Silkworm Growth and Silk Yield on Selected Supplemented/ Un-supplemented Mulberry Varieties

ABSTRACT Sericulture has been on developing foot hills till today in Pakistan. It is for the past few years that this industry has received recognition through greater research on the nutritional aspects. Various experiments i.e., nutritional supplementation and searching alternate food plants have been on way with varying degree of success. The present approach includes the very much neglected aspect of trying mulberry varieties and working out their impact on the silkworm growth/ development and silk yield. In Pakistan three varieties of mulberry Morus alba, M. nigra and M. indica are commonly found, these were selected for the present studies and the already established mineral dose 0.2 %N +0.1%P+0.3%K+0.1%Ca+0.15%Mg+0.15%Mn was supplemented to them through dipping method. Amongst the so designed nine treatments those in which the silkworm larvae were fed on M. nigra leaves in any form /combinati-yielded best results, as the larvae in these treatments consumed more food (73.71 gm/10 larvae) and converted maximum (77.22%) of it into body matter. This improved silkworm growth, a primary parameter for increasing the over all production and resulted in better cocoon size which weighed heavier (2.43 gm/cocoon and 1.10 gm/shell) as compared to rest of the test treatments (0.67 and 0.70 gm/shell on M. alba and M. indica , respectively).     

Recycling of urea associated with the host plant urease in the silkworm larvae,Bombyx mori

Urea concentration and urease activity in the midgut content were compared between larvae of the silkworm, Bombyx mori fed an artificial diet and those fed fresh mulberry leaves. A considerable amount of urea was found in the midgut content of the both larvae, however it was significantly lower in the larvae fed fresh mulberry leaves than in the larvae fed the artificial diet; average urea concentrations in the midgut content of the larvae fed fresh mulberry leaves and the artificial diet were 2.9 and 4.6 &mgr;mol/g, respectively. Urea in the midgut content seems to be secreted from the insect itself since the amount of urea in both diets were negligibly small. Urease activity was detected only in the midgut content of the larvae fed fresh mulberry leaves but not in other tissues of the larvae. On the other hand, no urease activity was detected in the midgut content of the larvae fed the artificial diet. Subsequently, to elucidate the role of mulberry leaf urease in the midgut lumen, larvae that had been reared on the artificial diet were switched to fresh mulberry leaves. The diet switch caused a rapid decrease in urea concentration in the midgut content and an increase in ammonia concentration in the midgut content, suggesting that secreted urea could be hydrolyzed to ammonia by mulberry leaf urease in the midgut lumen. Furthermore, to investigate the physiological significance of mulberry leaf urease on urea metabolism of the silkworm, (15)N-urea was injected into the hemocoel, and after 12 h the larvae were dissected for (15)N analysis. A considerable amount of (15)N was found to be incorporated into the silk-protein of the larvae fed fresh mulberry leaves, but there was little incorporation of (15)N into the silk-protein of the larvae fed the artificial diet. These data indicate that urea is converted into ammonia by the action of mulberry leaf urease in the midgut lumen and used as a nitrogen source in larvae fed mulberry leaves.     

Photoperiod induced developmental effects on silkmoth,Bombyx mori

Bombyx mori is most common silkmoth used for the commercial production of the silk. The different environmental conditions may affect development of silkmoth and hence the output of silkworm. Present study investigated effects of different photoperiodic conditions on the development of silkmoth. Three experiments were performed on newly hatched larvae. In experiment1, larvae were divided into three groups; group 1, 8L:16D (8 h light and 16 h dark), group 2, 12L:12D (12 h light and 12 h dark) and group 3, 16L:8D (16 h light and 8 h dark). In experiment 2, larvae were exposed to either continuous light or continuous dark. In experiment 3, larvae were divided into three groups. Each group received light of similar duration (16L:8D) and intensity (1.1 ± 0.1 PAR) but of different wavelengths; i.e. long wavelength red light (640 nm), short wavelength blue light (450 nm) and white light. Larvae were fed on young mulberry (Morus alba) leaves. Temperature and humidity were maintained at 26 (±2) °C and 75 (±5)%, respectively, throughout the study. Observations on change in body length of larvae were made on every alternate day. We also calculated the shell ratio, time taken in cocoon formation and emergence. Results from experiment show that the larvae underwent significant change in body length but with faster growth rate under 12L:12D photoperiod in comparison to 8L:16D and 16L:8D. Time taken in cocoon formation and hatching was significantly less, while shell ratio was significantly higher in 12L:12D in comparison to other groups. We did not observe much effect of monochromatic light on the development of silkmoth.     

Survival of Heleomyza borealis (Diptera, Heleomyzidae) larvae down to − 60°C

Heleomyza borealis Boh. (Diptera, Heleomyzidae) overwinters as larvae in Arctic habitats, where they may experience winter temperatures below ? 15°C. The larvae freeze at c.? 7°C but in acclimation experiments 80% survived when exposed to ? 60°C. Of the larvae exposed to between ? 4 and ? 15°C, only 3% pupated. However, when cooled to ? 20°C this increased to 44%, with 4% emerging as adults. Larvae maintained at 5°C contained low levels of glycerol, sorbitol and trehalose, which did not increase with acclimation to low temperatures. However, levels of fructose increased from 6.1 μg mg^?1 fw in control animals to 17 μg mg^?1 fw when exposed to ? 2°C for 1 week. Larval body water (2.2 ± 0.1 g/g dw, mean ± SD, n = 100) and lipid content (0.22 ± 0.002 g/g fw, mean ± SE) showed no significant change during acclimation to low temperatures. Larvae maintained at a constant 5°C survived for over 18 months with little loss of body mass (from 7.5 ± 1.2 to 7.0 ± 1.2 mg fw, mean ± SD, n = 20), but none pupated. Heleomyza borealis larvae appear to feed and grow until they reach a body mass of about 7.5 mg and then become dormant. They remain in this state until they experience a low temperature stimulus (< ? 15°C) followed by a warm period (≈ 5°C). This ensures that the larvae pupate and adults emerge in early summer, allowing the maximum growing period before the following winter. Heleomyza borealis are adapted to survive long winters in a dormant larval state. They have a low metabolic rate, can conserve body water even at subzero temperatures but do not synthesize large quantities of cryoprotectants.     

不同饲料饲养家蚕其肠道微生态优势菌群类型的组成及差异性

为探讨鳞翅目昆虫的生长发育及抗病性与肠道微生态状况的关系,以不同的桑科植物柘叶与桑叶分别饲养家蚕,采用纯培养分离检测技术、16S rDNA序列测定和系统发育分析方法,对4、5龄家蚕肠道优势菌群的类型进行了鉴定和差异性分析。结果表明:柘叶与桑叶饲养家蚕共有的优势菌群有短波单胞杆菌属(Brevundimonas)、寡养单胞菌属(Stenotrophomonas)、肠杆菌属(Enterobacter)和葡萄球菌属(Staphylococcus)4个类群。从桑叶饲养家蚕肠道中检索到的优势菌群还有气单胞菌属(Aeromonas)、短杆菌属(Brevibacterium)、柠檬酸杆菌属(Citrobacter)、埃希氏菌属(Escherichia)和克雷伯氏菌属(Klebsiella)5个类群,而从柘叶饲养家蚕肠道中检索到的优势菌群仅有假单胞菌属(Pseudomonas)和土壤杆菌属(Agrobacterium)2个类群。饲料的改变导致家蚕肠道微生态细菌种群组成的变化,从柘叶饲养家蚕肠道中分离出的优势菌群与桑叶饲养的家蚕相比,出现较大差异且不如桑叶饲养家蚕的菌群丰富。推测这种改变可能与柘叶饲养家蚕生长发育不良、容易患病具有相关性。     

Effect of different artificial diets on diapause induction under controlled temperature and photoperiod in the silkworm, Bombyx mori L.

ABSTRACT. Embryonic diapause of the silkworm, Bombyx mori , is generally induced by temperature and photoperiod during the egg stage of the previous generation and not in the larval stage. However, when silkworm larvae are reared on an artificial diet instead of mulberry leaves, their diapause is strongly affected by temperature and photoperiod experienced in the larval stage, with a distinct long-day response for diapause induction. Moreover when larvae which have been reared on artificial diet under long-day condition are fed mulberry leaves even for a short period of time, most of the resultant female adults lay diapause eggs. These results suggest that the photoperiodic response of larvae for diapause induction may be strongly suppressed by some components in mulberry leaves.     

大气氟污染源附近食桑昆虫中氟的积累和分布

对大气F污染源附近野桑蚕,桑赤蠖和桑蚕体内的氟化物含量和器官分布进行了研究,结果表明,污染源附近食桑昆虫体有较高的氟化物含量,且食桑昆虫体的氟化物含量随着离污染源的距离增大而降低,野桑蚕,桑赤蠖和桑蚕的氟化物含量与桑叶的氟化物含量有极显著的线性正相关性,食桑昆虫不同器官间的氟化物积累量也存在较大差异,其消化管是食桑昆虫的主要氟化物积累器官。     

Preventive effects of N‐acetyl‐l‐cysteine against imidacloprid intoxication on Bombyx mori larvae

Imidacloprid, a widely used neonicotinoid insecticide, is toxic to silkworm (Bombyx mori). To explore whether N‐acetyl‐l ‐cysteine (NAC) has an effect on preventing silkworm (B. mori) from toxification caused by imidacloprid, we fed the fifth‐instar larvae with mulberry leaves dipped in 200 mg/L NAC solution before exposing in imidacloprid, and investigated the silkworm growth, survival rate, feed efficiency, cocoon quality, and the activities of antioxidant enzymes in midgut. The results showed that addition of NAC could significantly increase body weight, survival rate, and feed efficiency of imidacloprid poisoned silkworm larvae (P < 0.05), as well as cocoon mass, cocoon shell mass, and the ratio of cocoon shell (P < 0.05). Furthermore, it could significantly promote the activities of the antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxide in the midgut of fifth‐instar larvae under imidacloprid exposure at the late stage of treatment. In addition, it also could downregulate the malondialdehyde content. The results of our findings proved that the added NAC may have some beneficial effects on protection or restoration of antioxidant balance in imidacloprid exposed larvae.     

Bromatological characterization of mulberry cultivars, Morus spp., and determination of nutritional indexes of Bombyx mori L. (Lepidoptera: Bombycidae)

The objective of this work was to assess, through consumption and utilization of natural food measurements, whether mulberry cultivars, Morus spp., could be recommended to rear the silkworm, Bombyx mori L., in a commercial scale. The mulberry cultivars Miura (standard), Korin and Tailandesa and the hybrids FM 3/3, FM 86, SK 1 and SK 4 were tested. Seventy five fifth-instar commercial hybrid larvae were individualized in gerbox? unities and maintained in a rearing room (25 ± 3oC and 80 ± 10% RH).The mulberry leaves of each of the five cultivars used to feed the silkworm larvae were submitted to bromatological analysis. The dry weight of larvae at the beginning and at the end of the fifth instar, the food consumed and the feces eliminated were recorded to determine the following indexes: relative consumption rate (RCR), relative metabolic rate (RMR), relative growth rate (RGR), approximate digestibility (AD), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD), metabolic cost (MC). The bromatological composition analysis of mulberry leaf cultivars revealed that the hybrid SK 4 presented superior nutritional quality compared to the standard cultivar Miura, due to its higher content in crude protein and ethereal extract, and lower detergent fiber content. The hybrid SK 4 was the most adequate food to the silkworm larvae because it was ingested in small amounts, thus providing good digestibility to the larvae, low metabolic cost, good growth rate and one of the highest efficiencies in the conversion of the ingested food and ingested and digested in biomass.     

Suppression of phototaxis in silkworm larvae

A study of the olfactory and visual organs of the larvae of the silkworm (Bombyx mori), using electrophysiological and surgical techniques, indicates that olfactory stimuli from mulberry leaves, conducted through the antennae or the maxillary palps, cause continuous suppression of the phototactic response, and that the central nervous system plays an important role in this ‘control’ of phototaxis. Such phototactic suppression lasts for 30 h in fifth instar larvae, even after mulberry leaves have been removed.     

Host plant urease in the hemolymph of the silkworm, Bombyx mori

Urease activity was detected in the hemolymph of the silkworm, Bombyx mori from the beginning of spinning to the pharate adult stage if the larvae were reared on mulberry leaves throughout the 5th-instar (the last larval instar). In contrast, no urease activity was detected in the hemolymph of insects fed artificial diets, resulting in accumulation of urea during the spinning stage. To identify the hemolymph urease, the enzyme was highly purified from the hemolymph of the spinning larvae that had been reared on mulberry leaves and the properties of the purified enzyme were compared with those of the mulberry leaf urease. Four out of six monoclonal antibodies raised against jack bean seed urease cross-reacted equally with the silkworm hemolymph urease and the mulberry leaf urease. Under reducing conditions, the hemolymph urease and the mulberry leaf urease migrated at 90.5 kDa on SDS-PAGE gels. The first 20 N-terminal sequence of the hemolymph urease revealed complete identity with that of the leaf urease. The optimum pH for activity and Km value for urea were almost the same for the two enzymes. In conclusion, these two ureases are very likely identical, strongly suggesting that the mulberry leaf urease passes through the larval gut wall into the hemolymph without being digested. In addition, oral administration of mulberry leaf urease just before spinning induced considerable urease activity in the hemolymph of the larvae, but the same treatment did not induce enzyme activity in the hemolymph of the larvae three days before the onset of spinning. These results suggest that the silkworm larvae acquire the host plant urease specifically at the end of the feeding stage in order to degrade urea accumulated in the hemolymph.     

Effects of mulberry leaves to replace rapeseed meal on performance of sheep feeding on ammoniated rice straw diet

This study was conducted to investigate effects of leaves of mulberry tree (Morus alba) as a protein supplement to isonitrogenously replace rapeseed meal (RSM) on performance of growing lambs offered ammoniated rice straw (ABRS) (Trial 1), and to evaluate the digestive characteristics of the ABRS supplemented with different ratios of RSM and mulberry leaves in terms of in vitro gas production (Trial 2). In Trial 1, 45 Huzhou lambs were divided into five equal groups according to their body weight and gender. Lambs in each group were kept in three pens (male, female and mixed (one male and two females)), and received one of the following dietary treatments: 100 g RSM (A), 75 g RSM plus 60 g mulberry leaves (B), 50 g RSM plus 120 g mulberry leaves (C), 25 g RSM plus 180 g mulberry leaves (D), and 240 g mulberry leaves (E). All animals were given ABRS ad libitum along with 100 g ground corn per head per day. The intake of ABRS was slightly increased with the supplementary level of mulberry leaves, and hence total intake increased with the increasing level of mulberry leaves. The growth rates were higher in diets A and E than those in other treatments (P<0.05), with little difference between diets A and E, and the slowest in C. Animals of all genders showed a similar trend, though male lambs was higher in weight gain than the female. While feed efficiency was higher in diet A, concentrate consumption per kilogram of weight gain was lower when higher level of mulberry leaves was supplemented (diets D and E). Feed cost per kilogram gain was lower in diets E and A compared to other treatments. Degradation of dry matter in the rumen of sheep were higher for mulberry leaves than for RSM, but crude protein was less degraded for mulberry leaves than for RSM. The potential GP was significantly higher in diet A than those in B, C and D (P<0.05), and higher in E than in C (P<0.05) (Trial 2), indicating a negative associate effect of mulberry leaves and RSM on digestion. It is inferred that mulberry leaves may be used as a protein supplement to ammoniated straw diets to fully substitute for RSM, but these two supplements should unlikely be supplemented together to avoid the negative associate effect.     

Esterification of cholesterol and cholestanol in the whole body,tissues, and frass of Heliothis zea

The quantity of free and esterified sterols in the whole body, intestine, hemolymph, fat body, and frass of 6th-instar larvae of H. zea, fed cholesterol or cholestanol, was measured in order to determine if there was a difference in the utilization of these two molecules. The principal sterol in the tissues of the larvae was cholestanol or cholesterol, when they were fed diet containing these two molecules, respectively; there was little, if any, metabolism of dietary cholestanol to cholesterol. There was little or no difference in the amount of total sterol in the whole body, tissues, or frass of larvae fed the two different diets, indicating that the absence of a Δ⁵-bond in cholestanol does not prevent the uptake or distribution of this sterol to various tissues. However, the relative percentage of steryl ester was significantly higher in prepupae reared on a diet containing cholestanol instead of cholesterol (6–7-, 4-, 13-, 4-, and 2-fold increase, for the whole body, intestine, hemolymph, fat body, and frass, respectively). The average percentage of total sterol that was esterified in the tissues was greater in the fat body (10.8 ± 15.4 and 44.2 ± 12.3%, respectively, for larvae fed cholesterol and cholestanol) than in the hemolymph (0.5 ± 0.1 and 6.3 ± 0.8%) and intestine (1.2 ± 0.1 and 4.7 ± 1.1%). The percentage of sterol that was esterified in the frass of larvae was large (26.9 ± 3.7 and 48.2 ± 0.5%, respectively, for larvae fed cholesterol and cholestanol). Therefore, the fact that larvae of H. zea fed cholestanol, instead of cholesterol, contain this saturated molecule as their principal tissue sterol and preferentially esterify it may explain, at least in part, why their rate of growth on cholestanol is slower than on cholesterol.     

Identification of Enterococcus mundtii as a pathogenic agent involved in the "flacherie" disease in Bombyx mori L. larvae reared on artificial diet

Enterococcus mundtii was shown to be directly correlated with flacherie disease of the silkworm larvae reared on artificial diet supplemented with chloramphenicol. Its identification was carried out by means of light and electron microscopy and nucleotide sequencing of 16S gene. The bacterium is capable of rapidly multiplying in the silkworm gut and of invading other body tissues, as demonstrated by deliberate infection of germfree larvae and by subsequent TEM observations. E. mundtii can endure alkaline pH of the silkworm gut and it has been proved to adapt in vitro to commonly applied doses of chloramphenicol, whose use can further contribute to reduce competition by other bacteria in Bombyx mori alimentary canal.The modality of transmission of the infection to the larvae was among the objectives of the present research. Since contamination of the progeny by mother moths can be avoided through routine egg shell disinfection, a trans-ovarian vertical transmission can be ruled out. On the other hand the bacterium was for the first time identified on mulberry leaves, and therefore artificial diet based on leaf powder could be a source of infection. We showed that while microwaved diet could contain live E. mundtii cells, the autoclaved diet is safe in this respect. Being E. mundtii also part of the human-associated microbiota, and since B. mori is totally domestic species, a possible role of man in its epidemiology can be postulated.     

Bioaccumulation of cobalt in silkworm (Bombyx mori L.) in relation to mulberry, soil and wastewater metal concentrations

The present study was planned to evaluate Co(II) toxicity in silkworm population. The soil was irrigated using synthetic wastewater to determine the effects of pH and initial cobalt concentration in its bioaccumulation in silkworm (Bombyx mori L.) food chain. The amount of cobalt in wastewater, soil, mulberry and silkworm was determined by atomic absorption spectrophotometer (AAS) analysis. The obtained results clearly indicate that silkworm can be used as template to indicate local cobalt pollution as its body length, body weight and mortality rate was found to be strongly related to cobalt concentration. Higher the cobalt amount in mulberry leaves more the toxicity to silkworm population. At 400 mg/L Co concentration and pH 4 there was maximum deposition of Co in the soil from the synthetic effluent. However, in plants and silk worm the accumulation of Co was maximum at pH 4.5 at an initial Co concentration of 400 mg/L in the synthetic effluent. The maximum cobalt found in wastewater, soil, mulberry and silkworm was 400 ± 0.01, 273.5 ± 0.04, 42.85 ± 0.01, 36.62 ± 0.22 mg/kg, respectively.     

Insect food for astronauts: gas exchange in silkworms fed on mulberry and lettuce and the nutritional value of these insects for human consumption during deep space flights

In this study, silkworm moth (Bombyx mori L.) larvae were regarded as an animal protein source for astronauts in the bioregenerative life support system during long-term deep space exploration in the future. They were fed with mulberry and stem lettuce leaves during the first three instars and the last two instars, respectively. In addition, this kind of environmental approach, which utilised inedible biomass of plants to produce animal protein of high quality, can likewise be applied terrestrially to provide food for people living in extreme environments and/or impoverished agro-ecosystems, such as in polar regions, isolated military bases, ships, submarines, etc. Respiration characteristics of the larvae during development under two main physiological conditions, namely eating and not-eating of leaves, were studied. Nutrient compositions of silkworm powder (SP), ground and freeze-dried silkworms on the 3rd day of the 5th instar larvae, including protein, fat, vitamins, minerals and fatty acids, were measured using international standard methods. Silkworms' respiration rates, measured when larvae were eating mulberry leaves, were higher than those of similar larvae that hadn't eaten such leaves. There was a significant difference between silkworms fed on mulberry leaves and those fed on stem lettuce in the 4th and 5th instars (P<0.01). Amounts of CO2 exhaled by the silkworms under the two physiological regimes differed from each other (P<0.01). There was also a significant difference between the amount of O2 inhaled when the insects were under the two physiological statuses (P<0.01). Moreover, silkworms' respiration quotient under the eating regime was larger than when under the not-eating regime. The SP was found to be rich in protein and amino acids in total; 12 essential vitamins, nine minerals and twelve fatty acids were detected. Moreover, 359?kcal could be generated per 100?gram of SP (dry weight).     

蜕皮激素诱导下家蚕CYP3基因家族的表达变化

为了研究家蚕Bombyx mori CYP3家族基因经蜕皮激素诱导后的表达变化, 用蜕皮激素溶液（2×10^-3 μg/μL）浸泡的桑叶喂食家蚕B. mori 5龄幼虫, 以不用蜕皮激素处理的桑叶喂食家蚕为对照, 采用双跟踪标定定量PCR（dual-spike-in qPCR）方法, 检测在蜕皮激素诱导下家蚕中肠和脂肪体内CYP3基因家族的转录水平。结果表明: 与对照相比, 在蜕皮激素诱导下家蚕幼虫体内脂肪体中CYP302, CYP306和CYP339的转录水平分别上升了191.4, 7.4和421倍, 在中肠中变化不显著; 其余基因转录水平变化不明显或检测不到转录活性。结果说明CYP339基因有可能参与家蚕蜕皮激素的代谢, 这为进一步研究P450基因与内源物质的关系提供理论基础。     

Determination of iminosugars in mulberry leaves and silkworms using hydrophilic interaction chromatography-tandem mass spectrometry

Mulberry 1-deoxynojirimycin (DNJ, a potent α-glycosidase inhibitor) has been investigated thoroughly for its analytical methods and therapeutic potential against diabetes, whereas little attention has been given to other iminosugars such as 2-O-α-d-galactopyranosyl-DNJ (GAL-DNJ) and fagomine. For instance, concentration and composition of these iminosugars in mulberry leaves as well as sericulture products have not been fully characterized due to lack of suitable analytical methods. Here we developed a simultaneous determination method for DNJ, GAL-DNJ, and fagomine using hydrophilic interaction chromatography (HILIC) with tandem mass spectrometry (MS/MS). When mulberry leaf extracts were subjected to HILIC-MS/MS with multiple reaction monitoring (MRM), individual iminosugars could be separated and detected. The developed method is sufficiently sensitive for determining iminosugars in mulberry leaves as well as silkworms, providing new information (e.g., different amounts of iminosugars in mulberry leaf varieties; high DNJ and low GAL-DNJ in the silkworm body, especially in the blood) that is useful for producing iminosugar-rich products for nutraceutical purposes.     

Bt水稻"克螟稻”花粉对家蚕生长发育的影响

本试验以家蚕为供试对象研究Bt水稻"克螟稻”花粉对家蚕生长发育的影响.结果发现,与正常无花粉处理相比,无论是非Bt水稻花粉处理,还是Bt水稻花粉处理,对初孵家蚕幼虫的致死率无多大影响,而对家蚕的体重有较大影响,其中三龄期家蚕体重存在极显著差异.还发现Bt水稻花粉处理组家蚕在3龄时期大小很不一致,最轻体重为18.1mg,而最重体重为183.8mg.这是由于采用人工抖粉桑叶上花粉浓度不均匀造成的.鉴于实际生态条件下桑叶上的花粉浓度可能远远低于试验条件,因此,在实际稻桑共作环境下,Bt水稻"克螟稻”花粉对家蚕生长发育可能不会造成太大的影响。 Abstract:The effect on the development of silkworm larvae of Bt transgenic rice pollen containing cry1 Ab gene from Bacillus thuringiensis were investigated.Compared with normal treatment,mortality of newly hatched silkworm lar vae in either Bt rice pollen or susceptible rice pollen treatment were not significantly different,while the variances of silkworm larvae weight at third instar were significant at 0.01level.In addition,the weight of each silkworm larva at third instar in Bt rice pollen treatment showed a big difference,the biggest and smallest silkworm larvae were 183.8rug and 18.lmg respectively,which was probably caused by the difference of Bt rice pollen concentration on the mulberry leaves.As pollen concentration on the mulberry leaves in actual field was lower than in lab,the influence on the development of newly hatched silkworm larvae of Bt rice pollen is not likely significant in actual ecological cnvi ronment.     

Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus‐oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 μg/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 μg/ml) resulted in high rates of maturation to the MII stage (92.6 ± 2.5 and 98.5 ± 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 ± 4.2 and 45.1 ± 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 ± 2.9%). Finally, the addition of 0.1 μg/ml iAC and 0.5 mM 3‐isobutyl 1‐methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 μg/ml iAC (31.9 ± 5.5 vs. 12.1 ± 1.6 and 45.5 ± 2.9 vs. 19.1 ± 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86–91,1999. © 1999 Wiley‐Liss, Inc.