From the ethyl acetate extract of Murraya koenegii (Rutaceae) leaves, isomahanine (1) and mahanine (2) were isolated that showed antibacterial activity towards Flavobacterium columnare and Streptococcus iniae which ca...From the ethyl acetate extract of Murraya koenegii (Rutaceae) leaves, isomahanine (1) and mahanine (2) were isolated that showed antibacterial activity towards Flavobacterium columnare and Streptococcus iniae which caused columnaris disease and streptococcosis respectively. Isomahanine was found to have the strongest activity against F. columnare (isolate ALM-00-173) and S. iniae (isolate LA94-426) based on 24-h 50% inhibition concentration (IC50) and minimum inhibition concentration (MIC). Although compound (7), a nicotinamide isolated from Amyris texana had the lowest MIC (2.8 ± 0 mg/L) of any of the test compounds against F. columnare, the 24-h IC50 of 14.8 ± 0.6 mg/L was higher than that of isomahanine and subsequently the 24-h IC50 RDC values for (7) were almost a magnitude of order higher than those obtained for isomahanine. Isomahanine also had the strongest activity against S. iniae, with a 24-h IC50 of 1.3 ± 0.1 mg/L and MIC of 3.5 ± 0 mg/L, respectively.展开更多
The effect of penicillin-G on Flavobacterium psychrophilum, the cause of bacterial coldwater disease, was evaluated in 15 min and overnight exposures. Separate tests evaluated the effect of increasing doses of penicil...The effect of penicillin-G on Flavobacterium psychrophilum, the cause of bacterial coldwater disease, was evaluated in 15 min and overnight exposures. Separate tests evaluated the effect of increasing doses of penicillin-G to rainbow trout Oncorhynchus mykiss eggs, fingerlings, and adults. Rainbow trout eggs were exposed for 1 h to penicillin doses of 0, 250, 500, 1,000, 10,000, 50,000, or 100,000 IU/mL. Mean percent hatch ranged from 64.6% to 75.1%, and did not significantly differ among the treatments. Fingerlings were injected intraperitoneally (i.p.) with 200 μL of 0, 1, 10, 100, 1,000, 10,000, 100,000, or 500,000 IU/mL. Probit analysis resulted in a LD10 (lethal dose for 10% of injected fish) of 52,868 IU/mL (95% confidence limits: 31,522 - 69,490 IU/mL) and a LD50 of 131,466 IU/mL (113,095 - 157,878 IU/mL). Brood stock were injected (i.p.) with 200 μL of penicillin at concentrations of 0 (control), 10,000, 50,000, 100,000, 200,000, 400,000, or 800,000 IU/mL. Mortality ranged from 0% to 7% (50,000 IU/mL treatment) and did not significantly differ among treatments. In 28 h exposure tests, penicillin concentrations of ≥ 333 IU/mL were required to completely suppress growth of F. psychrophilum. In 15 min exposures, ≥ 10,000 IU/mL were needed to achieve the same result. Tests indicated that rainbow trout eggs can tolerate 1 h exposures to penicillin-G concentrations as high as 100,000 IU/mL, and that brood fish can tolerate injections of at least 800,000 IU/mL. Fingerling data however, suggested that injection of doses greater than 10,000 IU/mL can be toxic. The data suggests rainbow trout eggs or brood can tolerate high doses of penicillin-G that could be used for controlling the transfer of F. psychrophilum to hatcheries receiving eggs.展开更多
From time immemorial, human beings have used pigments made from vegetables, fruits, superior plants, animal tissues and cereals. One of the greatest sources of pigments is the bacterium that, with the use of the moder...From time immemorial, human beings have used pigments made from vegetables, fruits, superior plants, animal tissues and cereals. One of the greatest sources of pigments is the bacterium that, with the use of the modern technology, has increased the production of metabolites of interest. The microbiological production of carotenoids has not been optimized to obtain pigment production quantities of pigments and carotenoids recovery that lower production costs. The aim of this work was to design a Zeaxanthin production process with Flavobacterium sp. immobilized cells in a fluidized bed bioreactor. An optimum culture medium for Zeaxanthin production in stirred flasks (2.46 g·L–1) was obtained. Furthermore, optimum process conditions for a maximum yield of Zeaxanthin production, by fluidized bed bioreactor, were established. A statistical analysis showed that the most significant factors were air flow, pH and NaCl concentration (4.5 g·L–1). In this study a maximum Zeaxanthin production of 3.8 g·L–1 was reached. The highest reported yield to date was 0.329 g·L–1.展开更多
Columnaris (caused by Flavobacterium columnare) is one of the most common bacterial diseases affecting the pond-raised channel catfish (Ictalurus punctatus) in the southeastern United States of America resulting in an...Columnaris (caused by Flavobacterium columnare) is one of the most common bacterial diseases affecting the pond-raised channel catfish (Ictalurus punctatus) in the southeastern United States of America resulting in annual losses of millions of dollars. As part of our continuing effort to discover environmentally benign compounds for the control of columnaris disease, acyl derivatives of phloroglucinol were synthesized and tested against F. columnare using a rapid bioassay. Among the analogs that were tested, diacyl analogs showed very high antibacterial activity against F. columnare in the laboratory bioassay. Diisovaleryl and diisobutyryl analogs were found to have the strongest activity against F. columnare (isolate ALM-00-173) based on 24-h 50% inhibitory concentration (IC50) and minimum inhibitory concentration (MIC). Diisovaleryl and diisobutyryl analogs had IC50 values 0.82 mg/L and 0.80 mg/L, respectively, whereas the drug control florfenicol had an IC50 value of 0.81 mg/L. Diisovaleryl and diisobutyryl analogs also had 24-h relative-to- drug-control IC50 values around 1.0 indicating activities similar to florfenicol, which is included in medicated feed and is one of the current management approach for columnaris.展开更多
Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermo...Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.展开更多
Flavobacterium columnare, the etiological agent of columnaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable marker...Flavobacterium columnare, the etiological agent of columnaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers (chloramphenicol and spectinomycin resistance) for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.展开更多
Flavobacterium columnare is the pathogenic agent of columnaris disease in aquaculture. Using a recently developed gene deletion strategy, two genes that encode the Glyco_hydro_19 domain(GH19 domain) containing protein...Flavobacterium columnare is the pathogenic agent of columnaris disease in aquaculture. Using a recently developed gene deletion strategy, two genes that encode the Glyco_hydro_19 domain(GH19 domain) containing proteins, g hd-1 and ghd-2, were deleted separately and together from the F. columnare G 4 wild type strain. Surprisingly, the single-, Δg hd-1 and Δg hd-2, and double-gene mutants, Δ ghd-1 Δ ghd-2, all had rhizoid and non-rhizoid colony morphotypes, which we named Δ ghd-1, Δ ghd-2, Δ ghd-1 Δ ghd-2, and NΔ ghd-1, NΔ ghd-2, and NΔ ghd-1 Δ ghd-2. However, chitin utilization was not detected in either these mutants or in the wild type. Instead, skimmed milk degradation was observed for the mutants and the wild type; the non-rhizoid strain NΔg hd-2 exhibited higher degradation activity as revealed by the larger transparent circle on the skimmed milk plate. Using zebrafish as the model organism, we found that non-rhizoid mutants had higher LD5 0 values and were less virulent because zebrafish infected with these survived longer. Transcriptome analysis between the non-rhizoid and rhizoid colony morphotypes of each mutant, i.e., NΔg hd-1 versus(vs) Δ ghd-1, NΔ ghd-2 vs Δ ghd-2, and NΔ ghd-1 Δ ghd-2 vs Δ ghd-1 Δ ghd-2, revealed a large number of dif ferentially expressed genes, among which 39 genes were common in three of the pairs compared. Although most of these genes encode hypothetical proteins, a few molecules such as phage tail protein, rhs element Vgr protein, thiol-activated cytolysin, and Ton B-dependent outer membrane receptor precursor, expression of which was down-regulated in non-rhizoid mutants but up-regulated in rhizoid mutants, may play a role F. columnare virulence.展开更多
Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide.Many studies have focused on the identification of protective antigens to aid in the development of nov...Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide.Many studies have focused on the identification of protective antigens to aid in the development of novel vaccines against the disease.In this study,an immunoblotting approach was employed to identify immunogenic outer membrane proteins(OMPs)from F.columnare in two-dimensional electrophoresis(2-DE)map gels using antibacterial sera obtained from grass carp(Ctenopharyngodon idella),and anti-grass carp-recombinant Ig(rIg)monoclonal antibodies.Five unique immunogenic proteins,including the gliding motility lipoprotein GldJ(GldJ),hypothetical protein FCOL_13420(Fco1),lipoprotein(Lip),F0F1 ATP synthase subunit beta(F0f1)and outer membrane efflux protein precursor(Omep),were characterized.Over-expression of these proteins in Escherichia coli DE3,and their immunogenicity and protective efficacy were evaluated in grass carp.The relative percent survival(RPS)of the groups immunized separately with recombinant GldJ,Lip and Omep was 72%,64%and 68%,respectively when compared to control fish.Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F.columnare.The results suggest that GldJ,Lip and Omep are major protective antigens and may be considered as novel candidates in the development of vaccines against columnaris disease in fish.展开更多
Male-killing is 1 of 4 known strategies that inherited parasitic endosymbionts have evolved to manipulate their host＇s reproduction. In early male-killing, infected male offspring are killed early in embryogenesis. W...Male-killing is 1 of 4 known strategies that inherited parasitic endosymbionts have evolved to manipulate their host＇s reproduction. In early male-killing, infected male offspring are killed early in embryogenesis. Within the Insecta, male-killing bacteria have been found in a wide range of hosts. The Coccinellidae families of beetles, better known as ladybirds, are particularly prone to male-killer invasion. In samples of the coccinellid, Coc- cinula crotchi, from Japan, a new male-killing bacterium was revealed by phenotypic assay. Molecular genetic analysis revealed the identity to be a tetracycline-sensitive Flavobac- terium that causes female-biased offspring sex ratio. Furthermore, that Flavobacterium strain was found to be closely related to the Flavobacterium causing male-killing in the congeneric Japanese coccinellid, Coccinula sinensis, which was collected from the same region. However, we found slightly different Flavobacterium strains infecting C. sinen- sis from regions with different environmental conditions. This may be an indication of horizontal transmission of male-killing Flavobacterium between these 2 ladybird spices. Finally, environmental conditions may affect the spread of male-killing bacteria among their hosts.展开更多
文摘From the ethyl acetate extract of Murraya koenegii (Rutaceae) leaves, isomahanine (1) and mahanine (2) were isolated that showed antibacterial activity towards Flavobacterium columnare and Streptococcus iniae which caused columnaris disease and streptococcosis respectively. Isomahanine was found to have the strongest activity against F. columnare (isolate ALM-00-173) and S. iniae (isolate LA94-426) based on 24-h 50% inhibition concentration (IC50) and minimum inhibition concentration (MIC). Although compound (7), a nicotinamide isolated from Amyris texana had the lowest MIC (2.8 ± 0 mg/L) of any of the test compounds against F. columnare, the 24-h IC50 of 14.8 ± 0.6 mg/L was higher than that of isomahanine and subsequently the 24-h IC50 RDC values for (7) were almost a magnitude of order higher than those obtained for isomahanine. Isomahanine also had the strongest activity against S. iniae, with a 24-h IC50 of 1.3 ± 0.1 mg/L and MIC of 3.5 ± 0 mg/L, respectively.
文摘The effect of penicillin-G on Flavobacterium psychrophilum, the cause of bacterial coldwater disease, was evaluated in 15 min and overnight exposures. Separate tests evaluated the effect of increasing doses of penicillin-G to rainbow trout Oncorhynchus mykiss eggs, fingerlings, and adults. Rainbow trout eggs were exposed for 1 h to penicillin doses of 0, 250, 500, 1,000, 10,000, 50,000, or 100,000 IU/mL. Mean percent hatch ranged from 64.6% to 75.1%, and did not significantly differ among the treatments. Fingerlings were injected intraperitoneally (i.p.) with 200 μL of 0, 1, 10, 100, 1,000, 10,000, 100,000, or 500,000 IU/mL. Probit analysis resulted in a LD10 (lethal dose for 10% of injected fish) of 52,868 IU/mL (95% confidence limits: 31,522 - 69,490 IU/mL) and a LD50 of 131,466 IU/mL (113,095 - 157,878 IU/mL). Brood stock were injected (i.p.) with 200 μL of penicillin at concentrations of 0 (control), 10,000, 50,000, 100,000, 200,000, 400,000, or 800,000 IU/mL. Mortality ranged from 0% to 7% (50,000 IU/mL treatment) and did not significantly differ among treatments. In 28 h exposure tests, penicillin concentrations of ≥ 333 IU/mL were required to completely suppress growth of F. psychrophilum. In 15 min exposures, ≥ 10,000 IU/mL were needed to achieve the same result. Tests indicated that rainbow trout eggs can tolerate 1 h exposures to penicillin-G concentrations as high as 100,000 IU/mL, and that brood fish can tolerate injections of at least 800,000 IU/mL. Fingerling data however, suggested that injection of doses greater than 10,000 IU/mL can be toxic. The data suggests rainbow trout eggs or brood can tolerate high doses of penicillin-G that could be used for controlling the transfer of F. psychrophilum to hatcheries receiving eggs.
文摘From time immemorial, human beings have used pigments made from vegetables, fruits, superior plants, animal tissues and cereals. One of the greatest sources of pigments is the bacterium that, with the use of the modern technology, has increased the production of metabolites of interest. The microbiological production of carotenoids has not been optimized to obtain pigment production quantities of pigments and carotenoids recovery that lower production costs. The aim of this work was to design a Zeaxanthin production process with Flavobacterium sp. immobilized cells in a fluidized bed bioreactor. An optimum culture medium for Zeaxanthin production in stirred flasks (2.46 g·L–1) was obtained. Furthermore, optimum process conditions for a maximum yield of Zeaxanthin production, by fluidized bed bioreactor, were established. A statistical analysis showed that the most significant factors were air flow, pH and NaCl concentration (4.5 g·L–1). In this study a maximum Zeaxanthin production of 3.8 g·L–1 was reached. The highest reported yield to date was 0.329 g·L–1.
文摘Columnaris (caused by Flavobacterium columnare) is one of the most common bacterial diseases affecting the pond-raised channel catfish (Ictalurus punctatus) in the southeastern United States of America resulting in annual losses of millions of dollars. As part of our continuing effort to discover environmentally benign compounds for the control of columnaris disease, acyl derivatives of phloroglucinol were synthesized and tested against F. columnare using a rapid bioassay. Among the analogs that were tested, diacyl analogs showed very high antibacterial activity against F. columnare in the laboratory bioassay. Diisovaleryl and diisobutyryl analogs were found to have the strongest activity against F. columnare (isolate ALM-00-173) based on 24-h 50% inhibitory concentration (IC50) and minimum inhibitory concentration (MIC). Diisovaleryl and diisobutyryl analogs had IC50 values 0.82 mg/L and 0.80 mg/L, respectively, whereas the drug control florfenicol had an IC50 value of 0.81 mg/L. Diisovaleryl and diisobutyryl analogs also had 24-h relative-to- drug-control IC50 values around 1.0 indicating activities similar to florfenicol, which is included in medicated feed and is one of the current management approach for columnaris.
基金Supported by the National Basic Research Program of China(973 Program)(No.2009CB118703)the Science and Technology Program of Xiamen Southern Oceanographic Center(No.14PYY050SF03)the National Science and Technology Support Program Project of China(No.2012BAD25B02)
文摘Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.
基金Supported by the National Basic Research Program of China(973Program)(No.2009CB118703)
文摘Flavobacterium columnare, the etiological agent of columnaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers (chloramphenicol and spectinomycin resistance) for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.
基金Supported by the Chinese Academy of Sciences(No.XDA08010207)the National Key Technology R&D Program of China(No.2012BAD25B02)the State Key Laboratory of Freshwater Ecology and Biotechnology(No.2016FBZ04)
文摘Flavobacterium columnare is the pathogenic agent of columnaris disease in aquaculture. Using a recently developed gene deletion strategy, two genes that encode the Glyco_hydro_19 domain(GH19 domain) containing proteins, g hd-1 and ghd-2, were deleted separately and together from the F. columnare G 4 wild type strain. Surprisingly, the single-, Δg hd-1 and Δg hd-2, and double-gene mutants, Δ ghd-1 Δ ghd-2, all had rhizoid and non-rhizoid colony morphotypes, which we named Δ ghd-1, Δ ghd-2, Δ ghd-1 Δ ghd-2, and NΔ ghd-1, NΔ ghd-2, and NΔ ghd-1 Δ ghd-2. However, chitin utilization was not detected in either these mutants or in the wild type. Instead, skimmed milk degradation was observed for the mutants and the wild type; the non-rhizoid strain NΔg hd-2 exhibited higher degradation activity as revealed by the larger transparent circle on the skimmed milk plate. Using zebrafish as the model organism, we found that non-rhizoid mutants had higher LD5 0 values and were less virulent because zebrafish infected with these survived longer. Transcriptome analysis between the non-rhizoid and rhizoid colony morphotypes of each mutant, i.e., NΔg hd-1 versus(vs) Δ ghd-1, NΔ ghd-2 vs Δ ghd-2, and NΔ ghd-1 Δ ghd-2 vs Δ ghd-1 Δ ghd-2, revealed a large number of dif ferentially expressed genes, among which 39 genes were common in three of the pairs compared. Although most of these genes encode hypothetical proteins, a few molecules such as phage tail protein, rhs element Vgr protein, thiol-activated cytolysin, and Ton B-dependent outer membrane receptor precursor, expression of which was down-regulated in non-rhizoid mutants but up-regulated in rhizoid mutants, may play a role F. columnare virulence.
文摘Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide.Many studies have focused on the identification of protective antigens to aid in the development of novel vaccines against the disease.In this study,an immunoblotting approach was employed to identify immunogenic outer membrane proteins(OMPs)from F.columnare in two-dimensional electrophoresis(2-DE)map gels using antibacterial sera obtained from grass carp(Ctenopharyngodon idella),and anti-grass carp-recombinant Ig(rIg)monoclonal antibodies.Five unique immunogenic proteins,including the gliding motility lipoprotein GldJ(GldJ),hypothetical protein FCOL_13420(Fco1),lipoprotein(Lip),F0F1 ATP synthase subunit beta(F0f1)and outer membrane efflux protein precursor(Omep),were characterized.Over-expression of these proteins in Escherichia coli DE3,and their immunogenicity and protective efficacy were evaluated in grass carp.The relative percent survival(RPS)of the groups immunized separately with recombinant GldJ,Lip and Omep was 72%,64%and 68%,respectively when compared to control fish.Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F.columnare.The results suggest that GldJ,Lip and Omep are major protective antigens and may be considered as novel candidates in the development of vaccines against columnaris disease in fish.
文摘Male-killing is 1 of 4 known strategies that inherited parasitic endosymbionts have evolved to manipulate their host＇s reproduction. In early male-killing, infected male offspring are killed early in embryogenesis. Within the Insecta, male-killing bacteria have been found in a wide range of hosts. The Coccinellidae families of beetles, better known as ladybirds, are particularly prone to male-killer invasion. In samples of the coccinellid, Coc- cinula crotchi, from Japan, a new male-killing bacterium was revealed by phenotypic assay. Molecular genetic analysis revealed the identity to be a tetracycline-sensitive Flavobac- terium that causes female-biased offspring sex ratio. Furthermore, that Flavobacterium strain was found to be closely related to the Flavobacterium causing male-killing in the congeneric Japanese coccinellid, Coccinula sinensis, which was collected from the same region. However, we found slightly different Flavobacterium strains infecting C. sinen- sis from regions with different environmental conditions. This may be an indication of horizontal transmission of male-killing Flavobacterium between these 2 ladybird spices. Finally, environmental conditions may affect the spread of male-killing bacteria among their hosts.
