Optimal silkworm larva host for high-level production of Mus musculus IL-4 using a baculovirus expression vector system

Interleukine-4 (IL-4) is a cytokine that plays an important role in the immune system and recognized as a biological medicine. Therefore, there is a demand for the production of IL-4 with high performance. The expression of a recombinant IL-4 protein in the prokaryotic system usually results in the formation of an inclusion body. To date, the solution to obtain those active products without the refolding process remains to be established. In this study, we tried to acquire a biologically active recombinant Mus musculus IL-4 (rMmIL-4) using a silkworm-baculovirus expression vector system (silkworm-BEVS). We constructed two recombinant baculoviruses coding rMmIL-4 with the distinct location of affinity purification tags and succeeded in the expression and purification of rMmIL-4 proteins directly without the refolding process. Both purified proteins displayed comparable biological activity to the commercial proteins produced by the E. coli expression system. Besides, we performed screening of silkworm strains to seek optimal hosts for the mass-production of rMmIL-4. Intriguingly, we found that some silkworm strains showed significantly higher secretion levels of rMmIL-4 in silkworm sera. Our study provides meaningful insights into the industrial-scale production of rMmIL-4 with high productivity for pharmaceutical applications in the future.     

Attraction of Lutzomyia longipalpis to human skin odours

Abstract. Male and female Lutzomyia longipalpis sandflies showed attraction to human skin emanations placed on warmed glass Petri dishes. Unfed virgin females were more strongly attracted than males, which also showed attraction. Four human subjects were tested and significant variation was found between the numbers of sandflies attracted to their skin emanations. This suggests that some individuals were more attractive than others. There was a significant difference between the response shown by sandflies from the Jacobina and Lapinha regions of Brazil, suggesting that sandflies from the Jacobina region were more anthropophilic. In addition, sandflies from Jacobina had a significantly higher level of activity than those from Lapinha. The role of sandfly attraction to humans as a risk factor in Leishmania transmission is considered.     

Combination of antibiotics and chitin synthesis inhibitors for the control of Microcerotermes diversus (Isoptera: Termitidae)

The symbiotic microorganisms in the gut of termites play a key role in the digestion of cellulose and nitrogen metabolism. Therefore, disruption of these symbioses activity can open a door toward termite management. Thus, the current study aimed to exploit termite gut bacterial capacity in order to utilize it for efficient termite control. So, in the current study, gut bacteria of Microcerotermes diversus have been extracted, cultivated on both liquid and solid media, and screened with a range of antibiotics to find the most effective antibiotics. Results showed that chloramphenicol and nalidixic acid were the most effective antibiotics in preventing the colony unit formation of the gut bacteria amongst 16 antibiotics tested. Also, from two chitin synthesis inhibitors tested hexaflumuron with an LC₅₀ of 613.9 µg ml⁻¹ was more toxic than lufenuron with an LC₅₀ of 1414.5 µg ml⁻¹. Thus, two antibiotics (chloramphenicol and nalidixic acid) were used simultaneously with a sub-lethal concentration of hexaflumuron against the termite and a variety of factors such as wood consumption rate, running speed, body water content, and tunneling activity were evaluated under laboratory conditions. Results showed that the combination of these two antibiotics (chloramphenicol and nalidixic acid) with hexaflumuron caused a significant decrease in termite consumption rate, running speed, and tunneling behavior, but didn’t affect the body water contents of termites. These results suggest that using a combination of antibiotic/s and hexaflumuron is a promising management practice to get a suitable control measure for the studied termite.     

Population consequences of huge nesting aggregations ofRopalidia plebeiana (Hymenoptera: Vespidae)

Nest survival rates and reproductive rates of females of the Australian paper wasp, Ropalidia plebeiana, in nest aggregations under a concrete bridge were studied. The annual colony cycle commenced in August with the reutilization of old nests and by the founding of new nests, mainly by associations of foundresses. Distribution patterns of the number of foundresses per nest was nearly random on new nests while contagious on old nests. About one-third of the increase in number of nest was achieved by dividing large, old nests and two-thirds by founding new nests. Nest survival rates for old nests (August to April) and new nests (November to April) were 89.6% and 88.8% respectively, far higher rates than those of other polistine wasps so far reported. The numbers of nests and reproductive females increased during a colony cycle by 2.30 and 10.98 times, respectively. Thus, huge aggregations of nests are probably beneficial for this species by enabling the realization of high reproductive rates. We could find no density-dependent effect in nest growth rate or production of female reproductives. Ratio of cells parasitized by ichneumonid wasps was low. Nests in the central part of an aggregation were prasitized at significantly lower rates than nests on the periphery or scattered nests outside a dense aggregation, suggesting a selfish herd effect.     

Insecticidal activities of two fig tree leaf extracts against the cotton pest Spodoptera litura (Fab.) and their impact on the non-target red worm Eisenia foetida (Savigny)

Extracts of botanical origin naturally contain a complex mixture of chemicals considered effective in managing lepidopteran pests. Chemical screening of the ethanolic leaf extracts of two fig tree species, Ficus lyrata and Ficus auriculata, delivered 12 and 15 phyto-compounds with relatively high peak area percentages in phytol and flavone, respectively. Larvicidal activity against Spodoptera litura yielded higher mortality rates at maximum-concentration treatment (600 ppm) with Ficus lyrata (91.3%) and Ficus auriculata (98.5%) extracts during the second instar. Sub-lethal dosages (300 ppm) of both Ficus lyrata and Ficus auriculata extracts impeded the development and reproduction of lepidopteran pests. The enzyme-inhibition activity of both fig extracts elicited a significant reduction in the major digestive enzymes alkaline phosphatase, acid phosphatase, and lactate dehydrogenase dose-responsively. Mid-gut histological screening of Ficus lyrata and Ficus auriculata extracts displayed gut lumen disruptions, shape alterations of columnar cells, and brush-border membrane damage. Further, the non-target toxicity of fig extracts against the red worm Eisenia foetida was minimal compared with that of the chemical temephos. Overall, the Ficus extracts proved to be eco-friendly strategies for managing the polyphagous pest Spodoptera litura and are potentially more sustainable and less harmful to non-target earthworms. Nonetheless, in silico predictions suggest that the active compounds in fig extracts are predominantly toxic against honeybees (16 compounds) and violate TICE rules (10 compounds). Therefore, the biological actions of fig extracts' individual novel chemistries need to be examined on target and non-target species to develop better pest management strategies.     

CRISPR/Cas9 mediated genome editing of Helicoverpa armigera with mutations of an ABC transporter gene HaABCA2 confers resistance to Bacillus thuringiensis Cry2A toxins

High levels of resistance to Bt toxin Cry2Ab have been identified to be genetically linked with loss of function mutations of an ABC transporter gene (ABCA2) in two lepidopteran insects, Helicoverpa armigera and Helicoverpa punctigera. To further confirm the causal relationship between the ABCA2 gene (HaABCA2) and Cry2Ab resistance in H. armigera, two HaABCA2 knockout strains were created from the susceptible SCD strain with the CRISPR/Cas9 genome editing system. One strain (SCD-A2KO1) is homozygous for a 2-bp deletion in exon 2 of HaABCA2 created by non-homologous end joining (NHEJ). The other strain (SCD-A2KO2) is homozygous for a 5-bp deletion in exon 18 of HaABCA2 made by homology-directed repair (HDR), which was produced to mimic the r2 resistance allele of a field-derived Cry2Ab-resistant strain from Australia. Both knockout strains obtained high levels of resistance to both Cry2Aa (>120-fold) and Cry2Ab (>100-fold) compared with the original SCD strain, but no or very limited resistance to Cry1Ac (<4-fold). Resistance to Cry2Ab in both knockouts is recessive, and genetic complementary tests confirmed Cry2Ab resistance alleles are at the same locus (i.e. HaABCA2) for the two strains. Brush border membrane vesicles (BBMVs) of midguts from both knockout strains lost binding with Cry2Ab, but maintained the same binding with Cry1Ac as the SCD strain. In vivo functional evidence from this study demonstrates knockout of HaABCA2 confers high levels of resistance to both Cry2Aa and Cry2Ab, confirming that HaABCA2 plays a key role in mediating toxicity of both Cry2Aa and Cry2Ab against H. armigera.     

A reduction in temperature induces bioactive red pigment production in a psychrotolerant Penicillium sp. GEU_37 isolated from Himalayan soil

Filamentous fungi are being globally explored for the production of industrially important bioactive compounds including pigments. In the present study, a cold and pH tolerant fungus strain Penicillium sp (GEU_37), isolated from the soil of Indian Himalaya, is characterized for the production of natural pigments as influenced by varying temperature conditions. The fungal strain produces a higher sporulation, exudation, and red diffusible pigment in Potato Dextrose (PD) at 15 °C as compared to 25 °C. In PD broth, a yellow pigment was observed at 25 °C. While measuring the effect of temperature and pH on red pigment production by GEU_37, 15 °C and pH 5, respectively, were observed to be the optimum conditions. Similarly, the effect of exogenous carbon and nitrogen sources and mineral salts on pigment production by GEU_37 was assessed in PD broth. However, no significant enhancement in pigmentation was observed. Chloroform extracted pigment was separated using thin layer chromatography (TLC) and column chromatography. The two separated fractions i.e., fractions I and II with Rf values 0.82 and 0.73, exhibited maximum light absorption, λ_max, at 360 nm and 510 nm, respectively. Characterization of pigments using GC–MS showed the presence of the compounds such as phenol, 2,4-bis (1,1-dimethylethyl) and eicosene from fraction I and derivatives of coumarine, friedooleanan, and stigmasterole in fraction II. However, LC-MS analysis detected the presence of derivatives of compound carotenoids from fraction II as well as derivative of chromenone and hydroxyquinoline as major compounds from both the fractions along with other numerous important bioactive compounds. The production of such bioactive pigments under low temperature conditions suggest their strategic role in ecological resilience by the fungal strain and may have biotechnological applications.     

Seasonal surveillance of deer and horse flies (Diptera: Tabanidae), Gyeonggi province,Republic of Korea

Deer and horse flies, Family Tabanidae, are biological, mechanical, and potential vectors of approximately 27 viral, bacterial, and helminthic pathogens of veterinary and medical importance. Biting fly surveillance was conducted using New Jersey light traps and Mosquito Magnet® traps from May-October during 2015–2018 at eight sites (villages and US military installations and training areas) near/in the Demilitarized Zone and at a US military installation in southern Gyeonggi province, Republic of Korea.A total of 9985 deer and horse flies, comprising 4 genera and 17 species, were collected. The predominant species collected was Chrysops mlokosiewiczi (95.39%), followed by Chrysops suavis (1.48%) and Haematopota koryoensis (1.07%), while the remaining species accounted for only 2.06% of the specimens collected. A bimodal peak was observed for C. mlokosiewiczi in June and late-July, but not for the other two predominant species during the whole of the study period. This study provides and insight into ecological behavior and seasonal abundance of deer and horse flies in Gyeonggi province, Republic of Korea.     

New data on lance flies (Diptera,Lonchaeidae) associated with figs (Moraceae,Ficus spp.) in Japan and Taiwan,with descriptions of two new species of the genus Silba Macquart

Larvae of the genus Silba Macquart, 1875 were obtained from the syconia of three different species of Ficus growing on the southern islands of Japan and Taiwan. Examination of the emerged adults identified four species, two of which are described as new to science: Silba erecta MacGowan and Arimoto sp. nov. and Silba thunbergii MacGowan and Arimoto sp. nov. Of the four species observed, the larvae of S. erecta, Silba ishigaki MacGowan and Okamoto, 2013 and S. thunbergii feed on the tissue of the fig syconia. Silba inubiwa MacGowan and Okamoto, 2013 was observed as a predator of three fig wasp species. The biogeographic distribution and biological data are provided for each of these fig-associated species.     

Predicted voltinism of the camellia spiny whitefly,Aleurocanthus camelliae (Hemiptera: Aleyrodidae), in major Japanese tea-producing districts based on life history parameters

We examined the temperature-dependent development, adult longevity, fertility, and sex ratio of the whitefly Aleurocanthus camelliae, which infests tea plants, under laboratory conditions. Eggs and nymphs were reared on tea shoots at 20, 22.5, 25, and 27.5 °C with a photoperiod of 15L:9D. The developmental zero of the egg, total nymphal stadia, and egg–adult duration of A. camelliae were 12.6, 11.6, and 11.9 °C, respectively, with thermal constants of 176.7, 394.7, and 569.9 d-degrees, respectively. The mean longevities of adult female and male A. camelliae were 4.4 and 2.7 d, respectively. These results suggested A. camelliae voltinism of two to three generations per year in Kanto, Tokai, and Kinki districts and three to five generations per year in the Shikoku and Kyushu districts, Japan.