Constancy of ascending projections in the metamorphosing brain of the meal-beetle Tenebrio molitor L. (Insecta: Coleoptera)

Summary The fate of ascending projections of thoracic interneurons in the metamorphosing brain of Tenebrio molitor is described. Persistent brain neurons were identified and their fate is described during metamorphosis. The projection sites of ascending elements are invariable throughout metamorphosis both in quantitative and in qualitative terms. Some of these ascending neurons are serotonin-immunoreactive and this set of neurons maintains a constant projection site within the metamorphosing brain. The alterations in the projection sites of these and other ascending neurons in the ventral nerve cord were analysed experimentally. The central projection sites of these persistent ascending neurons are not important for the maintenance of their nerve cord projections throughout metamorphosis. Experimental deletion of ascending neurons which project into the suboesophageal ganglion varies the shape of persistent central neurons.     

Crustacean cardioactive peptide-immunoreactive neurons in the ventral nerve cord and the brain of the meal beetle Tenebrio molitor during postembryonic development

Summary By use of an antiserum against the crustacean cardioactive peptide (CCAP) several types of bilaterally symmetrical neurons have been mapped quantitatively in the ventral nerve cord and in the brain of the meal beetle, Tenebrio molitor. The general architecture of these neurons was reconstructed from peroxidase-antiperoxidase-labelled whole-mount preparations. From the subesophageal to the seventh abdominal ganglia two types of neurons show a repetitive organization of contralateral projection patterns in each neuromere. The first type has few branches in the central neuropil and a distinct peripheral projection. The second type is characterized by an elaborate central branching pattern, which includes ascending and descending processes. Some of its peripheral branches were found to supply peripheral neurohemal areas. In the protocerebrum, 10 CCAP-immunoreactive neurons occur with projections into the superior median protocerebrum and the tritocerebrum. Immunopositive neurons were mapped in larval and various pupal stages, as well as in the adult. All types of identified neurons were found to persist throughout metamorphosis maintaining their essential structural and topological characteristics. The CCAP-immunoreactive neurons of T. molitor are compared with those described for the locust. Putative structural homologies of subsets of neurons in both species are discussed.     

Biochemical and biophysical characterization of collagen in Tenebrio molitor L. (Coleoptera, Tenebrionidae)

The collagen from the mesenteric sheath of the tenebrionid insect Tenebrio molitor was extracted by limited pepsin digestion and purified. This collagen was characterized using CM-cellulose chromatography, sodium-dodecylsulfate disc-gel electrophoresis and aminoacid analysis. This molecule was found to be assembled from three identical alpha chains and could be represented by the formula (alpha) 3. The amino acid composition is characteristic of collagen (one-third glycine, high iminoacid content), with high content of hydroxylysine and low content of alanine. Cyanogen bromide digests of these chains indicated that they are not related to any of the known invertebrate or vertebrate chains of interstitial collagens. The molecular weight (M = 280000D) and length (290 nm) were typical, and the banding patterns of the segment-long-spacing crystallites (SLS) and of the reconstitued fibrils were very similar to type I collagen. The denaturation temperature (Td) was 30.7 degrees C and correlated with the total pyrrolidine content as observed in other collagens (von Hippel & Wong's relation). It was concluded that the collagen from this insect showed the classical biochemical and biophysical features of other invertebrate interstitial "primitive" collagens.     

The structure and evacuation of the spermatophore of Tenebrio molitor L. (Coleoptera: Tenebrionidae)

Sperm are transferred from male to female mealworm beetles within a spermatophore. The highly organized spermatophore can be visualized as a tube, blind at both ends, and invaginated at the anterior. The wall of the tube is composed of two layers, one of fibrous protein and the other of lipoprotein which appears frothy in electron micrographs. After being deposited in the bursa of the female, or placed in saline, the spermatophore undergoes sequential stereotyped expansions at the anterior tip and then bursts, releasing semen within the female bursa. The ‘evacuated’ spermatophore becomes coated by a transluscent layer and is ejected from the female 18 hr after copulation.     

The fate of persisting thoracic neurons during metamorphosis of the meal beetle Tenebrio molitor (Insecta: Coleoptera)

Summary A set of motor neurons and interneurons in the thoracic nervous system of the meal beetle Tenebrio molitor L. is described that persist during metamorphosis. The motor neurons under discussion innervate the thoracic ventral longitudinal muscles and were identified by retrograde transport of intramuscularly injected horseradish peroxidase. Persisting motor neurons exhibit a complex repetitive pattern that changes only slightly during development. Additionally, the characterization of serotonin-immunoreactive neurons defines a complex set of interneurons that also persist throughout development. The fate of these identified neurons is outlined in detail with special reference to variations in their dendritic arborizations. All motor and interneurons are affected by a similar change in their shape during development. The larval neurons lack the contralateral arborization that is found in the adult beetle and is already distinguishable in the prepupa. Essentially only quantitative changes of the neuronal shape were observed during the pupal instar. No pupa-specific degeneration of certain axo-dendritic structures of these neurons was found. Removal of descending interneurons by sectioning the promesothoracic connectives causes specific degeneration of the dendritic tree of an identified serotonin-immunoreactive interneuron.     

Proctolin-immunoreactive neurons persist during metamorphosis of an insect: A developmental study of the ventral nerve cord of Tenebrio molitor(Coleoptera)

Summary Proctolin-immunoreactive neurons in all neuromers of the ventral nerve cord of Tenebrio molitor L. have been quantitatively demonstrated and mapped throughout metamorphosis. Each neuromer contains an anterior and a posterior group of neurons with light and dark staining properties as revealed by peroxidase-antiperoxidase labeling. Serial homologous subsets of dark staining neurons with central and peripheral projections have been identified and found to persist during morphogenetic changes from the larva to the adult. Most neurons maintain their topological and structural characteristics throughout metamorphosis. The identified proctolin-immunoreactive neurons exhibit structures similar to those described in other insect species; some may correspond known motoneurons.     

Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle,Tenebrio molitor (Coleoptera,Tenebrionidae)

The genome of Tribolium castaneum encodes two allatostatin [AS type B; W(X)₆Wamide and AS type C; PISCF‐OH] and one allatotropin (AT) precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing Consortium, 2008: Nature 452:949–955). Here we studied the activity (in vitro) of peptides derived from these precursors on the synthesis/release of juvenile hormone (JH) III. The corpora cardiaca‐corpora allata (CC‐CA) complexes of adult females of another tenebrionid beetle, the mealworm Tenebrio molitor, were used. Incubating the gland complexes in a medium containing Trica‐AS B3 peptide, we showed that the peptide has allatostatic function in T. molitor. The activity of the type C AS depended on the age of the test animals and their intrinsic rate of JH III biosynthesis. The Trica‐AS C peptide inhibited the JH release from CA of 3‐day‐old females with a high intrinsic rate of JH synthesis, but activated JH release from the CA of 7‐day‐old females with a lower intrinsic rate of JH production. The allatotropin peptide (Trica‐AT) also activated the JH release from the CA of 7‐day‐old females in a dose‐dependent and reversible manner. Unexpectedly, a type A AS derived from the precursor of the American cockroach Periplaneta americana (Peram‐AS A2b) inhibited the JH release from the CA of younger and older females in the concentration range of 10^?8 to 10^?4 M, and the effects were fully reversible in the absence of peptide. These data suggest a complex role of allatoactive neuropeptides in the regulation of JH III biosynthesis in beetles. © 2010 Wiley Periodicals, Inc.     

Density-dependent prophylaxis in the mealworm beetle Tenebrio molitor L. (Coleoptera: Tenebrionidae): cuticular melanization is an indicator of investment in immunity

If there are costs involved with the maintenance of pathogen resistance, then higher investment in this trait is expected when the risk of pathogenesis is high. One situation in which the risk of pathogenesis is elevated is at increased conspecific density. This paper reports the results of a study of density-dependent polyphenism in pathogen resistance and immune function in the mealworm beetle Tenebrio molitor. Beetles reared at high larval densities showed lower mortality when exposed to a generalist entomopathogenic fungus and a higher degree of cuticular melanization than those reared solitarily. The degree of cuticular melanization was a strong indicator of resistance, with darker beetles being more resistant than lighter ones regardless of rearing density. No differences were found between rearing densities in the levels of phenoloxidase, an enzyme key to the insect immune response. The results show that pathogen resistance is phenotypically plastic in T. molitor, suggesting that the maintenance of this trait is costly.     

Diversity of digestive proteinases in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae

The spectrum of Tenebrio molitor larval digestive proteinases was studied in the context of the spatial organization of protein digestion in the midgut. The pH of midgut contents increased from 5.2-5.6 to 7.8-8.2 from the anterior to the posterior. This pH gradient was reflected in the pH optima of the total proteolytic activity, 5.2 in the anterior and 9.0 in the posterior midgut. When measured at the pH and reducing conditions characteristic of each midgut section, 64% of the total proteolytic activity was in the anterior and 36% in the posterior midgut. In the anterior midgut, two-thirds of the total activity was due to cysteine proteinases, whereas the rest was from serine proteinases. In contrast, most (76%) of the proteolytic activity in the posterior midgut was from serine proteinases. Cysteine proteinases from the anterior were represented by a group of anionic fractions with similar electrophoretic mobility. Trypsin-like activity was predominant in the posterior midgut and was due to one cationic and three anionic proteinases. Chymotrypsin-like proteinases also were prominent in the posterior midgut and consisted of one cationic and four anionic proteinases, four with an extended binding site. Latent proteinase activity was detected in each midgut section. These data support a complex system of protein digestion, and the correlation of proteinase activity and pH indicates a physiological mechanism of enzyme regulation in the gut.     

Balanced intake of protein and carbohydrate maximizes lifetime reproductive success in the mealworm beetle,Tenebrio molitor (Coleoptera: Tenebrionidae)

Recent developments in insect gerontological and nutritional research have suggested that the dietary protein:carbohydrate (P:C) balance is a critical determinant of lifespan and reproduction in many insects. However, most studies investigating this important role of dietary P:C balance have been conducted using dipteran and orthopteran species. In this study, we used the mealworm beetles, Tenebrio molitor L. (Coleoptera: Tenebrionidae), to test the effects of dietary P:C balance on lifespan and reproduction. Regardless of their reproductive status, both male and female beetles had the shortest lifespan at the protein-biased ratio of P:C 5:1. Mean lifespan was the longest at P:C 1:1 for males and at both P:C 1:1 and 1:5 for females. Mating significantly curtailed the lifespan of both males and females, indicating the survival cost of mating. Age-specific egg laying was significantly higher at P:C 1:1 than at the two imbalanced P:C ratios (1:5 or 5:1) at any given age throughout their lives, resulting in the highest lifetime reproductive success at P:C 1:1. When given a choice, beetles actively regulated their intake of protein and carbohydrate to a slightly carbohydrate-biased ratio (P:C 1:1.54–1:1.64 for males and P:C 1:1.3–1:1.36 for females). The self-selected P:C ratio was significantly higher for females than males, reflecting a higher protein requirement for egg production. Collectively, our results add to a growing body of evidence suggesting the key role played by dietary macronutrient balance in shaping lifespan and reproduction in insects.     

A nuptially transmitted ichthyosporean symbiont of Tenebrio molitor (Coleoptera: Tenebrionidae)

The yellow mealworm, Tenebrio molitor, harbors a symbiont that has spores with a thick, laminated wall and infects the fat body and ventral nerve chord of adult and larval beetles. In adult males, there is heavy infection of the epithelial cells of the testes and between testes lobes with occasional penetration of the lobes. Spores are enveloped in the spermatophores when they are formed at the time of mating and transferred to the female's bursa copulatrix. Infection has not been found in the ovaries. The sequence of the nuclear small subunit rDNA indicates that the symbiont is a member of the Ichthyosporea, a class of protists near the animal-fungi divergence.     

Management of entomopathogenic fungi in cultures of Tenebrio molitor (Coleoptera: Tenebrionidae)

Larvae of mealworms Tenebrio molitor L. (Coleoptera: Tenebrionidae) have been used as animal feed, but fungal pathogens rapidly downsize the populations, resulting in economic losses. In this work, we established an effective management strategy for fungal pathogens. An entomopathogenic fungus, Beauveria bassiana, was isolated from mealworm cadavers. The bioassay of some isolates of this species at >90% relative humidity revealed that the ERL1575 isolate had the highest virulence. At 20–30% RH, ERL1575 conidia when ingested produced 80% mortality but when sprayed topically produced only <10% mortality. Mealworms that had ingested conidia were exposed to 20, 25, 30 and 35°C and high humidity (>95%) for 5 days. This experiment produced about 90% mortality except at 35°C where mortality was <20%. When 40 fungicides were assayed against ERL1575, fluazinam (1000‐fold) and mancozeb (667‐fold) significantly inhibited conidial germination and/or hyphal growth. When fluazinam and mancozeb were added to the mealworm diet of conidia‐inoculated wheat bran, most were alive 3 days post application. However, 100% mortality resulted 3 days post application in the conidia‐inoculated wheat bran without any fungicides. In conclusion, B. bassiana isolates are pathogenic at <30°C when they are ingested by mealworms but fluazinam and mancozeb can be used for management to control the pathogen in their cultures.     

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.     

Spermatogenesis in Tenebrio molitor (Tenebrionidae,Coleoptera): A Fine Structure and Anti-tubulin Immunofluorescence Study

Spermatogonia and both generations of spermatocytes of Tenebrio molitor possess conventional bipolar spindles with only few aster MTs. Spindles in metaphase spermatogonia are surrounded by fenestrated two-layered cisternae and do not contain intraspindle membranes. In metaphase spermatocytes, a spindle envelope is missing, but intraspindle membranes are abundant. Mitochondria form long threads lateral to the nucleus in prophase I of meiosis. The elongated mitochondria also align parallel to the spindle apparatus in prometaphase I. As a consequence, the spindles reside in a cage formed of mitochondria. This arrangement may guarantee proper bisection of the chondriome during division. Cells are tightly packed during spermatogonial divisions and in prophase I, but large intercellular spaces develop when the first meiotic spindle assembles. Then, cytoplasmic bridges which persist between the cells as a result of incomplete cytokinesis appear as slender tubes. Anti-tubulin immunofluorescence using an antibody against acetylated α-tubulin revealed intense acetylation throughout spermatogonial mitosis but a low degree of α-tubulin acetylation in meiotic spindles prior to telophase. This may indicate a high microtubule turnover in meiosis.     

黄粉虫幼虫体壁硬化过程中酚氧化酶活性的变化

为研究酚氧化酶(PO)在昆虫蜕皮过程中的功能和作用, 采用微量测定法研究了黄粉虫Tenebrio molitor体壁硬化过程中血淋巴和表皮中的PO活性变化。结果表明：初蜕皮幼虫血淋巴中PO活性较高, 但随着体壁的不断黑化与硬化, 其活性呈现下降趋势, 在3～4 h内达到最低点, 而后PO活性逐渐上升, 7 h左右活性上升至最高, 并接近于正常幼虫的水平;在刚蜕完皮后的1 h内, 体壁中 PO活性基本无变化, 但随后即开始下降, 3 h左右降到最低点, 然后开始回升, 6~7 h左右恢复到正常水平, 并趋于稳定;以L-DOPA为底物, 通过双倒数曲线作图法求得黄粉虫血淋巴PO的Km＝1.176 mmol/L, 体壁PO的Km＝0.881 mmol/L, 表明体壁PO与底物L-DOPA的亲和力要高于血淋巴PO。研究表明两种来源的酚氧化酶均参与了黄粉虫幼虫的体壁硬化过程, 但在作用方式及与底物的亲和力方面存在差异。     

基于转录组数据高通量发掘黄粉甲微卫星引物（英文）

黄粉虫Tenebrio molitor作为理想的模式研究生物, 虽然已围绕该昆虫在多个研究领域开展了诸多研究, 但是有关其分子和遗传方面的研究仍知之甚少。为此, 本研究基于前期构建的黄粉甲转录组数据库, 成功发掘获得1 249个微卫星序列。其中, 单碱基或三碱基序重复列最多, 分别占44.44%和41.15%; A/T型重复序列出现频率最高, 占42.70%。除单核苷酸重复序列外, 重复单元的重复次数以5次最多, 占30.90%。基于鉴定获得的微卫星序列, 共设计获得1 004对微卫星引物, 而且每对引物还设计了5对替代引物。研究获得的微卫星引物将有助于今后开展黄粉甲功能和比较基因组学方面的研究。     

Metabolic changes associated with active water vapour absorption in the mealworm Tenebrio molitor L. (Coleoptera, Tenebrionidae): a microcalorimetric study

Water vapour absorption (WVA) is an important mechanism for water gain in several xeric insects. Theoretical calculations indicate that the energetic cost of WVA should be small (5-10% of standard metabolic rate) assuming realistic efficiencies. In this study we explored the relationship between WVA, metabolic heat flux (HFmet.) and CO2 release in larvae of Tenebrio molitor using microcalorimetry. By comparing metabolic heat flux with the catabolic rate estimated from VCO2 , we were able to differentiate anabolic and catabolic rates prior to and during WVA, while simultaneously monitoring water exchange. Three to four hours before the onset of WVA, larvae showed clear increases in HFmet. and catabolic flux, and a simultaneous decrease in anabolic flux. Following the onset of WVA, HFmet. decreased again until indistinguishable from control (non-absorbing) values. Possible factors contributing to the "preparatory phase" are discussed, including mobilization of Malpighian tubule transporters and muscular activity in the rectum. Absorbing larvae reduced the water activity of the calorimetric cell to 0.906, agreeing with gravimetric estimates of the critical equilibrium activity. Periods of movement during WVA coincided with decreased uptake fluxes, consistent with the animal's hydrostatic skeleton and the need to close the anus to generate pressure increases in the haemocoel.     

用替代寄主繁殖的川硬皮肿腿蜂的学习行为

采用Y型嗅觉仪进行双向选择实验,研究了用替代寄主黄粉虫蛹繁殖的川硬皮肿腿蜂寄主搜索过程中的学习行为。结果表明,川硬皮肿腿蜂羽化期和成虫初期经历松枝皮、松针、松节油、杉枝皮等的挥发物后,雌蜂对这些挥发物的选择性明显提高,但对杉叶挥发物无明显的学习行为。羽化期和成虫初期是否投放新鲜寄主,对雌蜂的气味选择性没有明显的影响。成蜂取食黄粉虫蛹和蜂蜜时均可联系性学习松枝和杉枝气味。取食并经历杉枝和松枝挥发物4天和8天的寄生蜂之间对相应气味的选择性无显著差异。