In recent years, initiatives aimed at mitigating eutrophication have successfully reduced nitrogen and phosphorus concentrations in numerous lakes across China. Notably, the management of total nitrogen levels has prompted a shift in the dominant genera responsible for harmful algal blooms. Among these,
Dolichospermum, a typical diazotrophic cyanobacterium, exhibits the ability to differentiate heterocysts for atmospheric N
2 fixation under nitrogen-limited conditions. However, the underlying mechanisms driving heterocyst differentiation in response to the absence of specific nitrogen compounds remain poorly understood. This study analyzed the driving factors influencing heterocyst frequency using field data from Lake Chaohu collected between January and June 2022. Furthermore, an experiment was conducted utilizing NH
4Cl, NaNO
3 and urea as nitrogen sources, with specific nitrogen deficiencies created to investigate the response mechanisms of
Dolichospermum under these conditions. The results indicated significant monthly variations in heterocyst frequency in Lake Chaohu, which were associated with the interaction of multiple driving factors. Nutrient changes emerged as the most intuitive driving factor, with heterocyst frequency showing a significant negative correlation with total nitrogen and dissolved total nitrogen levels. Experimental results demonstrated that the absence of NO
3N promoted both the biomass and heterocyst frequency of
Dolichospermum. When NH
4N was limited, the proliferation of
Dolichospermum was inhibited, leading to an extended period of heterocyst development. Although a lack of urea eventually increased heterocyst frequency in
Dolichospermum, there was no significant increase in biomass. The concentrations of the three nitrogen sources exhibited a negative correlation with heterocyst differentiation, with the effects of NO
3N and urea deficiency on heterocyst differentiation being significantly stronger than those of NH
4N. Moreover, heterocyst differentiation frequency was positively correlated with photosynthetic efficiency, which indicated that the acquisition and distribution of photosynthetic energy between heterocysts and vegetative cells also influence the differentiation process of heterocysts to some extent. The findings highlight the differing responses of heterocyst differentiation to various forms of nitrogen, emphasizing the importance of prioritizing NH
4N removal in nutrient control. However, further research is needed to determine the key threshold concentrations of different nitrogen sources that trigger heterocyst differentiation.