen endoplasmic reticulum (ER) membranes in the midgut and nuclear and cellular membranes in the fat body, after application of blended fenitrothion and extract. An increased amount of heterochromatin in the fat body was observed, too. In the second variant, pre-treatment of the extract increased the lethality of larvae, decreased the level of glycogen and lipids in the fat body and disrupted integrity of midgut cellular membranes. S. nigrum extract, applied prior to fenitrothion treatment can be a factor increasing fenitrothion toxicity in T. molitor larvae. Thus, this strategy may lead to decreased emission of synthetic insecticides to the environment.

Protease IV, a quorum sensing-dependent protease of Pseudomonas aeruginosa modulates insect innate immunity.

In Pseudomonas aeruginosa, quorum sensing (QS) plays an essential role in pathogenesis and the QS response controls many virulence factors. Using a mealworm, Tenebrio molitor as a host model, we found that Protease IV, a QS-regulated exoprotease of P. aeruginosa functions as a key virulence effector causing the melanization and death of T. molitor larvae. Protease IV was able to degrade zymogens of spätzle processing enzyme (SPE) and SPE-activating enzyme (SAE) without the activation of the antimicrobial peptide (AMP) production. Since SPE and SAE function to activate spätzle, a ligand of Toll receptor in the innate immune system of T. molitor, we suggest that Protease IV may interfere with the activation of the Toll signaling. Independently of the Toll pathway, the melanization response, another innate immunity was still generated, since Protease IV directly converted Tenebrio prophenoloxidase into active phenoloxidase. Protease IV also worked as an important factor in the virulence to brine shrimp and nematode. These results suggest that Protease IV provides P. aeruginosa with a sophisticated way to escape the immune attack of host by interfering with the production of AMPs.

[Modulating effect of weak combined magnetic fields on duration of mealworm beetle Tenebrio molitor metamorphosis stage].

It is shown that an exposure of pupae of the mealworm beetle Tenebrio molitor to the combined static (42 µT) and very weak alternating (250 nT) magnetic fields exerts different influence, depending on the frequency of the alternating magnetic field, on duration of metamorphosis processes in these insects. For instance, an exposure of pupae to weak combined magnetic fields, adjusted to the frequency of ion cyclotron resonance for glutaminic acid (4,4 Hz), stimulates metamorphosis process--a transitional stage from pupae to imago lasts shorter. An inhibiting effect was observed when adjusted to the frequency of ion cyclotron resonance for Ca2 (32,2 Hz). At some frequencies this effect is not seen. For instance, an exposure at a frequency of ion cyclotron resonance for K+ (16,5 Hz) exerts no noticeable effect on the duration of the pupal metamorphosis stage.

Opposite effect of capsaicin and capsazepine on behavioral thermoregulation in insects.

Transient receptor potential channels are implicated in thermosensation both in mammals and insects. The aim of our study was to assess the effect of mammalian vanilloid receptor subtype 1 (TRPV1) agonist (capsaicin) and antagonist (capsazepine) on insect behavioral thermoregulation. We tested behavioral thermoregulation of mealworms larvae intoxicated with capsaicin and capsazepine in two concentrations (10(-7) and 10(-4) M) in a thermal gradient system for 3 days. Our results revealed that in low concentration, capsaicin induces seeking lower temperatures than the ones selected by the insects that were not intoxicated. After application of capsazepine in the same concentration, the mealworms prefer higher temperatures than the control group. The observed opposite effect of TRPV1 agonist and antagonist on insect behavioral thermoregulation, which is similar to the effect of these substances on thermoregulation in mammals, indicates indirectly that capsaicin may act on receptors in insects that are functionally similar to TRPV1.

Immunogold localization of RNA polymerase II and pre-mRNA splicing factors in Tenebrio molitor oocyte nuclei with special emphasis on karyosphere development.

Ultrastructural and immunomorphological characteristics of the developing karyosphere and extrachromosomal nuclear bodies (NBs) in Tenebrio molitor oocytes are presented. Three consecutive stages of karyosphere development were identified: reticular, compact and ring-shaped. At the beginning of the karyosphere development (reticular and compact stages), condensed chromosomes are associated with a fibrogranular material (FGM). The successive karyosphere development is accompanied by the reorganization of FGM into fibrogranular NBs. Special attention was given to the nuclear distribution of hyperphosphorylated and non-phosphorylated forms of RNA polymerase II (pol II) and pre-mRNA splicing factors (snRNPs and SC35 protein) during karyosphere development and NB formation. The immunoelectron microscopy revealed that two forms of pol II and splicing factors being assembled in FGM are deposited in appropriate NBs. Some NBs were also shown to contain coilin, a marker protein for Cajal (coiled) bodies. We suggest that different types of NBs appearing in T. molitor oocyte nuclei along with the cessation of transcriptional activity during the karyosphere development represent storage domains for inactive RNA transcription/processing machinery to later usage in early embryogenesis.

The pro-phenoloxidase of coleopteran insect, Tenebrio molitor, larvae was activated during cell clump/cell adhesion of insect cellular defense reactions.

To characterize the proteins involved in cell clump/cell adhesion of insect cellular defense reactions, we induced the cell clump/cell adhesion reaction in vitro with the hemolymph of larvae of the coleopteran insect, Tenebrio molitor. The 72 kDa protein was specifically enriched in the residues of cell clump/cell adhesion and was purified to homogeneity. A cDNA clone for the 72 kDa protein was isolated. We found that the 72 kDa protein was an activated phenoloxidase from Tenebrio pro-phenoloxidase. We suggest that activated phenoloxidase is involved in the cell clump/cell adhesion reaction as well as in the synthesis of melanin.

Development of locustatachykinin immunopositive neurons in the central complex of the beetle Tenebrio molitor.

Locustatachykinin-immunoreactive (LomTK-IR) interneurons were found to be associated with the central complex, a prominent neuropil region of the insect brain. The structures and development of this set of brain interneurons was studied from the embryo onward in the beetle Tenebrio molitor, showing individual neurons that persist from the late embryo to the adult stage. Their essential structural characteristics were already present in the late embryo, but distinct parts of their arborization patterns became newly formed throughout development. Using a combination of immunohistochemistry and single-cell injection, we demonstrated minute structural changes, allowing a characterization of structural plasticity of identifiable, persistent, neuropeptidergic neurons throughout ontogenesis. Furthermore, this study has provided new information about basic principles of central brain neuroanatomy and the development of a distinct midbrain region of the insect brain, the central complex. The development of its basic connections, the connections between the fan-shaped body and the protocerebral bridge, and the compartmentation of these neuropil regions were shown, using LomTK-IR neurons as marker structures. These basic features of the central complex-associated LomTK-immunopositive neurons were formed in the embryonic brain, whereas in metamorphosis, reorganization of these persistent interneurons was restricted to the formation of a precisely defined projection of their side branches.

Embryonic and postembryonic development of serial homologous neurons in the subesophageal ganglion of Tenebrio molitor (Insecta: Coleoptera).

Neuroblast pattern, engrailed expression and proliferation in the subesophageal neuromers of the beetle Tenebrio molitor are characterized throughout embryogenesis. The proliferation of neuroblasts has been studied throughout postembyronic development. Serotonin, crustacean cardioactive peptide and tyrosine hydroxylase-like-immunoreactive neurons are characterized and their neuronal development has been studied. There is an initial posterior-anterior gradient in neuroblast segregation leading to a reduced number of neuroblasts in the frontal subesophageal neuromer. The study of the engrailed expression shows that only the anterior subfraction of the neuromeral neuroblast configuration is reduced, whereas the posterior two rows of engrailed-positive neuroblasts are not affected during the first 40% of embryogenesis. The overall number of proliferations in the first subesophageal neuromer reaches only 30-50% of the value found in each of the other two neuromers. The analysis of serotonin and crustacean cardioactive peptide immunoreactivity allows the identification of serial homologous neurons which persist from the early embryo to the adult stage. In the different gnathal neuromers, these neurons form structurally highly similar projection patterns, but show different extensions of their arborizations, corresponding to the relative size of each neuromer. Structural homologies between subesophageal and thoracic neuromers are discussed.

Regeneration in the anterior-posterior axis of the insect thoracic segment.

After removal of a transverse strip of ventral thorax from the beetle, Tenebrio molitor, interaction occurred between epidermis posterior to the mesothoracic leg and that anterior to the metathoracic leg. Depending on the size and position of the excision, this interaction resulted in either the regeneration of the extirpated tissue or its replacement by an A/P reversed pattern of sclerites and supernumerary leg. By either route, local pattern continuity was restored between the normal meso- and metathoracic legs. Similarly, when a leg plus adjacent tissue was extirpated, continuity was restored by leg regeneration or by formation of an A/P reversed duplication of sclerites. The results of these strip excisions can be understood in terms of two current models of the ventral thorax (the Boundary Model and the Polar Coordinate Model), each of which postulates a distinct compartment or region intervening between the epidermis surrounding the bases of successive legs. However, the models do not explain the large differences in the frequency of formation of the duplication/deletion pattern after excisions of different widths. The results are also compatible with a different model, involving an A-P sequence of positional values similar to that proposed for the abdominal segment. Regeneration would restore continuity within the sequence by the shortest route, forming either the midsegment (including the leg) or the intersegmental region. The meso- and metathorax differ in the structure of the ventral sclerites and in the segmentation of the tarsus of the leg. The structures regenerated after the various excisions show that the segment border is not crossed during regeneration and indicate that an A/P compartment border running through the leg is usually also respected. There is no sign, however, of a third line of lineage restriction that would indicate a subdivision of the segment into three compartments (as proposed in the Boundary Model).