Research progress of risk factors of Leber’s hereditary optic neuropathy / 中华眼底病杂志

Leber’s hereditary optic neuropathy (LHON) is a paradigm maternal hereditary eye disease, mainly involving the retinal and macular fibers of the optic disc in the anterior ethmoid plate of the sclera. LHON has the characteristics of sex bias among males and incomplete penetrance. Primary mitochondrial DNA mutations m.11778G>A, m. 14484T>C, m.3460G>A are the molecular basis of LHON. However, other risk factors, such as secondary mitochondrial DNA mutations, mitochondrial haplotypes, nuclear modification genes, estrogen, vitamin B12 and environmental factors, work together to affect its phenotypic expression. The clinical diagnosis of LHON mainly limited to the detection of the primary mutation site of mitochondrial DNA. Therefore, comprehensive analysis of multiple risk factors of LHON will facilitate to construct multi-dimensional model of prevention, diagnosis and treatment system, which provide accurate and individualized medical services for patients. These may alleviate the incidence in LHON families. It also provides new ideas and different angles for the in-depth study of the pathogenesis of LHON.

The Characteristics of T Lymphocyte Reconstitution after Haploid Hematopoietic Stem Cell Transplantation in SAA and Its Relationship with aGVHD / 中国实验血液学杂志

OBJECTIVE@#To investigate the recovery characteristics of T cell subsets in patients with severe aplastic anemia (SAA) who received haploid hematopoietic stem cell transplantation(HSCT) and its relationship with acute graft-versus-host disease(aGVHD).@*METHODS@#The clinical data of 29 SAA patients who received haploid hematopoietic stem cell transplantation in the department of hematology, Shanxi Bethune Hospital from June 2018 to January 2022 were retrospectively analyzed. The absolute counts of CD3+T, CD4+T, CD8+T lymphocytes and the ratio of CD4+T/CD8+T lymphocytes in all patients before transplantation, 14, 21, 30, 60, 90 and 120 days after transplantation were analyzed. The proportion of T lymphocytes was compared in the non-aGVHD group, the grade Ⅰ-Ⅱ aGVHD group and the grade III-IV aGVHD group.@*RESULTS@#The counts of all T cells in 27 patients were far below the normal level at 14 and 21 days after transplantation, but there was obvious heterogeneity. There was a certain relationship between T cell immune reconstitution and conditioning regimen, age, and immunosuppressive treatment before transplantation. CD3+T cells showed a steady upward trend at 30, 60, 90, and 120 days after transplantation, and returned to the normal levels at 120 days after transplantation; faster recovery of CD4+T cells was closely related to aGVHD, which was at 30, 60, 90, 120 days after transplantation showed a slow upward trend, and which was still far below the normal level of 120 days after transplantation. CD8+T cell counts began to recover at 14 and 21 days after transplantation, and the recovery was earlier than the CD4+T cells, and its recovery speed was rapid 30 and 60 days after transptantation, which showed an upward trend and exceeded the normal levels 90 days after transplantation. Since CD8+ T cells reconstituted quickly, while the CD4+ T cells reconstitution was slowly, which made the long-term CD4+T/CD8+T cell ratio after transplantation was inverted . Compared with the non-aGVHD group, the absolute counts of CD3+T, CD4+T, and CD8+T cells in the aGVHD group were significantly higher than those in the non-aGVHD group at each time period after transplantation. In the aGVHD group, grade Ⅲ-Ⅳ aGVHD occurred more frequently in the early post-transplantation period (within 14-21 days), the grade Ⅰ-Ⅱ aGVHD group mostly occurred within 30-90 days after transplantation, and CD3+T, CD4+T, CD8+T cell counts in the grade Ⅲ-Ⅳ aGVHD group were significantly higher than those in the grade Ⅰ-Ⅱ aGVHD group; and the greater the proportion of CD4+T, the more severe the degree of aGVHD.@*CONCLUSION@#The speed of T cell immune reconstitution after SAA haploid transplantation is different, which is related to the conditioning regimen, age, and immunosuppressive therapy before transplantation. The rapid recovery of CD4+ T cells is closely related to the occurrence of aGVHD.

Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells

The use of two inhibitors of Mek1/2 and Gsk3β (2i) promotes the generation of mouse diploid and haploid embryonic stem cells (ESCs) from the inner cell mass of biparental and uniparental blastocysts, respectively. However, a system enabling long-term maintenance of imprints in ESCs has proven challenging. Here, we report that the use of a two-step a2i (alternative two inhibitors of Src and Gsk3β, TSa2i) derivation/culture protocol results in the establishment of androgenetic haploid ESCs (AG-haESCs) with stable DNA methylation at paternal DMRs (differentially DNA methylated regions) up to passage 60 that can efficiently support generating mice upon oocyte injection. We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations. Furthermore, we demonstrate that TSa2i-treated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation. Strikingly, AG-haESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells, in part through the enhanced proliferation of H19-DMR hypermethylated cells. Together, we establish AG-haESCs that can long-term maintain paternal imprints.

Clinical Study of Haploid Allogeneic Hematopoietic Stem Cell Transplantation Combined with Post-transplant Cyclophosphamide in Severe Aplastic Anemia Patients / 中国实验血液学杂志

OBJECTIVE@#To evaluate the clinical effect of haploid allogeneic hematopoietic stem cell transplantation(haplo-HSCT) in the treatment of severe aplastic anemia (SAA), and to explore the efficacy different between post-transplant cyclophosphamide (PT/Cy) and standard-dose ATG.@*METHODS@#The clinical data of 38 patients with SAA in our hospital from January 2012 to December 2019 were collected and retrospectively analyzed. The efficacy was evaluated. The patients with haplo-HSCT were divided into low-dose ATG combined with PT/Cy group and standard-dose ATG group, and the blood cell hematopoietic reconstruction time, GVHD incidence, mortality and survival time of the patients in the two groups was compared.@*RESULTS@#Among the 32 patients, hematopoietic reconstitution were detected in 9375%(30/32) recipients. The median time of neutrophil and platelet engraftment was 15(10-22) days and 13(7-30) days, respectively. The incidence of GVHD was 21.89%, the incidence of infection was 93.75%, and the 2-year overall survival rate was 84.38%. The hematopoietic reconstitution time, incidence of GVHD, mortality rate and survival time were no statistical differences between the patients in the two groups(all P>0.05).@*CONCLUSION@#Haplo-HSCT is an effective method for the treatment of SAA，low-dose ATG combined with PT/Cy can lighten the economic burden on patients, it would be a feasible treatment plan for SAA with light side effect.

Effect of modified FC/ATG preconditioning on allogeneic hematopoietic stem cell transplantation for severe aplastic anemia / 中华器官移植杂志

Objective:To evaluate the efficacy and safety of modified FC/ATG pretreatment in the treatment of severe aplastic anemia(SAA).Methods:From June 2012 to June 2020, clinical data of 64 patients with severe aplastic anemia undergoing allogeneic hematopoietic stem cell transplantation with modified FC/ATG(Flu 30 mg·m -2·d -l, -5~-2 d、CTX 50 mg·kg -1·d -1~-2 d、ATG: 2.5 mg·kg -1·d -1, -5~-2 d) pretreatment were retrospectively analyzed.There were MSD-HSCT ( n=29) and Haplo-HSCT ( n=35). Results:One patient died of intracerebral hemorrhage before transplantation and the remainders were completely implanted.During a median follow-up period of 14.5(1-95) months, overall survival (OS) rate of 92.2%.It was significantly higher than OS rate of 67.2% in the treatment of SAA by foreign pretreatment regimens containing low-dose TBI.And pretreatment scheme containing FC+ BU/TBI had an OS of slightly >91.3% in the treatment of SAA.The 3-year OS rates were 85.7% and 93.5% in Haplo-HSCT and MSD-HSCT groups ( P=0.058). The OS rate of SAA Haplo-HSCT/MSD-HSCT group was similar to that of " Beijing Protocol" (BU/CY+ ATG) (89%, 91%). The viral infection rates of EB and CMV were significantly higher in haplo-HSCT group than those in MSD-HSCT group and inter-group difference was statistically significant ( P<0.05). However, univariate analysis showed that two groups had no effect on survival time ( P=0.403, P=0.132). Univariate analysis showed that survival time was significantly associated with the presence of Ⅲ-Ⅳ° aGVHD and the presence of severe complications ( P=0.007, P=0.001). Further multivariate analysis revealed that severe complication was an independent risk factor for survival ( P=0.003). Conclusions:The efficacy of improved FC/ATG pretreatment in the treatment of SAA in MSD-HSCT or Haplo-HSCT is higher than other domestic and international pretreatment schemes in OS rate, safety and effectiveness.Onset of severe complication and association with Ⅲ ~ Ⅳ ° aGVHD are the influencing factors for patient survival.The efficacy of Haplo-HSCT group is similar to that of MSD-HSCT group.It may be employed as an alternative donor for SAA patients without fully congruent donors.

Genetic characterization of maize doubled haploid lines for Fusarium stalk rot caused by Fusarium verticillioides

Fusarium stalk rot disease (FSR) of maize caused by Fusarium verticillioides (Sacc.) Nirenberg is becoming an important biotic production constraint in many of the major maize growing areas causing substantial yield losses. Inbreds are preferred as parents in hybrid development owing to homozygous nature and high heterotic ability. Double haploid (DH) technology has emerged as a significant milestone. A total of 339 DH lines were generated from two inbred lines, VL1043 (susceptible) and CM212 (resistant), through in vivo haploid induction method. The 339 DH lines along with parents were phenotyped for their response to the FSR at the College of Agriculture, V. C. Farm, Mandya, India during summer, kharif and rabi seasons of the 2019–2020. Best linear unbiased predictors (BLUPs) were estimated for the FSR disease scores over three seasons. A wide range of BLUP scores of three to nine indicated the presence of higher variation for response of DH lines to FSR disease. The higher estimates of standardized range (1.31) and phenotypic coefficient of variation (19.80) also displayed higher variability. Nine lines were moderately resistant and 188 exhibited moderately susceptible reaction. The distribution of DH lines was positively skewed (1.34) and platykurtic (2.31) which suggested complementary epistasis and involvement of large number of genes in the disease expression.

Optimization and clinical application of the preconditioning regimen in the treatment of severe aplastic anemia with interfamilial haploidentical peripheral hematopoietic stem cell transplantation: A report of 26 cases / 解放军医学杂志

Objective To evaluate the safety and efficacy of an further optimized pretreatment protocol for the treatment of severe aplastic anemia (SAA) by haploidentical peripheral hematopoietic stem cell transplantation. Methods From July 2009 to July 2019, 26 SAA patients in the Affiliated Hospital of North China University of Technology were treated with haploidentical peripheral blood stem cell transplantation. The "Beijing protocol" of haploid transplantation for treating SAA is modified busulfan (BU)/cyclophosphamide (CY) + rabbit anti thymocyte globulin (ATG) regimen. We based on it and further refined it. Here's how it works: (1) According to the different volumes of hematopoietic tissue in bone marrow biopsy before transplantation, different methods for clearance of residual hematopoietic cells were established in SAA patients. If bone marrow hematopoietic tissue volume<10%, the bone marrow was pretreated with Beijing protocol of BU/CY+ATG. If 10% ≤ bone marrow hematopoietic tissue volume ≤25%, the dose of BU for 1 day was added on the basis of the original protocol: Increase the dose of BU to 9.6 mg/kg (intravenous drip in 3 days), which became a modified BU/CY+ATG transplantation preconditioning protocol. (2) According to the diagnosis of the disease, the transplantation pretreatment was designed: Patients with SAA-PNH syndrome or with PNH alone were treated with further modified BU/CY+ATG transplantation preconditioning protocol, and the BU dose was set as 9.6 mg/kg (intravenous drip in 3 days). Transplantation way: The "Beijing protocol" is use of bone marrow plus peripheral blood hematopoietic stem cell. Based on it, the protocol was modified to simple peripheral blood hematopoietic stem cell transplantation. Results 22 of 26 SAA patients underwent hematopoietic reconstruction. The patients were followed up until December 2019, and the results were as follows: During a median follow-up period of 48 (5-122) months, 5 patients died, 4 of whom suffered transplant-related deaths (15.4%, 4/26), and 1 of whom was due to central nervous system infection (3.8%, 1/26). The 3-year overall survival rate (OSR) was 84.2%, the 3-year progression-free survival (PFS) was 72.6% (SAA I: 100.0%, SAA-PNH: 100.0%, SAA II: 72.2%). Conclusions The further improved BU/CY+ATG transplantation preconditioning scheme is safe and effective in SAA with haploid peripheral blood stem cell transplantation. It is suitable not only for SAA I, SAA II, but also for patients with PNH clone, thus being worth wider clinical aplplication.

Construction, characteristics and high throughput molecular screening methodologies in some special breeding populations: a horticultural perspective

Advanced marker technologies are widely used for evaluation of genetic diversity in cultivated crops, wild ancestors, landraces or any special plant genotypes. Developing agricultural cultivars requires the following steps: (i) determining desired characteristics to be improved, (ii) screening genetic resources to help find a superior cultivar, (iii) intercrossing selected individuals, (iv) generating genetically hybrid populations and screening them for agro-morphological or molecular traits, (v) evaluating the superior cultivar candidates, (vi) testing field performance at different locations, and (vii) certifying. In the cultivar development process valuable genes can be identified by creating special biparental or multiparental populations and analysing their association using suitable markers in given populations. These special populations and advanced marker technologies give us a deeper knowledge about the inherited agronomic characteristics. Unaffected by the changing environmental conditions, these provide a higher understanding of genome dynamics in plants. The last decade witnessed new applications for advanced molecular techniques in the area of breeding,with low costs per sample. These, especially, include next-generation sequencing technologies like reduced representation genome sequencing (genotyping by sequencing, restriction site-associated DNA). These enabled researchers to develop new markers, such as simple sequence repeat and single- nucleotide polymorphism, for expanding the qualitative and quantitative information onpopulation dynamics. Thus, the knowledge acquired from novel technologies is a valuable asset for the breeding process and to better understand the population dynamics, their properties, and analysis methods

Polyploidy as a chromosomal component of stochastic noise: variable scalar multiples of the diploid chromosome complement in the invertebrate species Girardia schubarti from Brazil / Poliploidia como componente cromossômico de ruído estocástico: variações escalares múltiplas do componente cromossômico diploide do invertebrado Girardia schubarti ocorrente no Brasil

Abstract Chromosome stoichiometry, a form of genetic plasticity, specifically refers to variation in the standard diploid genomic composition of an individual or species. In the present work, freshwater planarians (Girardia schubarti) were analyzed to recognize variations in chromosomal stoichiometry especially of complete ploidal change between specimens, within specimens and between cells within specimens and any relations they might have with selected components of phenotypic plasticity. Homoploid polyploids for the group reached rational scalar multiples (e.g. tetraploids) or irrational scalar multiples (e.g. triploids). Karyotypic mosaics emerged where individual cells presented polyploid multiples in arithmetic and geometric progressions. Ploidal multiplicity, a chromosomal component of stochastic noise, had positive phenotypic effects (increased dimensions) on morphologic criteria of body length, body width and dorsal surface reflecting a significant genotypic plasticity (GP) and robust phenotypic plasticity (PP). Variable but significant association of genotypic plasticity with robust phenotypic variance suggests kinetics of phenotypic homeostasis that is species-specific permitting phenotypic adaptability to environmental variables by means of GP. That association is diminished, deactivated or lost in more advanced and more complex organisms.

Resumo A estequiometria cromossômica, uma forma de plasticidade genotípica, representa variações na composição genômica diploide de um indivíduo ou espécie. Planárias límnicas (Girardia schubarti) foram analisadas para verificar a estequiometria cromossômica, especialmente alterações na ploidia entre espécimes, em cada espécime e entre células do mesmo espécime, além de relações dessas alterações com a plasticidade fenotípica. Espécimes poliploides homoploides apresentaram múltiplos escalares racionais ou irracionais, tais como triploides. Mosaicos cariotípicos ocorreram quando células apresentaram poliploides múltiplos em progressões aritméticas e geométricas. Nas planárias estudadas, a multiplicidade ploidal, um componente cromossômico de ruído estocástico, apresentou efeitos fenotípicos positivos, causando aumento das dimensões dos indivíduos, tais como comprimento corporal, largura do corpo e superfície dorsal, indicando plasticidade genotípica (GP) significativa e plasticidade fenotípica (PP) robusta. Associações significativas da plasticidade genotípica com variâncias fenotípicas robustas, embora variáveis, sugerem que a homeostase fenotípica, a qual é espécie-específica, possibilita adaptações a variáveis ambientais através da GP. Tal associação apresenta-se reduzida, desativada ou perdida em organismos mais complexos.

Construction of a murine model of chronic graft-versus-host disease by haplo-identical spleen cell infusion / 中华器官移植杂志

Objective For providing experimental platform of chronic graft-versus-host disease (cGVHD),to establish a mouse model by haplo-identical spleen cell infusion.Methods The donor male mice (Balb/cH-2d) and the recipient (Balb/c C57BL/6) F1 H2-d/b (CB6F1) female mice were randomly divided into four groups:3 experimental groups injected with 3 107,6 107 and 9 107 spleen cells,respectively,while the control group received RPMI 1640 solution.H-2d and H-2b were checked to analyze the chimerism in bone marrow cells.Body mass,figure,cutaneous manifestation and survival of recipient mice were observed and scored every 3 days.Pathologic changes of target organs were observed and scored.Results Injection of 6 107 and 6 107 splenocytes in the recipient mice resulted in a chronic disease with a low level of parental cell engraftment steadily.As compared with 3 107 group,the incidence of cGVHD in 6 107 and 9 107 groups were significantly increased (P ＜0.01).But there was no significant difference between 6 107 and 9 107 groups (P＞0.05).Conclusion A murine model of cGVHD after haplo-identical spleen cell infusion of donor is successfully established by injection of 6 107 and 9 107 spleen cells.

Assessment of Arabidopsis thaliana CENH3 promoter in Brassica juncea for development of haploid inducer lines.

Centromeres are epigenetically specified by the centromeric histone H3 protein (CENH3). The timing and level of expression of CENH3 is tightly regulated to match the demands of the host cell. So far in plants, only CENH3 promoter of Arabidopsis thaliana (L.) Heynh. has been characterized. However, whether CENH3 promoters retain their characteristic mode of regulation in other species remains to be established. In the present study, activity of AtCENH3 promoter was investigated using reporter gene assay in Brassica juncea (L.) Czern. A 1156 bp promoter fragment of AtCENH3 gene (At1g01370) including the first 111 nucleotides of the coding sequence was amplified and cloned into the pORE-R2 binary vector to ensure translation fusion with the uidA coding sequences. The Agrobacterium tumefaciens strain GV3101 harbouring the recombinant construct was used to transform B. juncea cv. RLM198 hypocotyl explants. Histochemical assay of T0 and T1 transgenics showed GUS expression in shoot apical meristem, leaf, sepal, flower pedicel and root tip. Intense GUS expression was observed in meristematic tissues, particularly at shoot and root apices. However, mature leaves, flowers, pollen and ovules exhibited very low or no GUS expression. Our results showed that AtCENH3 promoter regulates cognate gene expression in Brassica juncea as it does in A. thaliana, and hence a suitable candidate for developing haploid inducer line in B. juncea.

Development of Maize Haploid Inducer Lines and Doubled Haploid Lines in Pakistan.

The work on maize doubled haploid development has started at the Maize Research Station, Ayub Agricultural Research Institute, Faisalabad, Pakistan during 2010 in collaboration with University of Agriculture Faisalabad, Pakistan. The aim of current study was to develop locally adopted maize haploid inducer lines utilizing cheap and easy source of Stock6 and indeterminate gametophyte mutant ig1 gene lines imported from Maize genetic Coop Stock Centre. The lines were later utilized to produce doubled haploid inbred lines. The environmental conditions of Faisalabad are extreme in nature. In spring crop during pollination period temperature may reach up to 45°C. Therefore, locally adopted haploid inducers are needed. Best performing local inbred lines were screened having branched heavy tassel, bold seed, good pollen shedding ability and vigor. These lines were utilized as female donor parent while imported lines as recurrent pollen parent in back cross breeding program until BC4 generation. These lines were selfed twice until BC4F2 and evaluated for haploid induction rate (HIR). Lines with high HIR were further intercrossed to achieve maximum transgressive segregation. Mass selection for the adaptation traits was exercised for individual F2 plants followed by ear-to-row plantation of selected progeny. Four best haploid inducer lines with HIR up to 5% having very good tassel size, height and heat stress tolerance were selected in 2014. Haploid seeds collected in different induction crosses until 2012, were used for colchicine doubling treatment. The doubling percentage of the haploid plants was very low 0.15% and out of 1000 treated seedling 15 survived and only 5 reached up to maturity, where only one D0 cob was harvested. This was successfully selfed and was grown in three different locations for next two seasons and there was no segregation in the successive generations. Developing countries that cannot afford costly haploid inducer lines can work on Stock6 and other cheap sources available free of cost and can develop their own haploid inducer lines well adapted to their own climatic conditions.

Factors Affecting Embryogenic Callus Production and Plant Regeneration in Anther Culture of Bupleurum chinense / 中草药·英文版

Objective To evaluate the influences of the genotypes,anther developmental stages,and cultural conditions on the efficiency of embryogenic callus induction and plant regeneration in the anthers culture of Bupleurum chinense.Methods The different effects such as four genotypes,plant growth regulators,and temperature condition were compared in the experiments.The histological study was performed with the process of the anther culture.Results The highest inducing rate of embryogenic calli were achieved for the genotypes Zhongcaiyihao(ZCYH),Z4,and Z5 at the early-to middle-uninucleate stages,except for genotype ZPM1 at the tetrad stage.Cold pretreatment increased the production of the embryogenic callus,in which 4-day cold pretreatment improved the production of embryogenic callus from 0% to 2.2% and 5.0% for genotypes ZPM1 and ZCYH,respectively.No embryogenic callus was induced in the medium containing less than 0.75 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D).The highest regeneration rate (34.6%)was obtained in 1/2 MS salts regeneration medium supplemented with 0.1 mg/L 6-benzylmaminopurine (BA).The low concentration of BA was able to promote the embryogenic callus formation and subsequent plantlet regeneration via somatic embryogenesis.Chromosome counting of regenerated plantlets showed mostly diploid plant (2n = 12)with only one haploid plant(n = 6).Because of the low rate of microspore embryo formation,we only tracked the process of embryogenesis from the connective tissue,instead of microspore by histological observations.Conclusion This study establishes an efficient system for embryogenic callus induction and plant regeneration system.This is the first report on the haploid plantlet through the anther culture orB.chinense.

Genotypic differences in proembryoid development and green plantlets regeneration through androgenesis in barley varieties / Diferenças genotípicas no desenvolvimento de pró-embrióides e regeneração de plântulas verdes via androgênese em genótipo de cevada

The development of in vitro haploid plants followed by spontaneous or induced genome duplication allows to achieve, in one generation, the recovery of total homozygosis. The efficiency of the haplodiploidization process through in vitro anther culture of barley is variable among genotypes. This study was aimed at determining the androgenetic response of nine barley genotypes from the breeding program of Embrapa Trigo, analysing proembryoid development and green plantlets regeneration in anthers cultivated in vitro. Cultivar 'BR2' presented the highest average of proembryoids (104/anther) and 'MN698' presented the highest average of green plantlets (0,41/anther). There was a significant variation among the average values of barley genotypes for embryo formation and green plantlets regeneration, making possible the selection to combine androgenetic capacity and good agronomic traits.

A obtenção, na cevada, de plantas haplóides in vitro e a posterior duplicação natural ou artificial do genoma permitem alcançar a homozigose completa, em uma geração. A eficiência da haplodiploidização pela cultura de anteras é variável entre os genótipos. Foi avaliada a resposta androgenética através da formação de pró-embrióides e da regeneração de plântulas verdes em nove cultivares do programa de melhoramento de cevada da Embrapa Trigo, em anteras cultivadas in vitro. A cultivar "BR2" apresentou maior média de pró-embrióides (104/antera), enquanto "MN698" mostrou a maior média de plântulas verdes (0,41/antera). Houve variação significativa entre os valores médios dos genótipos em relação à formação de pró-embrióides e à regeneração de plântulas verdes, indicando a possibilidade de seleção para combinar a capacidade androgenética com boas características agronômicas.

Characters that differ between diploid and haploid honey bee (Apis mellifera) drones

Diploid males have long been considered a curiosity contradictory to the haplo-diploid mode of sex determination in the Hymenoptera. In Apis mellifera, 'false' diploid male larvae are eliminated by worker cannibalism immediately after hatching. A 'cannibalism substance' produced by diploid drone larvae to induce worker-assisted suicide has been hypothesized, but it has never been detected. Diploid drones are only removed some hours after hatching. Older larvae are evidently not regarded as 'false males' and instead are regularly nursed by the brood-attending worker bees. As the pheromonal cues presumably are located on the surface of newly hatched bee larvae, we extracted the cuticular secretions and analyzed their chemical composition by gas chromatograph-mass spectrometry (GC-MS) analyses. Larvae were sexed and then reared in vitro for up to three days. The GC-MS pattern that was obtained, with alkanes as the major compounds, was compared between diploid and haploid drone larvae. We also examined some physical parameters of adult drones. There was no difference between diploid and haploid males in their weight at the day of emergence. The diploid adult drones had fewer wing hooks and smaller testes. The sperm DNA content was 0.30 and 0.15 pg per nucleus, giving an exact 2:1 ratio for the gametocytes of diploid and haploid drones, respectively. Vitellogenin was found in the hemolymph of both types of imaginal drones at 5 to 6 days, with a significantly lower titer in the diploids.