BACKGROUND
anacystis nidulans is a rod-shaped, unicellular prokaryotic cyanobacterium and plays an important role in aquatic ecosystems as a primary producer.
it is often used as an indicator for studying the toxic metabolic levels of heavy metals.
many heavy metal studies have been done using a. nidulans as the model system for the reaction of organisms to heavy metal stresses  <cit> .
in freshwater environments, dense algal blooms of cyanobacteria are usually caused by nutrient enrichment  from sewage, agricultural fertilizers and industrial run-off into waterways  <cit> .
algal blooms are considered threat to the water system  <cit> .
cyanophage are viruses that infect cyanobacteria and are ubiquitous in both freshwater and marine environments.
these phages play important roles in modulating cyanobacterial populations, affecting primary productivity, increasing water quality and may have a profound influence on global biogeochemical cycles  <cit> .
although the interaction between a cyanophage and its host organism is important in maintaining water quality in freshwater systems, little is known about how viruses regulate microbial mortality in natural waters.
recently, it was found that lysogenic infection was common in marine synechococcus sp.
<cit> .
cyanophage infecting a single strain of marine synechococcus sp.
can reach  <dig> to  <dig> per ml in seawater  <cit> .
suttle and chan  <cit>  have estimated that between 5– <dig> % of marine synechococcus cells were lysed by cyanophage daily.
the discovery of a high abundance of viral particles  in natural waters  <cit>  initiated the research on the ecological impact of the viral infection and lysis of marine microbes.
they also provided evidence that viruses can affect microbial populations by either going through a lytic cycle, causing destruction of the host cell, or maintaining a lysogenic stage, in which the viral genome is inserted and maintained as the prophage in its host cell  <cit> .
there was also evidence to suggest that seasonal changes can cause the prophage to enter a lytic cycle thus leading to the disappearance of algae blooms  <cit> .
lysogeny can also be induced to a lytic cycle by pollutants  <cit> .
although extensive research has been done on the interaction between cyanophage and cyanbacteria in marine systems, there are no significant reports that have focused on freshwater cyanophage/cyanobacteria interactions.
the possibility of temperate as- <dig> and lysogenic a. nidulans was suggested by bisen et al  <cit> , but there was no direct evidence provided.
it has been reported that uv, mitomycin c and heavy metals such as copper, cadmium can induce the release of cyanophage in marine water  <cit> .
in this study, different concentrations of copper sulfate were used to study a possible as- <dig> lysogenic life cycle in addition to the previously characterized lytic cycle.
addition of copper sulfate led to a significant increase in phage production, a characteristic of an organism with a lysogenic life cycle.
the study of lytic induction from temperate a. nidulans can provide a good model for studying the interaction between cyanophage and cyanobacteria in freshwater ecosystems.
RESULTS
in order to determine if heavy metals could cause induction of as- <dig> from temperate a. nidulans , cuso <dig> was added at concentrations of  <dig>  × 10- <dig> m,  <dig>  × 10- <dig> m,  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m at day  <dig> post innoculation, the exponential growth stage of the culture.
growth of an-t was severely inhibited at concentrations of  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m. growth was affected to a certain extent in  <dig>  × 10- <dig> m of cuso4; growth rate in  <dig>  × 10- <dig> m cuso <dig> was very similar to the control .
as results seen in marine cyanobacteria, there is consistent release of virus through out the lysogenic cycle in the normal as-1/a.
nidulans infective system.
the viral release was monitored by plaque forming units .
the representative pictures of the plaque forming units  after cuso <dig> induction were obtained from different treated conditions and illustrated as no plaques and the proper number of plaques as showed in figure  <dig>  the results of as- <dig> pfu study, showed that  <dig> hours after copper induction,  <dig>  ±  <dig>  pfus were observed from the control,  <dig>  ±  <dig>  pfus were observed from  <dig>  × 10- <dig> m of cuso4;  <dig>  ±  <dig>  pfus were formed from  <dig>  × 10- <dig> m of cuso <dig>   <dig>  ±  <dig>  pfus were formed from  <dig>  × 10- <dig> m of cuso <dig>   <dig> hours after induction,  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  pfus were formed compared to the control,  <dig>  × 10- <dig> m,  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m of cuso <dig> respectively.
<dig> hours after induction,  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  pfus were formed for the control,  <dig>  × 10- <dig> m,  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m of cuso <dig> induction respectively.
pfus per  <dig> a. nidulans was also calculated,  <dig> hours after induction, they were  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig> , and  <dig>  ±  <dig>  respectively for the control,  <dig>  × 10- <dig> m,  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m of cuso <dig>   <dig> hours after induction, they were  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  respectively for the control,  <dig>  × 10- <dig> m,  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m of cuso <dig>   <dig> hours after induction, they were  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  respectively for the control,  <dig>  × 10- <dig> m,  <dig>  × 10-5m and  <dig>  × 10- <dig> m of cuso <dig>
the comparison of the treated an-t with the control was summarized in table  <dig>  in the concentration of  <dig>  × 10- <dig> m, the pfus were obvious at  <dig> hours after addition of copper; it induced  <dig>  ±  <dig>   times the control.
at  <dig> hours and  <dig> hours after addition of copper, the pfus were significantly reduced, with only  <dig>  ±  <dig>   times and  <dig>  ±  <dig>   times of the control.
the pfu per  <dig> a. nidulans with copper of  <dig>  × 10- <dig> m was  <dig>  ,  <dig>   and  <dig>   times of the control at  <dig>   <dig> and  <dig> hours respectively.
with concentrations of  <dig>  × 10- <dig> m, pfus were  <dig>  ±  <dig>  times ,  <dig>  ±  <dig>  times  and  <dig>  ±  <dig>  times  of the control at  <dig>   <dig> and  <dig> hours respectively after the addition of copper.
the pfu per  <dig> a. nidulans with copper of  <dig>  × 10- <dig> m was  <dig>  ,  <dig>   and  <dig>   times of the control at  <dig>   <dig> and  <dig> hours respectively after the addition of copper.
with a concentration of  <dig>  × 10- <dig> m, the number of pfus was  <dig>  ±  <dig>  times ,  <dig>  ±  <dig>  times  and  <dig>  ±  <dig>  times  of the control at  <dig>   <dig> and  <dig> hours respectively after the addition of copper.
the pfu per  <dig> a. nidulans with copper of  <dig>  × 10- <dig> m was  <dig>  ,  <dig>   and  <dig>   times of the control at  <dig>   <dig> and  <dig> hours respectively after the addition of copper.
concentrations of  <dig>  × 10- <dig> m may be too high to be inducers for releasing of temperate cyanophage as- <dig> .
this concentration of copper may be toxic for the growth of the cells and induce lethality through different mechanisms .
from this study, the percentage of increase of release  was also calculated for different conditions.
the results indicated that  <dig> hours after addition of copper,  <dig>  ±  <dig>  %,  <dig>  ±  <dig>  % and  <dig>  ±  <dig>  % of increase of release were observed at  <dig>  × 10-4m,  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m of cuso <dig> respectively.
<dig> hours after the addition of copper, the induction efficiency was not of major consequence with  <dig>  × 10- <dig> m of cuso <dig>  the pir was negative with a value of - <dig>  ±  <dig>  %.
the pir was  <dig>  ±  <dig>  % and  <dig>  ±  <dig>  % at  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m of cuso <dig> respectively.
<dig> hours after addition of copper, the pir for  <dig>  × 10- <dig> m of cuso <dig> was - <dig>  ±  <dig>  %.
the pir was  <dig>  ±  <dig>  % and  <dig>  ±  <dig>  % at  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m of cuso <dig> respectively .
while there is no clear evidence to explain why induction decreases over time, it is possible that either the phage/host interaction stabilizes after the initial stress or the toxic effect of heavy metal on the host causes a disruption in phage production.
although the heavy metal induction rates varied depending on the concentrations of the heavy metal, the overall induction of copper compared to control is clear.
the results suggest that cu2+ is a significant inducer for temperate as- <dig> released from an-t. the results correlated well with the study of induction for marine cyanobacterial lysogen although as- <dig> release rate and induction rate by copper were much lower than the marine cyanophage/cyanobacterial lysogen studies  <cit> .
further study with other reported inducers mitomycin c and uv was also carried out to compare the pir of both mitomycin c and uv with copper studies.
the maximum pir for different factors are showed in table  <dig>  it is indicated that copper, uv and mitomycin c are able to induce the release of phage with pir of  <dig>  ±  <dig> ,  <dig>  ±  <dig> , and  <dig>  ±  <dig>  respectively.
CONCLUSIONS
these results suggest that as- <dig> lysogenic phage can be induced by copper with a concentration range from  <dig>  × 10- <dig> m to  <dig>  × 10- <dig> m. the best condition for phage induction occurred at  <dig> hours after addition of all these concentrations.
copper concentrations of  <dig>  × 10- <dig> m showed the highest level of viral induction.
cu2+ is an important inducer for lysogenic cyanobacterial cells and consequently could be a potential trigger in the cyanobacteria population in freshwater aquatic environments.

 anacystis nidulans is a rod-shaped, unicellular prokaryotic cyanobacterium and plays an important role in aquatic ecosystems as a primary producer. it is often used as an indicator for studying the toxic metabolic levels of heavy metals. many heavy metal studies have been done using a. nidulans as the model system for the reaction of organisms to heavy metal stresses. in freshwater environments, dense algal blooms of cyanobacteria are usually caused by nutrient enrichment from sewage, agricultural fertilizers and industrial run-off into waterways. algal blooms are considered threat to the water system. cyanophage are viruses that infect cyanobacteria and are ubiquitous in both freshwater and marine environments. these phages play important roles in modulating cyanobacterial populations, affecting primary productivity, increasing water quality and may have a profound influence on global biogeochemical cycles. although the interaction between a cyanophage and its host organism is important in maintaining water quality in freshwater systems, little is known about how viruses regulate microbial mortality in natural waters. recently, it was found that lysogenic infection was common in marine synechococcus sp.. cyanophage infecting a single strain of marine synechococcus sp. can reach to per ml in seawater. suttle and chan have estimated that between 5– % of marine synechococcus cells were lysed by cyanophage daily. the discovery of a high abundance of viral particles in natural waters initiated the research on the ecological impact of the viral infection and lysis of marine microbes. they also provided evidence that viruses can affect microbial populations by either going through a lytic cycle, causing destruction of the host cell, or maintaining a lysogenic stage, in which the viral genome is inserted and maintained as the prophage in its host cell. there was also evidence to suggest that seasonal changes can cause the prophage to enter a lytic cycle thus leading to the disappearance of algae blooms. lysogeny can also be induced to a lytic cycle by pollutants. although extensive research has been done on the interaction between cyanophage and cyanbacteria in marine systems, there are no significant reports that have focused on freshwater cyanophage/cyanobacteria interactions. the possibility of temperate as- and lysogenic a. nidulans was suggested by bisen et al, but there was no direct evidence provided. it has been reported that uv, mitomycin c and heavy metals such as copper, cadmium can induce the release of cyanophage in marine water. in this study, different concentrations of copper sulfate were used to study a possible as- lysogenic life cycle in addition to the previously characterized lytic cycle. addition of copper sulfate led to a significant increase in phage production, a characteristic of an organism with a lysogenic life cycle. the study of lytic induction from temperate a. nidulans can provide a good model for studying the interaction between cyanophage and cyanobacteria in freshwater ecosystems.  in order to determine if heavy metals could cause induction of as- from temperate a. nidulans, cuso was added at concentrations of × 10- m, × 10- m, × 10- m and × 10- m at day post innoculation, the exponential growth stage of the culture. growth of an-t was severely inhibited at concentrations of × 10- m and × 10- m. growth was affected to a certain extent in × 10- m of cuso4; growth rate in × 10- m cuso was very similar to the control. as results seen in marine cyanobacteria, there is consistent release of virus through out the lysogenic cycle in the normal as-1/a. nidulans infective system. the viral release was monitored by plaque forming units. the representative pictures of the plaque forming units after cuso induction were obtained from different treated conditions and illustrated as no plaques and the proper number of plaques as showed in figure the results of as- pfu study, showed that hours after copper induction, ± pfus were observed from the control, ± pfus were observed from × 10- m of cuso4; ± pfus were formed from × 10- m of cuso ± pfus were formed from × 10- m of cuso hours after induction, ±, ±, ± and ± pfus were formed compared to the control, × 10- m, × 10- m and × 10- m of cuso respectively. hours after induction, ±, ±, ± and ± pfus were formed for the control, × 10- m, × 10- m and × 10- m of cuso induction respectively. pfus per a. nidulans was also calculated, hours after induction, they were ±, ±, ±, and ± respectively for the control, × 10- m, × 10- m and × 10- m of cuso hours after induction, they were ±, ±, ± and ± respectively for the control, × 10- m, × 10- m and × 10- m of cuso hours after induction, they were ±, ±, ± and ± respectively for the control, × 10- m, × 10-5m and × 10- m of cuso the comparison of the treated an-t with the control was summarized in table in the concentration of × 10- m, the pfus were obvious at hours after addition of copper; it induced ± times the control. at hours and hours after addition of copper, the pfus were significantly reduced, with only ± times and ± times of the control. the pfu per a. nidulans with copper of × 10- m was, and times of the control at and hours respectively. with concentrations of × 10- m, pfus were ± times, ± times and ± times of the control at and hours respectively after the addition of copper. the pfu per a. nidulans with copper of × 10- m was, and times of the control at and hours respectively after the addition of copper. with a concentration of × 10- m, the number of pfus was ± times, ± times and ± times of the control at and hours respectively after the addition of copper. the pfu per a. nidulans with copper of × 10- m was, and times of the control at and hours respectively after the addition of copper. concentrations of × 10- m may be too high to be inducers for releasing of temperate cyanophage as-. this concentration of copper may be toxic for the growth of the cells and induce lethality through different mechanisms. from this study, the percentage of increase of release was also calculated for different conditions. the results indicated that hours after addition of copper, ± %, ± % and ± % of increase of release were observed at × 10-4m, × 10- m and × 10- m of cuso respectively. hours after the addition of copper, the induction efficiency was not of major consequence with × 10- m of cuso the pir was negative with a value of - ± %. the pir was ± % and ± % at × 10- m and × 10- m of cuso respectively. hours after addition of copper, the pir for × 10- m of cuso was - ± %. the pir was ± % and ± % at × 10- m and × 10- m of cuso respectively. while there is no clear evidence to explain why induction decreases over time, it is possible that either the phage/host interaction stabilizes after the initial stress or the toxic effect of heavy metal on the host causes a disruption in phage production. although the heavy metal induction rates varied depending on the concentrations of the heavy metal, the overall induction of copper compared to control is clear. the results suggest that cu2+ is a significant inducer for temperate as- released from an-t. the results correlated well with the study of induction for marine cyanobacterial lysogen although as- release rate and induction rate by copper were much lower than the marine cyanophage/cyanobacterial lysogen studies. further study with other reported inducers mitomycin c and uv was also carried out to compare the pir of both mitomycin c and uv with copper studies. the maximum pir for different factors are showed in table it is indicated that copper, uv and mitomycin c are able to induce the release of phage with pir of ±, ±, and ± respectively.  these results suggest that as- lysogenic phage can be induced by copper with a concentration range from × 10- m to × 10- m. the best condition for phage induction occurred at hours after addition of all these concentrations. copper concentrations of × 10- m showed the highest level of viral induction. cu2+ is an important inducer for lysogenic cyanobacterial cells and consequently could be a potential trigger in the cyanobacteria population in freshwater aquatic environments. 
in this study, the possible temperate a. nidulans was treated with different concentrations of heavy metal-copper. it has been reported that some marine cyanophage are temperate and can be induced from a lysogenic phase to a lytic phase by different agents such as heavy metals. however, to date no significant reports have focused on the temperate nature of freshwater cyanophage/cyanobacteria. from these results it appears that as- lysogenic phage can be induced by copper at concentrations from × 10- m to × 10- m. maximal phage induction occurred at hours after addition of copper, with an optimal concentration of × 10- m. cuso with concentrations of × 10- m, × 10- m, × 10- m and × 10- m were used to detect the induction of as- from a. nidulans. the ratio of as- release from a. nidulans was also determined. cu2+ is a significant inducer for lysogenic cyanobacterial cells and consequently would be a potential control agent in the cyanobacteria population in fresh water ecosystems. previous experiments with cyanophage as- and cyanobacteria anacystis nidulans have provided some evidence that as- may have a lysogenic life cycle in addition to the characterized lytic cycle.
 it has been reported that some marine cyanophage are temperate and can be induced from a lysogenic phase to a lytic phase by different agents such as heavy metals. however, to date no significant reports have focused on the temperate nature of freshwater cyanophage/cyanobacteria. previous experiments with cyanophage as- and cyanobacteria anacystis nidulans have provided some evidence that as- may have a lysogenic life cycle in addition to the characterized lytic cycle.  in this study, the possible temperate a. nidulans was treated with different concentrations of heavy metal-copper. cuso with concentrations of × 10- m, × 10- m, × 10- m and × 10- m were used to detect the induction of as- from a. nidulans. the population of the host, unicellular cyanobacteria anacystis nidulans, was monitored by direct count and turbidity while the amount of virus produced was derived from plaque forming units by a direct plating method. the ratio of as- release from a. nidulans was also determined. from these results it appears that as- lysogenic phage can be induced by copper at concentrations from × 10- m to × 10- m. maximal phage induction occurred at hours after addition of copper, with an optimal concentration of × 10- m.  cu2+ is a significant inducer for lysogenic cyanobacterial cells and consequently would be a potential control agent in the cyanobacteria population in fresh water ecosystems. 