CRISPR/Cas9 mediated knockout of Amyellow-y gene results in melanization defect of the cuticle in adult Apis mellifera.

Visible genetic markers are critical to gene function studies using genome editing technology in insects. However, there is no report about visible phenotypic markers in Apis mellifera, which extremely influences the application of genomic editing in honey bees. Here, we cloned and characterized the Amyellow-y gene in A. mellifera. Stage expression profiles showed that Amyellow-y gene was highly expressed in 2-, 4-day-old pupae, and newly emerged bees, and a high expression level was detected in the leg, thorax, wing and sting. To understand its functional role in pigmentation, Amyellow-y edited honeybees were created using CRISPR/Cas9, and it was found that the black pigment was decreased in the cuticle of mosaic workers and mutant drones. In particular, mutant drones manifested an overall appearance of yellowish cuticle in the body and appendages, including antennae, wings and legs, indicating that mutagenesis induced by disruption of Amyellow-y with CRISPR/Cas9 are heritable. Furthermore, the expression levels of genes associated with melanin pigmentation was investigated in mutant and wild-type drones using quantitative reverse transcription PCR. Transcription levels of Amyellow-y and aaNAT decreased markedly in mutant drones than that in wild-type ones, whereas laccase 2 was significantly up-regulated. Our results provide the first evidence, to our knowledge, that CRISPR/Cas9 edited G1 mutant drones of A. mellifera have a dramatic body pigmentation defect that can be visualized in adults, suggesting that Amyellow-y may serve as a promising visible phenotypic marker for genome editing in honey bees.

[Isolation and identification of a newbacterial pathogen infecting larvae of honeybee (Apismellifera ) Perish].

A bacteroidal disease of honeybee (Apis mellifera ) larvae was found in some regions of Zhejiang Province, China , in early spring 2005. The diseased larvae lost its shine, became yellow and rotted when serious. This symptom was different to any bacteroidal disease of honeybee larval been reported. So, it is considered to be a new bacteroidal disease of honeybee larval. Five pure cultures of bacteria were separated from ten collections of diseased honeybee larvae, named as L1, L2, L3, IA and L5. Among these five pure cultures, only L2 could make the healthy honeybee larvae become diseased in both field and lab test. The symptom caused by L2 was similar to the natural-infection. From the diseased larvae caused by L2 could isolate bacteria the same as L2. Thus 12 was determined as the causing agent of this bacteroidal disease of honeybee larval. L2 was identified according to the characteristics of morphology, physiological biochemical characteristics and 16S rRNA gene sequence. As a result, the morphology and physiological biochemical characteristics of L2 were similar to E. faecium. And its 16S rRNA sequences highly matched to E. faecium, the similarities between them were higher than 99%. The overall similarity values between L2 and the published 16S rRNA sequences of 41 typical species of Enterococcus were 93.9% - 99.5% , the top value was between 12 and E. faecium. In the phylogenetic tree, L2 and E. faecium were assembled in the same ramification. So 12 was identified as E. faecium. Although Enterococcus faecium was known as pathogen to many post, account for 12% of all nosocomial infections, only second to E. coli, but there is no report about this bacteria infects honeybee up to now. So it is a new pathogen to honeybee. The isolation and identification of pathogen of this new bacteroidal larvae disease, afford a good feasibility for available prevention and cure to this new disease.

Microsatellite analysis of royal jelly producing traits of Italian honeybee (Apis mellifera Liguatica).

Genetic variations at 10 microsatellite loci were surveyed to determine the evolutionary relationships and molecular characteristics of three different honeybee (Apis mellifera L.) populations from Italy and China, i. e., native Italian (Ee), Chinese-Italian (Eb) and selected high royal jelly producing bees (Ea). A total of 96 alleles,an average of 9.6 alleles per locus,were scored in Ee,Eb and Ea bees at 10 loci. Out of which 48 (5%) were different. This indicated a high degree of polymorphism and ever, some genetic differentiation among the three populations due to artificial selection and geographical isolation. The polymorphic information contents (PIC) and heterozyosity of the three populations at 10 loci were 0.57, 0.50, 0.57, and 0.60, 0.57, 0.61, for Ee, Eb, Ea populations respectively, neither of which were different. This indicated same gene diversity within the three populations. The genetic distance was shorter between Ee and Eb bees as well as between Eb and Ea bees. Whereas that between Ee and Eb bees was longer. Further analysis indicated that the allele frequency of seven alleles at six loci (159 bp at A29,100 bp and 104 bp at A24; 110 bp at A7; 126 bp at A43, 221 bp at A14 and 221 bp at A113) increased going from Ee to Eb to Ea bees. Paired tests showed significant higher allele frequency between Ea and Eb bees,as well as between Ea and Eb bees. This indicates that these seven alleles are likely molecular markers of the high royal jelly producing bees. In addition,the allele frequency of four alleles at four loci (106 bp at A24,140 bp at A43;215 bp at A113 and 219 bp at A14) decreased going from Ea bees to Eb to Ee. Paired tests indicated significant lower allele frequency between Ea and Ee bees,as well as between Ea and Eb bees. Those four alleles may be the genetic markers for low royal jelly production.

[Cloning and sequence analysis of mrjp1 cDNA from Apis cerana cerana].

A cDNA library was constructed from eight-day-old worker heads of Apis cerana cerana. A probe derived from part of Apis cerana genomic mrjp3 segment was used to screen this library. A total of 120 positive clones of mrjps were screened out from the cDNA library with DIG-probe. The positive clones were amplified and sequenced with T3/T7 primer. 12 sequences were similar to Apis cerana india and Apis mellifera mrjp1 gene by BLAST analysis. The cDNA sequence analysis indicated that the homology was 93.78% between Apis mellifera mrjp1 and Apis cerana mrjp1 while the homology was 99.36% between Apis cerana cerana and Apis cerana india. This result confirmed in molecular level that Apis cerana cerana and Apis cerana india had a common ancestor since the relationship between Apis cerana and Apis mellifera was more far.

[Genetic analysis for developmental behavior of honeybee colony's royal jelly production traits in western honeybees].

Analyses of genotypic effects for colony's royal jelly (RJ) yields and RJ in each queen cell as well as acceptances of queen cell cups of three lines of Western honeybees (Apis melliffera Lingistica) were conducted by using a genotypic model for analyzing the genetic and non-genetic effects. Analytis approaches of conventional and conditional variance and correlation were employed to evaluate the developmental behavior of honeybee colony's RJ producing ability. The results indicated that significant variance due to genotypic effect was detected for colony's RJ yields and RJ in each queen cell cup at all stages, while the acceptance of queen cell cups was found variance significantly at 10 stages of its total 11. It meant that the three traits were dominated by genotypic effect. Significant conditional variances were found at some stages when no unconditional one being detected. Correlation analysis between same trait at different stages indicated that the significant coefficients always existed due to genotypic effects for RJ yields and RJ in each queen cell cup. Although the significant coefficients of the acceptance of queen cell cup at different stages were found at most of the stages, the coefficients were not found in come of the stages. These results indicated that the genotypic effects dominated in early stages of colony's RJ yield and RJ in each queen cell cup influencing its late stages in the same way, but the acceptance of queen cells cup was not. The analysis of correlation between different pair of traits showed that the coefficients between colony's RJ yields and RJ in each queen cell cup were significant at all stages, and while the coefficients between colony's RJ yields and the acceptance of queen cell cups were not always significant at all stages. It was because the genotypic effects dominated the former pair of traits had harmonious effects, while the later pair of traits were not.

[Research on morphological genetic marker of honeybee (Apis melli fera ligustica) in royal jelly production performance].

The lengths of hypopharyngeal glands (HG) from the left and right side were determined in 19 workers of honeybee(Apis mellifera ligustica). There were no significant differences (P<0.05) in length between the left and the right in one worker's hypopharyngeal gland. Three hundred and thirty workers were collected from eleven colonies of "ZND No.1" Italian honeybee(Apis mellifera ligustica) respectively. Head weight, body weight, ratio between head weight and body weight,bursa number and length of hypopharyngeal gland(HG)were tested in these samples. Royal jelly productions were determined during the flowing period of rape and Chinese milk retch from March 30 to April 26 in Chun'an County of Zhejiang Province in 2001. The correlation analysis between royal jelly production and head weight, ratio between head weight and body weight, bursa number of HG,and length of HG were conducted. The correlation coefficient between royal jelly production and length of HG was the largest. The correlation coefficient between royal jelly production and bursa number was the second. It was suggested that the length of HG could be used as one of genetic markers for the production performance of royal jelly.

Expression of apalbumin1 of Apis cerana cerana in the larvae of silkworm, Bombyx mori.

Royal jelly (RJ) is a thick, milky material produced by both the hypopharyngeal and the mandibular glands of nurse honeybees. The main proteins of RJ, named apalbumins or major royal jelly proteins (MRJPs), have multiple biological functions. Apalbumin1 is the most abundant glycoprotein of RJ. In this study, Bacmid- apalbumin1 was constructed for Apis cerana cerana using the newly established Bac-to-Bac/BmNPV baculovirus expression system (BES). This procedure allowed us to obtain the recombinant A. cerana cerana ( Acc) apalbumin1 (r Accapalbumin1) from the hemolymph of silkworm larvae through the BmNPV bacmid system, 96 h postinfection. The r Accapalbumin1 was then purified by Ni-NTA spin columns and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A 55 kDa protein with good solubility was then obtained. The peptide Ile-Phe was identified from trypsin production of r Accapalbumin1. Such a peptide has been reported to have an antihypertensive ability. Our results have therefore potential applications in biomedical research and open new perspectives for the study of apalbumins.

Possible effect of 30K proteins in embryonic development of silkworm Bombyx mori.

The silkworm Bombyx mori possesses a 30K protein family of 3x10(4) Da, the biological functions of which have not been fully identified. The relationship between the 30K protein family and the embryonic development of temperature sensitive sex-linked mutant strain of silkworm was investigated by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results show that protein spots 1-5 of the 30K protein family, mainly existing in normal strain, are possibly related to embryonic development. The early consumption of a 30K protein named 6G1-30K-1 and the accumulation of 30K proteins named 6G1-30K-3 and 6G1-30K-4 are likely caused by the destruction of physiological balance in normal embryonic development, which may lead to lower hatchability of the temperature sensitive strain. The results suggest that reasonable metabolism of 30K proteins is a prerequisite for the embryo's normal development.