Morphology of the aedeagal gland of the male mealworm beetle (Tenebrio molitor L.)

The aedeagal gland of male Tenebrio molitor consists of numerous acini containing several secretory units (organules) of three epithelial cells in series. The distal cortical cell and intermediate cell are secretory cells. Secretory products are passed into microvilli-lined extracellular reservoirs. From these storage areas products flow through minute canaliculi and into the efferent ductule. Canaliculi, cuticular trabeculae, and fibrillar material are characteristic features of the efferent ductules within the extracellular reservoirs of secretory cells. After passing from the secretory cells, the efferent ductule penetrates the basal ductule cell. The thin epicuticle that comprises the wall of the ductule is confluent with the epicuticle of the cuticular sheath forming the wall of the genital pocket. Secretory products flow from the cortical cell ductule into the intermediate cell and eventually empty into the genital pocket. A chemical reaction apparently takes place in the intermediate cell ductule, resulting in a frothy secretion product. When released from the ductule, this frothy product forms a foam-like layer that coats the inner wall of the genital pocket. Ultrastructural and probable functional aspects of this gland are described and discussed.     

Cytodifferentiation in the accessory glands of Tenebrio molitor. X. Ultrastructure of the tubular gland in the male pupa

The tubular accessory gland consists of a simple secretory epithelium surrounded by a muscular coat. Over the pupal instar, the gland increases ten-fold in volume and 15-fold in length. Pupal development is divisible into a phase of mitosis and one of cell growth. During the mitotic phase, cytoplasmic membranes are sparse and nuclei move toward the luminal face of the epithelium to undergo division. In the cell growth phase, the cells become more columnar, a few stacks of rough endoplasmic reticulum are formed, and small dense secretory vesicles appear near the apical surface. The hormonal control of the developmental sequence is discussed.     

Trehalase from the bean-shaped accessory glands and the spermatophore of the male mealworm beetle,Tenebrio molitor

Summary InTenebrio molitor, male adults transfer sperm to the female via a spermatophore or sperm sac. The spermatophore is formed from secretions of the bean-shaped accessory glands (BAGs) and the tubular accessory glands (TAGs) of the male beetle. Trehalase is found in the adult BAGs. During the pupal stage, the activity in the BAGs was very low. After adult ecdysis, the total activity increased 100-fold from 0 days to 6 days and reached maximum levels at 9 days. The specific activity increased 20-fold from the time of ecdysis to 6 days thereafter. In the 10 day adult, trehalase levels in testes, seminal vesicles, vas deferens, TAGs, or ejaculatory ducts, were lower by two orders of magnitude than in the BAGs. However, the specific activity in the spermatophore was similar to that in the BAGs. Trehalases in the BAGs and the spermatophores showed very similar properties (soluble, optimum pH of 5.75 andK _m value of 5.4 mM for trehalose). Thus trehalase appears to be secreted from the BAGs and becomes incorporated into the spermatophores.Abbreviations BAG bean-shaped accessory gland - TAG tubular accessory gland     

Trehalase in the spermatophore from the bean-shaped accessory gland of the male mealworm beetle,Tenebrio molitor: purification,kinetic properties and localization of the enzyme

Trehalase from the bean-shaped accessory glands of the male mealworm beetle, Tenebrio molitor, was purified by acid treatment, with subsequent chromatography on columns of DEAE-cellulofine and Sephacryl S-300. The molecular masses of the native and the denatured forms were estimated to be 43 and 62 kDa by gel filtration and SDS-PAGE, respectively, an indication that the trehalase may be composed of a single polypeptide. The optimum pH of the reaction catalyzed by trehalase was 5.6–5.8. The K _m for trehalose was 4.4 mmol·l^–1. Immunohistochemical experiments with trehalase-specific antiserum showed that the enzyme was localized in one specific type of secretory cell in the bean-shaped accessory gland epithelium and within the semisolid secretory mass that was a precursor to the wall of spermatophore. SDS-PAGE and immunoblotting analysis revealed the presence of a polypeptide of about 62 kDa in the spermatophore, Immunohistochemical observations showed that the trehalase was located at the outgrowth in the anterior portion of the spermatophore. When a fresh spermatophore was immersed in phosphate-buffered saline it discharged sperm in the same manner as in the bursa copulatrix of the female. Before the rupture of the expanded bulb of the spermatophore, almost all of the trehalase had dissolved in the phosphate-buffered saline. The addition of validoxylamine A to the saline, a specific inhibitor of trehalase, did not affect the expansion and evacuation of the spermatophore. These results demonstrate that trehalase, synthesized by a specific type of secretory cell in the bean-shaped accessory gland epithelium, is actively passed into the lumen of the bean-shaped accessory gland and then incorporated into the spermatophore. Trehalase appears to be one of the structural proteins of the spermatophore, although the possibility can not yet be completely ruled out that the trehalase-trehalose system functions for the nourishment and/or activation of the sperm in the bursa copulatrix of the female.Abbreviations BAG bean-shaped accessory gland(s) - DEAE diethylaminoethyl - Kpi buffer K₂HPO₄/KH₂PO₄ buffer (pH 7.0) - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SDS sodium dodecy sulphate - Spph spermatophore(s) - TAG tubular accessory gland(s)     

20-Hydroxyecdysone control of the synthesis of putative calcium-binding proteins in the epidermis of Tenebrio molitor

The altered pattern of synthesis of putative calcium-binding proteins (pCaBPs) in the mid-instar epidermis following exposure to 20-hydroxyecdysone (20HE) in vitro was followed through the incorporation of [³⁵S]methionine into newly synthesized polypeptides. pCaBPs were separated from other epidermal polypeptides by Ca²⁺-dependent hydrophobic interaction chromatography, followed by polyacrylamide gel electrophoresis and fluorography. The dominant effect of 20HE is to depress pCaBP synthesis. Of the 17 newly-synthesized pCaBPs consistently detected in total cell lysates, the synthesis of ten was depressed strongly and that of the remaining seven was unaltered by exposure to 20HE. Most newly-synthesized pCaBPs identified were found in the cytosolic fraction of the epidermis. One pCaBP was identified as Tenebrio calmodulin based on its altered electrophoretic mobility in the absence of calcium ions, its isoelectric focusing point, its binding to phenyl-Sepharose and phenothiazine and its binding to antibodies against purified mammalian calmodulin. The synthetic rate of this pCaBP was not affected by 20HE. The distribution of another pCaBP (32 kDa) shifted from the cytosolic to the microsomal fraction on adding Ca²⁺ (or reversed by adding EGTA) to the cell extract before fractionation. The synthesis of this protein was depressed by 20HE. These findings suggest that 20HE influences epidermal behaviour, at least in part, through its ability to modulate the synthetic rate of several pCaBPs.     

Presynaptic modulation by octopamine at a single neuromuscular junction in the mealworm (Tenebrio molitor)

The effect of octopamine on the neuromuscular junction of the mealworm (Tenebrio molitor) was examined. Octopamine potentiated excitatory junctional potentials (EJPs) recorded intracellularly and extracellularly from ventral longitudinal muscle fibers. The potentiating action of octopamine was blocked in the presence of the alpha-adrenergic blocking agent, phentolamine, but not in the presence of another alpha-blocker, phenoxybenzamine, or the beta-blockers propranolol and dichloroisoproterenol. The resting membrane potential, membrane input resistance, reversal potential of EJP, glutamate potentials, and spontaneous miniature EJPs were found to be unaffected by octopamine. In contrast, quantal contents estimated by the extracellularly recorded EJP failures were greatly increased by octopamine. These results suggest that octopamine acted on the presynaptic terminals via alpha-adrenoceptor-like receptors (octopamine receptors) at the Tenebrio neuromuscular junctions to enhance the transmitter release associated with the motor nerve impulses.     

Cytodifferentiation in the accessory glands of Tenebrio molitor I. Ultrastructure of the tubular gland in the post-ecdysial adult male

The tubular accessory reproductive glands of the male mealworm beetle consist of a secretory epithelium surrounded by a thin muscular sheath. Each columnar secretory cell is divisible into three zones: basal which is adjacent to the muscle layer and contains rough endoplasmic reticulum and Golgi, intermediate, which contains endoplasmic reticulum and Golgi zones in the immature gland and is filled with secretory vesicles in the mature gland, and apical. Maturation also involves proliferation and organization of the rough endoplasmic reticulum in the basal and intermediate zone. The process appears to be complete at four days after ecdysis. Parallels with other insect glands and with the mammalian prostate are striking.     

Selective breeding and characterization of a black mealworm strain of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae)

Larvae of Tenebrio molitor Linnaeus are edible insects and are approved as a food ingredient in Korea. They are typically yellow; however, rare black larvae have been found in breeding boxes at insect farms. It is not clear whether black larvae represent a different species that invaded and hybridized with the yellow larvae of T. molitor or whether T. molitor shows intraspecific color variation. In this study, we characterized and identified black larvae for applications in industrial fields as well as accurate breeding and management. First, in a comparative analysis, we did not detect differences in the morphological characteristics of yellow and black larvae and adults, with the exception of larval body color. For accurate species identification, molecular analyses (p-distances and neighbor-joining) were performed based on partial COI sequences of 33 yellow and seven black larvae. Genetic divergence between yellow and black larvae ranged from 0.0% to 2.1%, revealing intraspecific variation. A neighbor-joining analysis strongly supported the classification of the two morphs as a single species. Black larvae were separated from yellow larvae and maintained by selective breeding. As a result, black larvae were completely fixed in the F2 generation (F1 = 96% and F2 = 100%). Yellow and black larvae showed no significant differences in developmental characteristics and fecundity. These findings improve our understanding of diversity within an important edible insect species and contribute to quality assurance in the food industry based on clear species identification.     

Isolation,partial characterization,and localization of the A and B proteins from the tubular accessory gland of male Tenebrio molitor

In its fully differentiated state, the tubular accessory gland of the male mealworm beetle, Tenebrio molitor, synthesizes five groups of proteins (A, B, C, D₁ and D₂) which are easily distinguished from one another on two-dimensional pI-SDS polyacrylamide gels (Black et al., 1982: Devl Biol. 94, 106–115). In the present work, the A and B proteins have been isolated by preparative gel electrophoresis and Amicon ultrafiltration. The isolation procedure provided two fractions of interest: one contained a mixture of A and B proteins (A/B) and the other consisted of only B proteins. The major proteins in the A class have a molecular weight of 17,900 while those of the B class are 19,000 daltons in size.Antibodies have been produced to the A/B mixture and to the B fraction. Ouchterlony immunodiffusion and straight line immunoelectrophoresis show that the A and B proteins share common immunological characteistics. The proteins from the tubular accessory gland were displayed on one dimensional SDS gels and electrophoretically blotted onto nitrocellulose paper. The antibodies to the A/B mixture recognize A and B bands on these gels. In addition, these antibodies show affinity for C proteins and another band of lower molecular weight.Using the anti-A/B with techniques of immunodiffusion, straight line immunoelectrophoresis, and immunoblotting, we have identified the A and B protein in extracts of soluble proteins in the spermatophore. Furthermore, the A/B proteins have been localized by immunohistochemical techniques within the apical portions of the secretory cells of the tubular gland and also in the lumen of the spermatophore.     

Extracardiac versus cardiac haemocoelic pulsations in pupae of the mealworm (Tenebrio molitor L.)

Pulsations in mechanical pressure of the pupal haemocoele were investigated by means of simultaneous recording from multiple sensors. It has been determined that cardiac and extracardiac haemocoelic pulsations are each regulated by substantially different and quite independent physiological mechanisms. At the beginning and in the middle of the pupal interecdysial period the anterograde heartbeat and extracardiac pulsations occur in similar, but not identical periods. During the advanced pharate adult stage, there appear almost uninterrupted pulsations from different sources: cardiac, extracardiac, intestinal, and the ventral diaphragm.Extracardiac pulsations are associated with pressure peaks of 200-500 Pa, occurring at frequencies of 0.3-0.5 Hz. The effect of heartbeat on haemocoelic pressure is very small, 100- to 500-fold smaller, comprising only some 1 or 2 Pa during the vigorous anterograde systolic contractions. Accordingly, extracardiac pulsations are associated with relatively large abdominal movements from 30-90 μm whereas heartbeat produces movements of only 100-500 nm. This shows that extracardiac pulsations can be easily confused with the anterograde heartbeat. It does not seem realistic to assume that the relatively weak insect heart, and not the 100- to 500-fold more powerful extracardiac system of abdominal pump, could be at all responsible for selective accumulation of haemolymph in anterior parts of the body, for inflation of wings or enhancement of tracheal ventilation.It has been established that thermography from the pericardial region is not specific for the heartbeat. It records subepidermal movement of haemolymph resulting from the actions of both dorsal vessel and extracardiac pressure pulses as well. Shortly before adult eclosion the cardiac and extracardiac pulsations occasionally strike in concert, which profoundly increases the flow of haemolymph through pericardial and perineural sinuses. The relatively strong extracardiac pulsations cause passive movements of various visceral organs, tissue membranes, or tissue folds, giving thus a false impression of an authentic pulsation of tissues. In addition, extracardiac pulsations cause rhythmical movements of haemolymph between various organs, thus preventing haemolymph occlusion at the sites where the heart does not reach. It has been emphasized, finally, that the function of the autonomic nervous system (coelopulse), which integrates extracardiac pulsations, depends on homeostatic moderation of excessive or deficient conditions in insect respiration and haemolymph circulation.     

Effects of processing methods on nutritional composition and antioxidant activity of mealworm (Tenebrio molitor) larvae

We examined the effects of different processing methods on the nutritional composition and antioxidant activity of mealworms. After processing with nine methods, we calculated the contents of protein, fat, ash, carbohydrate, minerals (P, Ca, K, Fe, Na), vitamin B group (B₁, B₂, B₃), moisture, and calories. When processed by freeze drying among freeze drying, hot air drying, oven broiling, roasting, pan frying, deep frying, boiling, steaming, and microwaving, the contents of protein, some minerals, and vitamins were the highest. The content of total minerals was lowest after deep frying, and those of vitamin B₁ and B₃ were the lowest after microwaving. Antioxidant activity was then evaluated using DPPH and ABTS radical scavenging assays. DPPH assays showed that microwaving, freeze drying, deep frying, steaming, boiling, and oven broiling of mealworms yielded scavenging activities of 20.9–29.0% at 2,000 μg/mL, which was similar to the activity level (22.7–33.2%) of 40–60 μM tocopherol. ABTS assays confirmed that only freeze‐dried mealworms at 2,000 μg/mL exhibited higher activity than 10 μM tocopherol. Interestingly, similar trends were found for antioxidant activity levels and total phenolic contents in mealworms.     

Cytodiffrentiation in the accessory glands of Tenebrio molitor. VI. A congruent map of cells and their secretions in the layered elastic product of the male bean-shaped gland

The morphology of the bean-shaped accessory glands (BAGs) of males of Tenebrio molitor is described. All cells in the secretory epithelium are long and narrow (300–400 mμ × 5 mμ). The seven types of secretory cells are distinguished from one another by the morphology of their secretory granules. Granule substructure varies from simple spheres with homogeneous electrondense contents to complex forms with thickened exterior walls or with crystalline and membranous contents. Individual cell types were mapped by staining whole glands with Oil Red O, and the cell distributions were confirmed by wax histology and ultramicroscopy. The secretions of all seven cell types form a secretory plug composed of seven layers. During mating, the secretory plug from each BAG is forced into the ejaculatory duct by contractions of a sheath of circular muscle. The mirror image plugs from symmetrical BAGs fuse and are transformed into the wall of the spermatophore.     

Developmental and environmental regulation of antifreeze proteins in the mealworm beetle Tenebrio molitor.

The yellow mealworm beetle, Tenebrio molitor, contains a family of small Cys-rich and Thr-rich thermal hysteresis proteins that depress the hemolymph freezing point below the melting point by as much as 5. 5 degrees C (DeltaT = thermal hysteresis). Thermal hysteresis protein expression was evaluated throughout development and after exposure to altered environmental conditions. Under favorable growth conditions, small larvae (11-13 mg) had only low levels of thermal hysteresis proteins or thermal hysteresis protein message, but these levels increased 10-fold and 18-fold, respectively, by the final larval instar (> 190 mg), resulting in thermal hysteresis > 3 degrees C. Exposure of small larvae (11-13 mg) to 4 weeks of cold (4 degrees C) caused an approximately 20-fold increase in thermal hysteresis protein concentration, well in excess of the less than threefold developmental increase seen after 4 weeks at 22 degrees C. Exposure of large larvae (100-120 mg) to cold caused 12-fold and sixfold increases in thermal hysteresis protein message and protein levels, respectively, approximately double the maximum levels they would have attained in the final larval instar at 22 degrees C. Thus, thermal hysteresis increased to similar levels (> 4 degrees C) in the cold, irrespective of the size of the larvae (the overwintering stage). At pupation, thermal hysteresis protein message levels decreased > 20-fold and remained low thereafter, but thermal hysteresis activity decreased much more slowly. Exposure to cold did not reverse this decline. Desiccation or starvation of larvae had comparable effects to cold exposure, but surprisingly, short daylength photoperiod or total darkness had no effect on either thermal hysteresis or message levels. As all environmental conditions that caused increased thermal hysteresis also inhibited growth, we postulate that developmental arrest is a primary factor in the regulation of T. molitor thermal hysteresis proteins.     

Biosynthesis of 4-methyl-1-nonanol: Female-produced sex pheromone of the yellow mealworm beetle,Tenebrio molitor (Coleoptera: Tenebrionidae)

The objective of this study was to elucidate the biosynthetic route to 4-methyl-1-nonanol, the female-produced sex pheromone of the yellow mealworm beetle, Tenebrio molitor L. The biosynthetic route to the pheromone was examined by (i) allowing the females to feed on defatted bran coated with a stable isotope-labeled putative precursor ([1-¹³C]acetate, [1-¹³C]propionate, [1-¹³C]pentanoate, [1-¹³C]2-methylheptanoic acid, or [²H₂]4-methylnonanoic acid); (ii) determining if the precursors were incorporated by analyzing the emitted pheromone by gas chromatography/selected ion monitoring-mass spectroscopy (GC/SIM-MS); (iii) where the pheromone was isotopically-enriched, determining the position of the isotopic label(s) through comparison of the MS fragmentation pattern with that of unlabelled 4-methyl-1-nonanol. Although the incorporation of [1-¹³C]acetate into 4-methyl-1-nonanol could not be detected, relatively large proportions of the pheromone were produced from the other precursors tested: 81% from [²H₂]4-methylnonanoic acid, 45% from [1-¹³C]2-methylheptanoic acid, 16% from [1-¹³C]pentanoate, and 35% from [1-¹³C]propionate (27% from only one unit, and 7.8% from two units). The results indicate that 4-methyl-1-nonanol is produced through a modification of normal fatty acid biosynthesis: initiation of the pathway with one unit of propionate results in the uneven number of carbons in the chain; incorporation of another unit of propionate during elongation provides the methyl branch; reduction of 4-methylnonanoic acid produces the alcohol pheromone. The elucidation of the biosynthetic pathway of 4-methyl-1-nonanol biosynthesis in the yellow mealworm is the first step towards understanding the biochemistry of sex pheromone production in this species.     

Gonadoinhibitory effects of Neb-colloostatin and Neb-TMOF on ovarian development in the mealworm, Tenebrio molitor L

The gonadostatic action of the peptides Neb-colloostatin (SIVPLGLPVPIGPIVVGPR) and Neb-TMOF (NPTNLH) from Neobellieria bullata was studied in female mealworm Tenebrio molitor. Both peptides potently inhibit ovarian development and terminal oocyte maturation of mated females during their first reproductive cycle. Injection of 4 mug of Neb-colloostatin or Neb-TMOFNeb-TMOF reduced, at day 4 of the cycle, the size of the terminal oocytes to about half or one third of the normal size in saline-injected controls. In addition, follicular patency was arrested. The injections of Neb-colloostatin and Neb-TMOF also caused a delay to the first ovulation and oviposition as well as a reduction of the number of eggs by about 50% in the first 3 days of the oviposition period. At 4 days after adult emergence, none of the peptides had caused significant changes in protein concentration or composition of the haemolymph. However, both peptides reduced total protein content in ovaries and induced qualitative changes in ovarian protein patterns. Electrophoretic analyses indicated that Neb-colloostatin and Neb-TMOF caused a loss of two proteins (150, 180 kDa) and a drastic reduction of 4 others (39, 43, 47, 130 kDa), which are the most abundant ones in ovaries of control females. On the other hand, they increased the concentration of 2 other polypeptides (65, 70 kDa), which normally occur in insignificant quantities in ovaries. Our results indicate that both peptides have a very similar mode of action despite the differences in their amino acid sequence. They seem to interfere with vitellogenin production by the fat body as well as with vitellogen uptake by the oocytes through modification of patency.