The nesting behavior, reproduction, fruit set and shape of O. cornifrons varied significantly with the released sex ratio of O. cornifrons. A female : male sex ratio of 1 : 2 was resulted in a 3.4 to 6.7 fold higher than other sex ratio in a nesting behavior. A ratio of 1 : 2 resulted in a 1.2-fold nesting rate, which was slightly higher than other nesting rates. Releasing only males resulted in a 2.4-fold greater amount of fruit set in non-pollinated sites. A sex ratio of 1 : 2 gave a slightly higher shape index and a 1.2 to 1.6-fold lower asymmetric index than other sex ratios. There was no significant difference between female release numbers in fruit set, and 100 to 200 females gave a slightly higher shape index than 400 females. Thus, we determined that 200 females should be released per 2,000㎡ and that the sex ratio of females to males should be 1 : 2.

We collected four species of Osmia spp. consisting of O. cornifrons, O. pedicornis, O. taurus and O. satoi in Yeongwol, Bonghwa and Yechon locations. O. pedicornis was the dominant species, which accounts for 59.6% to 76.4% in the three locations. The sex ratio of Osmia spp. were from 1:1 to 1: 2.8 in O. cornifrons and from 1:2.8 to 1: 5.5 in O. pedicornis. The sex ratio of O. cornifrons and O. pedicornis was different in 3 locations. The female and male cocoons of O. cornifrons were the long elliptical-shaped and grey-brown color. The female and male cocoons of O. pedicornis were the long elliptical-shaped and polished dark-brown color. In both species of O. cornifrons and O. pedicornis, the cocoon size of the females was significantly larger than that of the male. Between those two species, the latter had a relatively bigger size than the former.

In the age of sexual maturity of queen, mating occurred at 6.7% immediately after eclosion and it was the highest as 85.0% at 10 days after emergence. However, a remarkable decrease was occurred at 20 days after the emergence. With regard to oviposition rate, the highest rate was observed as 81.3-81.8% at 6 to 10 days of eclosion. The rate of colony foundation and progeny-queen production were the highest as 43.8% and 37.5%, respectively, at 8 days of eclosion. In the age of sexual maturity of male, mating rate was as high as 38.3% immediately after the eclosion, the highest as 80.0% at 25 days of eclosion, and 76.6% at 8 days of eclosion. The oviposition rate was the highest as 76.9% at 6 days of eclosion, and decreased to 75.0% at 8 days and 72.7% at 10 days of eclosion. Summarized, our results indicate that sexual maturity for mating of B. terrestris is most favorable 6-8 days after eclosion for queen and 6-10 days after for male.

We investigated pollination and foraging activities of honeybee (Apis mellifera L.) and bumblebee (Bombus terrestris L.) during flowering season of the asian pear (Pyrus pyrifolia N.) under different weather conditions. There was no significant statistical difference about the pollination activities of two species. However, the pollination activities of bumblebee were more active than those of honeybee under low temperature and rainfall period. The activities of honeybee and bumblebee were more influenced by temperature than other factors (i.e. illumination and wind velocity). Honeybee was more sensitive to temperature and illumination than bumblebee. At low temperatures (<20℃) on cloudy days (<30,000 lux) with a certain wind velocity (>4.0 m/s), the pollination activity of the honeybee was lower twice than that of bumblebee.

Distillers dried grain (DDG) and makgeolli spent grain (MSG) are agricultural by-product to produce alcoholic beverage. However, they are known to contain enough nutrients. Mealworm is a promising insect resource for an animal feed ingredient as well as alternative human food. With low cost, DDG and MSG were investigated as a feed ingredient for rearing high quality mealworms. DDG and MSG were mixed with wheat bran and compared to control feed (only wheat bran) for its effects on larval survivorship, larval weight, duration for larval development, pupation rate, and pupal weight. Adding DDG on wheat bran showed positive results for larval weight, duration for larval developmental period, and pupation rate. However, adding MSG made longer duration for larval development, but it also improved larval weight, pupal weight with more than 90% pupation rate. We confirmed that adding 30～50% of DDG or MSG to conventional wheat bran have a strong potential to replace the conventional wheat bran insect feed for quality insect production.

Osmia cornifrons is a cavity-nesting solitary species used as an apple pollinator in Korea. To elucidate the developmental characteristics of O. cornifrons, we investigated its development from the egg to adulthood, including a dormant prepupal phase and mating through indoor rearing (25 °C, 65% R.H.). The egg durations of the female and male bees were 3.6 ± 0.8 days and 3.1 ± 1.3 days, respectively. During larval development, the head widths of the 1st to 5th instars ranged from 0.7 ± 0.1 mm to 1.3 ± 0.1 mm. The peak of the growth in head width was the 2nd instar. The larval lengths ranged from 3.7 ± 0.6 mm to 13.6 ± 1.3 mm. The peak of growth was the 4th instar. The larval weights ranged from 4.5 ± 1.2 mg to 78.3 ± 16.1 mg. The peak of growth was the 3rd instars. The total larval durations of from the 1st to 5th instars for the females and males were 14.0 ± 6.0 days and 13.2 ± 5.8 days, respectively. The spinning durations of the females and males were 2.2 ± 0.7 days and 2.3 ± 0.8 days, the prepupation durations were 55.5 ± 5.9 days and 55.8 ± 2.9 days, and the pupation durations were 26.4 ± 2.1 days and 25.3 ± 2.3 days, respectively. The average longevity of the female adults and male adults was 21.8 ± 8.7 days and 24.4 ± 12.4 days, respectively. The total duration of from the egg to an adult bee of the O. cornifrons females and males was 123.5 days and 124.1 days, respectively. Mating consisted of the three following phases: the precopulatory (courtship and attempting copulation), copulation and postcopulatory phases. The mating times of the precopulatory, copulation and postcopulatory phases were 159.6 ± 288.9, 8.4 ± 7.1, 12.9 ± 4.5, and 198.8 ± 69.8 seconds.

Serine protease inhibitors play a critical role in physiological processes and immune responses by regulating serine protease activities. Here we describe the molecular cloning and antimicrobial activities of a serine protease inhibitor from the mason bee, Osmia cornifrons (OcSPI). OcSPI consists of 405 amino acid residues and contains a potential reactive center loop (RCL) region in its C-terminus. Recombinant OcSPI was produced as a 64-kDa glycoprotein in baculovirus-infected insect cells and exhibited inhibitory activity against chymotrypsin. Additionally, OcSPI demonstrated inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, but not against tissue plasminogen activator, thrombin, or plasmin. Recombinant OcSPI bound directly to Escherichia coli, Bacillus subtilis, and Beauveria bassiana and exhibited antimicrobial activity against both bacteria and fungi. Our results demonstrated the antimicrobial functions of OcSPI and suggest a role for OcSPI in the immune response of O. cornifrons.

The honeybee inhibitor cysteine knot (ICK) peptide acts as an antifungal peptide and insecticidal venom toxin. However, the ICK peptide from bumblebees has not been characterized. Here, we report the molecular cloning and antifungal activity of a bumblebee (Bombus ignitus) ICK peptide (BiICK). We identified a BiICK that contains an ICK fold. The BiICK was expressed in the epidermis, fat body, and venom gland of B. ignitus worker bees. A 6.7-kDa recombinant BiICK peptide was expressed in baculovirus-infected insect cells. Recombinant BiICK peptides directly bound to Beauveria bassiana, Ascosphaera apis, and Fusarium graminearum, but they did not bind to Escherichia coli, Paenibacillus larvae, or Bacillus thuringiensis. Consistent with this finding, BiICK exhibited antifungal activity against fungi. These results demonstrate that BiICK acts as an antifungal peptide.

The Osmia cornifrons plays an important role in pollinating fruit trees, such as apple trees. To better understand diapause and oviposition in O. cornifrons, we investigated the correlation between the ovarian development and secretion level of OcVg protein in hemolymph. During ovarian development in wintering the number of oocytes progressively increased in comparison with the length of the ovaries and the oocytes. After emergence, the oocyte and ovary sizes developed until 6 days after emergence and declined after 6 days, but the number of oocytes decreased gradually. The secretion level of OcVg protein in hemolymph revealed that during wintering, the secretion level increased from 1 month to 2 months and then stagnated after 2 months. After diapause, the secretion level increased gradually until day 6 of the newly emerged adult from cocoon stage, and thereafter gradually declined, remaining detectable until day 30 of the adult stage. The correction analysis between ovarian development and OcVg secretion level in hemolymph found that in wintering, the number of oocytes was positively correlated to OcVg secretion level. After diapause, the ovary and first oocyte lengths and the number of oocytes showed significant changes in OcVg secretion level and positively correlated with OcVg secretion level, respectively. These results suggest that there is a significant interaction between ovarian development and the secretion level of OcVg protein and the pattern of ovarian development and secretion of OcVg protein are stage-specific in the O. cornifrons female.

Bees of the genus Osmia are cavity-nesting solitary species that play important roles as pollinators. Nest traps are one of the most common methods to study Osmia bees. To elucidate the optimal environmental conditions of nesting sites, we investigated the effects of location, direction, altitude, and sites of nest traps on the rate of trap-nesting Osmia spp. During the collection period, the average rate of trap-nesting Osmia spp. collected within 90 days after the installation of traps was 17.0±20.0%. This percentage was 2.7-fold higher than that of trap-nesting bees in 30 days after the installation of traps. The Jeongseon location exhibited the highest rates of trap-nesting Osmia spp. collected in 30 and 90 days, representing 11.1 ±17.6% and 23.2±22.5%, respectively. The direction of the nest traps did not affect the rate of trap-nesting Osmia spp. The altitude ranges of the traps were 0-199 m, 200-399 m, 400-599 m, 600-799 m and over 800 m. Interestingly, the altitude range of 600-799 m showed the highest rate of trap-nesting bees, which was 40.4±3.9%. Higher altitudes seemed to correspond to higher rates of trap-nesting bees. With regards to the sites where the nests were placed, the rate of trap-nesting bees in a mud wall of an old house was 45.1±25.2%, which was 3-fold higher than that of a nest in an apple orchard. The flowering plants collected at different locations during the trap-nesting activity of Osmia bees belonged to 18 families and 34 species. In conclusion, the rates of trap nests colonized by Osmia spp. were affected by altitude, site, and plant diversity.

The post-hibernated queens of Korean native bumblebee, Bombus hypocrita saporoensis were collected in Jeong-Sun, Korea to investigate their ecological characteristics and reared under the laboratory conditions; 27℃, 65% R.H. and darkness. The average weight of collected queens was 0.87±0.14g and 0.8g class was most abundant (34.7%) among eight weight classes. The rates of colony initiation, colony foundation and progeny-queen production were 76.3%, 60.0% and 42.5%, respectively. The durations up to colony initiation and colony foundation were 12.0±6.7 days and 49.3±2.4 days, respectively. The duration up to first worker, male and queen emergence were 22.2±3.0 days, 63.4±6.5 days and 63.5±8.7 days, respectively. The number of progenies was 108.5±26.0 workers, 186.7±70.7 males and 39.1±30.3 queens. The longevity of colony founded queen was 90.2±19.9 days whereas that of non-colonized queen was 35.9±30.3 days. Copulation time was 29.5±9.6 min. The number of eggs per egg cell laid new queen was 9.3±2.3 grains. An egg shaped as banana and it’s size was 1.12±0.10 mm in a width, 3.54± 0.16 mm in a length and 2.26±0.34 mg in a weight.

Insect is an important player in the ecosystem as a prey for animals. Moreover, they are a valuable candidate food source for rearing animal. Tenebrio molitor (Coeloptera: Tenebrionidae) larvae are known as a good food source with high protein, unsaturated fatty acid, minerals. Therefore, it has strong potential to substitute the conventional meat consumption. To utilize T. molitor as a feed, the standard mass-rearing protocol is required. To make standard mass-rearing protocol, we tested different temperature(17.5, 20, 22.5, 25, 27.5 and 30°C) conditions for egg, larvae, pupae and adult T. molitor to identify the optimal rearing condition. Hatching was occurred within 15~32.5°C range. However, 17.5~27.5°C was required to get more than 70 % hatching rete. When the eggs were treated in 22.5~27.5°C, all eggs were hatched within 10 days. As larval development, shorter developmental period, higher pupation and eclosion rates were observed within 25~27.5°C temperature range. In addition, we compared the number of egg, oviposition duration and time required to start egg-laying. The minimum egg-laying(258.40±10.86) was observed at 17.5°C, but the maximum(749.10±7.45) was at 27.5°C. The maximum oviposition duration (137.00±12.73 day (mean±S.D.)) was achieved at 27.5°C, but the minimum (87.50±3.54 day (mean±S.D.)) was at 30°C. The time required to start egg-laying was less than 10 days at 17.5, 27.5, and 30°C. To consider all the factors, we concluded that the optimal temperature is 27.5 °C.

We investigated the distribution, sex ratio, and natural enemies and nesting competition insects of mason bees, Osmia spp. in 32 apple orchards during 2006 to 2008. The rate of bee nested was 14.0±1.7%. The rate of fauna nested were Osmia spp., 66.0%, natural enemies, 21.7% and nesting competition insects, 11.6%. We collected five species of Osmia spp. consisting of Osmia cornifrons, O. pedicornis, O. taurus, O. satoi and O. jacoti. In case of collected five Osmia spp, O. cornifrons was 69.6%, which was the dominant species and O. pedicornis was 13.0% and O. taurus was 11.2%, respectively. The collecting rate of Osmia spp. was significant difference among each year and 10 locations. The sex ratio of Osmia spp. were 1:1.9 in O. cornifrons, 1:5.1 in O. pedicornis and 1:2.2 in O. taurus. The sex ratio of O. cornifrons was significant difference among each year and 10 locations. Seven species of natural enemies that attack Osmia spp. were collected in apple orchard. Trogoderma and Chaetodactylus showed the highest rates with 48% and 38% among 7 natural enemies. Also, we collected nine species of nesting competition insects. Anterhynchium flavomaginatum showed the highest rates with 38.6% among 9 nesting competition insects.

Artificial insemination is a technique to transfer instrumentally sperm from the male into the female’s reproductive system. A key factor among artificial insemination techniques is gathering sperms. Here, a method for collecting sperm of bumblebee male by pressing was firstly developed. This method has stage of separating reprodutive tract of male, separating accessory testis, separating vas deferens, pressing vas deferens with cover-glass and collecting sperm with syringe. The developed method was 2.8 fold higher in rate of collecting sperm than that of existing method. Among 1M NaCl-, Insect ringer solution-, Phosphate buffered saline(PBS)-collecting sperm solutions, PBS was exhibited the best result as 17.2 sperms/cell, which corresponded to 1.6-1.8 fold increased over the result of other collecting sperm solution. Consequently, we think that this method is very important technique to save time for collecting sperm, to keep activity of sperm and to reduce contamination of sperm.

Osmia cornifrons plays a major role in the pollination of orchards, but basic information on vitellogenin and the oocyte development is limited. To better understand vitellogenin in hymenopteran insects, we cloned a cDNA encoding vitellogenin from the hornfaced bee O. cornifrons. O. cornifrons vitellogenin cDNA contains 5477 bp with an open reading frame of 1,783 amino acid residues, and has a predicted molecular mass of approximately 200.21 kDa and a pI of 6.55. O. cornifrons vitellogenin possesses four consensus (RXXR/S) cleavage sites and has conserved DGXR and GL/ICG motifs in the C-terminus. The deduced amino acid sequence of the O. cornifrons vitellogenin cDNA showed a 66% identity with Megachile rotundata, 53% to Apis mellifera, 51% to Bombus ignitus, and 42%-30% with other hymenopteran insect vitellogenins. Phylogenetic analysis showed that O. cornifrons vitellogenin clustered with vitellogenins from Megachildae, Apidae, Vespidae, and Formicidae species but not with those from Pteromalidae, Aphelinidae or Ichneumonidae species.

Bumblebees are important pollinators of crops and wildflowers. Bumblebees generally produce one generation per year. One of the key stages for year-round rearing of bumblebees is breaking diapause. To evaluate the effects of a combination method of CO2-narcosis and cold treatment to break the diapause of B. ignitus and B. terrestris queens, we determined whether this method affected their ability to establish a colony after the diapause break. The diapause treatment regimes that were utilized were CO2(CO2– narcosis), CT-1M(cold treatment at 5°C for 1 month), CT-1M-CO2(CO2–narcosis after cold treatment for 1 month), CT-2M-CO2(CO2–narcosis after cold treatment for 2 months), CT-2M (cold treatment for 2 months), CT-2.5M-CO2(CO2–narcosis after cold treatment for 2.5 months) and CT-2.5M (cold treatment at 5°C for 2.5 months). In view of the effects on the colony developmental characteristics of B. ignitus queens, the most favorable diapause treatment was CT-1M-CO2. A combination method of CO2– narcosis and cold temperature treatment yielded better results than that of single CO2–narcosis or cold temperature treatment on the colony development of diapause-broken B. ignitus queens. In the case of B. terrestris queens, we concluded that a combination method of CO2 and cold temperature treatment yielded better results than that of a single cold-temperature(up to2 months) treatment. In conclusion, the findings of the present study indicated that the combined application of CO2 and cold temperature was a favorable method for the colony development of diapause-broken B. ignitus and B. terrestris queens compared with only CO2–narcosisorcoldtemperaturetreatments. A combination method of CO2 and cold treatment reduced the side effect of CO2–narcosis and shortened the duration of cold treatment by at least 1month.

Recently, the flies are used to pollinate onion for seed production in Korea. However, the use of flies for pollination has caused complaints by local residents in onion seed production owing to sanitation issues and smell. Therefore, we studied the effects of pollination with bumblebee (Bombus terrestris) and flies(e.g., houseflies, flesh flies, blue bottle flies and blowflies) at small net(1.8m×2.3m×1.8m) in the greenhouse(66m2) for onion seed production. Although the number of flies was about 70 times than number of bumblebee workers, flies and bumblebees showed equivalent pollination efficiency. Therefore, pollination by B. terrestris can be used as an alternative approach in seed production of onion. An analysis of the most appropriate colony of B. terrestris for seed production of onion in small net showed that colony composed of fifty to eighty workers per small net was the most efficient level. When early maturing onion cultivars ‘Salad-5’ and ‘Yeonsinwhang’, and mid-late maturing onion cultivar ‘Colossus’ were pollinated by B. terrestris and files in small net, B. terrestris showed rather efficient pollination efficiency than flies or was equivalent to flies. In addition, difference among onion cultivars was affecting pollination effect by flies but was not affecting pollination effect by B.terrestris.

Bumblebee, Bombus terrestris is widely used to pollinate various crops, especially tomato, in greenhouses. Mason bee, Osmia cornifrons, which is using as apple pollinator. Here we investigated characteristics on pollinating activity of B. terrestris and O. conifrons at different weather conditions in apple orchard. The foraging activity of O. conifrons was more sensitive than that of B. terrestris at weather conditions including temperature, illumination intensity and wind velocity. The pollinating activity of O. conifrons was very low than that of B. terrestris at a low temperature, cloudy, rainy and windy weather. Therefore, Our results indicated that B. terrestris was suitable for apple pollinator than O. conifrons, especially in low temperature and bad weather.

To improvement the fruit quality of the korean raspberry fruit, 3 species of bee, Bombus terrestris, Apis mellifera and Osmia cornifrons were used for pollination in korean raspberry fruit field from Korea. This study was also conducted using 3 kinds of methods for pollination effect, natural condition, net-house and control method. Blooming time of korean raspberry fruit shows always in the middle of the May, foraging behavior of bees used in this examination showed different tendency each other. Especially pollinating time of B. terrestris was 2.73sec and transfer time between flower was 2.64sec. Rate of korean raspberry fruit setting pollinated with B. terrestris, A. mellifera, O. cornifrons were showed 83.1%, 80.5%, 77.5% respectively. And, rate of fruit setting of the untreated control and the control (natural condition pollination) were 36.2% and 76.0%. Therefore, korean raspberry fruit set in the case of B. terrestris compared control was superior to 230%, 109% in contrast to natural pollination showed, A. mellifera and O. cornifrons were no significant difference over natural pollination. The quality of the products of korean raspberry fruit pollinated with B. terrestris showed the highest quality as a result fruit diameter 19.5±0.85, fruit weight 2.7±0.32, sugar content 11.2±1.48 Brix%, seed no. of 86.8±10.15. The rate of productivity increase using the Bombus terrestris was 21% in contrast to natural pollination.

Bumblebee, Bombus terrestris is widely used to pollinate various crops, especially tomato, in greenhouses. Mason bee, Osmia cornifrons, which is using as apple pollinator. Here we investigated characteristics on pollinating activity of B. terrestris and O. conifrons at different weather conditions in apple orchard. The foraging activity of O. conifrons was more sensitive than that of B. terrestris at weather conditions including temperature, illumination intensity and wind velocity. The pollinating activity of O. conifrons was very low than that of B. terrestris at a low temperature, cloudy, rainy and windy weather. Therefore, Our results indicated that B. terrestris was suitable for apple pollinator than O. conifrons, especially in low temperature and bad weather.