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。     

Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.     

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.     

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.     

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.     

Self-selection of two diet components by Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and its impact on fitness

We studied the ability of Tenebrio molitor L. (Coleoptera: Tenebrionidae) to self-select optimal ratios of two dietary components to approach nutritional balance and maximum fitness. Relative consumption of wheat bran and dry potato flakes was determined among larvae feeding on four different ratios of these components (10, 20, 30, and 40% potato). Groups of early instars were provided with a measured amount of food and the consumption of each diet component was measured at the end of 4 wk and again 3 wk later. Consumption of diet components by T. molitor larvae deviated significantly from expected ratios indicating nonrandom self-selection. Mean percentages of dry potato consumed were 11.98, 19.16, 19.02, and 19.27% and 11.89, 20.48, 24.67, and 25.97% during the first and second experimental periods for diets with 10, 20, 30, and 40% potato, respectively. Life table analysis was used to determine the fitness of T. molitor developing in the four diet mixtures in a no-choice experiment. The diets were compared among each other and a control diet of wheat bran only. Doubling time was significantly shorter in groups consuming 10 and 20% potato than the control and longer in groups feeding on 30 and 40% potato. The self-selected ratios of the two diet components approached 20% potato, which was the best ratio for development and second best for population growth. Our findings show dietary self-selection behavior in T. molitor larvae, and these findings may lead to new methods for optimizing dietary supplements for T. molitor.     

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.     

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.     

Response of Galleria mellonella (Lepidoptera: Pyralidae) and Tenebrio molitor (Coleoptera: Tenebrionidae) to Spiroplasma citri inoculation

Injections into 4th instar larvae of Galleria mellonella using either in vitro or in vivo inoculum of the BR-6 isolate of Spiroplasma citri, propagated for one to nine passages, caused 5.7 to 24.7% mortality. Weight gain of the larvae injected at their 4th, 5th, and 6th instar was reduced in the first 4 days after inoculation but final pupal weight of the survivors was not significantly affected. Fourth instar larvae pupated within 10 days after the injection, but more larvae (6–13%) injected with 5th- to 9th-passage cultures pupated 5 or more days later than did larvae injected with 1st to 4th-passage cultures (0–3%). As many as one-third of the injected larvae developed into deformed pupae, with some external appendages missing or with a reduced and distorted thorax or abdomen with uneven tanning of the integument. Spiroplasma multiplied to dense concentrations (10⁸ to 10⁹/ml) in hemolymph smears from injected larvae incubated under oil. Larvae of Tenebrio molitor were not susceptible to S. citri by injection or feeding and G. mellonella were not susceptible by feeding. Transmissibility of S. citri by leafhopper vector to celery and periwinkle plants was retained after propagation for nine successive passages during 7 months in a nonhost insect such as Galleria.     

温度对黄粉虫幼虫生长发育及体液免疫防御能力的影响

【目的】探究饲养温度对黄粉虫Tenebrio molitor幼虫生长发育和体液免疫防御的影响。【方法】测定了不同温度（18, 22, 26和30℃）下饲养的黄粉虫幼虫的发育历期、蛹重、化蛹率;采用抑制区分析法测定了不同温度下饲养的免疫（用生理盐水将大肠杆菌Escherichia coli配制成1×104个菌体/μL悬浮液,用微量注射器将其注入虫体腹部的背面,每头幼虫注射1 μL）和非免疫(注射生理盐水)黄粉虫幼虫血淋巴的抑菌和溶菌酶活性,通过分光光度法测定了其酚氧化酶活性。【结果】结果显示,黄粉虫幼虫发育历期随饲养温度的上升而明显缩短（P<0.0001）,而不同温度下蛹重（P=0.067）与化蛹率（P=0.869）差异不显著。免疫组黄粉虫幼虫血淋巴的抑菌、酚氧化酶和溶菌酶活性随饲养温度上升而降低：抑菌和酚氧化酶活性随温度变化差异极显著（P<0.0001）,溶菌酶活性差异显著（P=0.013）。【结论】本研究结果表明,温度对黄粉虫的生长发育和免疫防御具有较大的影响,低温下黄粉虫幼虫的发育历期延长,但其体液免疫防御能力明显增强。     

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.     

Use of Tenebrio molitor (Coleoptera: Tenebrionidae) to recycle organic wastes and as feed for broiler chickens

Several dried waste materials from different origins were used as a substrate to grow Tenebrio molitor L. Nutrient/amino acid values differed depending on both larval size/weight and substrate. These larvae were experimentally used as a broiler feedstuff. Seven-day-old chicks of a commercially available strain with an average weight of 126 g were randomly distributed into nine six-broiler groups. Three levels of Tenebrio molitor larvae (0, 5, and 10% dry weight) were used in a 19% protein content sorghum-soybean meal basal diet, to evaluate feed intake, weight gain, and feed efficiency. Results after 15 d showed no significant differences among treatments. These data indicate that Tenebrio molitor has the potential to be used as protein source for raising broilers.     

Individual and population effects of eugregarine, Gregarina niphandrodes (Eugregarinida: Gregarinidae), on Tenebrio molitor (Coleoptera: Tenebrionidae)

Gregarines are single-celled parasites in the phylum Apicomplexa that infect invertebrates. They are highly abundant on three levels: among a large diversity of invertebrates, in the proportion of population of organisms they infect, and within individually infected organisms. Because of their remarkable prevalence, we hypothesize that they play an important role in support of their hosts. However, studies done to date on the impact of gregarines on their host are conflicting. Therefore, we studied the impact of gregarines on their host using a model Gregarina niphandrodes infection in Tenebrio molitor. The impact of infection was measured by comparing beetles with no or low infection to those with artificially induced high infection. The numbers of individuals in each of the three easily visible developmental stages of the T. molitor (larva, pupa, and adult) were censused weekly. From these observations, fertilities and probabilities of survival with transition between stages were estimated. These estimated vital rates were used to construct a stage-classified projection matrix model. We also measured the longevity of individual beetles with low and high infection that were grown in isolation. The results indicate that there is no significant difference in the population dynamics of beetles with low and high infection. However, the longevity was significantly different between beetles with low infection than the deliberately highly infected group.     

Nutritional analysis of alternative feed ingredients and their effects on the larval growth of Tenebrio molitor (Coleoptera: Tenebrionidae)

Alternative feeds comprising only brewer's spent grain (BSG) or distillers dried grain (DDG), both agricultural byproducts, and 50% BSG or 50% DDG mixed with 50% wheat bran (WB) were compared to the standard 100% WB feed provided to Tenebrio molitor larvae to evaluate their effects on the nutritional quality and economic viability of mass‐producing this insect. Crude protein content was highest in larvae fed on 100% WB and 100% DDG, and the latter had 2.32 and 1.88 times more essential and non‐essential amino acids than the 100% WB group, respectively. Larvae fed on 50% DDG + 50% WB had 1.30 times more saturated fatty acids but 1.08 times less unsaturated fatty acids and 1.03–1.23 times less potassium, phosphorus, and magnesium than larvae fed on 100% WB. Traces of mercury were detected in larvae fed on 100% WB and 50% DDG + 50% WB, although at levels below the General Food and Feed Standard (Ministry of Food and Drug Safety, Republic of Korea), but neither Escherichia coli or Salmonella spp. were detected in the feeds. Larval and pupal weight, larval development and survival, and pupation rate were higher in larvae fed on 50% BSG or 50% DDG mixed with 50% WB groups than in larvae fed on 100% WB. Therefore, using 50% BSG or 50% DDG mixed with 50% WB as an alternative feed in the mass production of T. molitor provides higher quality larvae at a lower cost than does using the conventional 100% WB feed.     

Potential of Tenebrio molitor (Coleoptera: Tenebrionidae) as a bioassay probe for Metarhizium brunneum (Hypocreales: Clavicipitaceae) activity against Ixodes scapularis (Acari: Ixodidae)

The yellow mealworm, Tenebrio molitor L., has been used to indicate qualitatively the presence of entomopathogenic fungi in the soil or as a model for evaluating stress and other factors on fungal activity. Although this beetle appears highly susceptible to many of these fungi, little quantitative information is available on the sensitivity of T. molitor to a specific fungus and, therefore, fungal presence or as an indicator for pathogenicity to other species. The purpose of this study was to establish the suitability of T. molitor larvae as a bioassay probe for Metarhizium brunneum for comparison against the blacklegged tick, Ixodes scapularis. Nine concentrations of M. brunneum strain F52 ranging from 1.0 x 10(1) to 8.4 x 10(8) conidial/ml were simultaneously tested against T. molitor larvae and I. scapularis adults. Larvae of yellow mealworm were less sensitive to M. brunneum than I. scapularis adults (LC50's 4.4 x 10(7) and 1.7 x 10(5) conidia/ml, respectively, 4-wk post-treatment). The greater sensitivity of I. scapularis to the fungus suggests that the detection of fungal mycosis in mealworms would indicate sufficient inoculum to be pathogenic to I. scapularis and make this insect a suitable probe for evaluation of the presence and activity of M. brunneum against the blacklegged tick in field applications.     

两种色型黄粉虫的生长发育、存活与饲料利用效率比较研究（英文）

黄粉虫Tenebrio molitor L.是一种重要的资源昆虫, 根据60日龄幼虫的体壁颜色, 通过连续12代的自然选育, 获得了黄、 黑2种色型黄粉虫。为进一步给黄粉虫优良品种培育提供科学的理论依据, 本实验首先对选育出的2种色型黄粉虫的生长发育、 存活及饲料利用效率进行了比较研究。结果表明： 2种色型黄粉虫的孵化率和化蛹率均约为83%和97%, 无显著差异; 但黄色型成虫的羽化率（♀：95%, ♂：91%）却明显高于黑色型成虫的羽化率（♀： 82%, ♂： 83%）。2种色型黄粉虫的卵和蛹历期均约为7 d和10 d, 亦无显著差异; 但黑色型幼虫较黄色型幼虫发育更快、 更整齐。黑色型幼虫共历经12~15龄, 黄色型幼虫历经12~17龄, 并且2种色型黄粉虫幼虫均以历经14龄居多, 历经14龄的黄、 黑2色型黄粉虫幼虫的比例分别为27%和53%。以14龄计, 黄、 黑2色型黄粉虫从卵至成虫的总历期分别为171 d和151 d。此外, 黄色型幼虫对饲料的平均利用效率明显高于同日龄黑色型幼虫, 黄色型幼虫的平均饲料消化率（AD）、 转化率（ECD）和利用率（ECI）分别为66.5%, 50.6%和33.6%, 而黑色型幼虫的平均饲料消化率（AD）、 转化率（ECD）和利用率（ECI）分别为62.1%, 46.8%和29.1%; 但2种色型黄粉虫同日龄幼虫的生长率和存活率差异不显著。综合上述结果可知, 黑色型黄粉虫较黄色型黄粉虫发育更快、 更整齐, 而黄色型黄粉虫对饲料的平均利用效率明显高于黑色型黄粉虫。这为进一步培育黄粉虫优良品种提供了有益参考。     

The response of Gregarina niphandrodes (Apicomplexa: Eugregarinida: Septatina) to host starvation in Tenebrio molitor (Coleoptera: Tenebrionidae) adults.

Numerous studies of host starvation have emphasized pathological effects of parasites on their insect host, but little attention has been focused on the effects of host starvation on the parasites. This study addressed the possibility that parasite life-cycle events could be manipulated by withholding food from the host. The system used was Gregarina niphandrodes (Apicomplexa: Eugregarinida) in Tenebrio molitor (Coleoptera: Tenebrionidae) adults. Gregarine gametocyst formation and shedding ceased after 1 day in starved beetles but continued in fed controls. There were no statistically significant differences between total lengths of associated (3 of 5 trials) or unassociated (5 of 5 trials) gregarines found between experimental and control groups, but average numbers of the 2 life cycle events were generally higher in fed hosts than in starved ones. If infected, fed control beetles continued to form gametocysts throughout the 7-day trial periods, and gametocysts could be observed in the gut. Starved experimental beetles had no gametocysts in their guts. Refeeding of starved beetles after 4 days resulted in resumption of gametocyst formation and shedding. The studies demonstrated that the gregarine life cycle could be stopped and then started at the gametocyst formation stage like an off/on switch, simply by withholding food from, then refeeding, the host.