As specialized bacteriophages, cyanophages exhibit two dominant life cycles: lytic and lysogenic. In both cases, cyanophages replicate using the host DNA machinery, which involves the following stages: attachment, penetration, biosynthesis, maturation, and release (lytic phase) [27,28]. In the lytic cycle, the mature cyanophage progeny are released after host cell lysis either through an endolysin-mediated mechanism or holin-mediated lysis ([29] and references therein). The lysogenic phage, or temperate phage, can have both lytic and/or lysogenic lifecycles. In the lysogenic cycle, cyanophage DNA is integrated into the host genome and replicated by host machinery for multiple generations to produce prophages ([30] and references therein) which are essentially in a preformed “dormant” state. Lysogenic prophages can rapidly enter the lytic cycle and be released through host cell lysis when host intracellular conditions change, such as when the host cell is stressed [28]. The lifecycle that temperate cyanophages follow depends on both intra- and extracellular factors and their interdependence, including but not limited to the impact of changing nutrient levels, ultraviolet radiation levels, and the presence of virophages (natural predators for phages), as well as any natural mutations in both the host cyanobacteria and the cyanophage [31].

Cyanophages have shown tremendous diversity in their structure, habitat and host range [32,33,34]. Numerous cyanophages have been isolated from freshwater and marine environments and are divided into three different virus families based on their morphologies: Note that viral nomenclature through the International Committee on Taxonomy of Viruses (ICTV) is moving away from morphological nomenclature, however given the breadth of studies reported herein using morphological nomenclature this was adapted throughout the review article: Myoviridae, Podoviridae, and Siphoviridae [35] (Table 1). While all cyanophages have been classified as having a single piece of double stranded DNA and the characteristic head shape of the bacteriophage, each family can be distinguished by their unique tail morphologies (Table 1). Cyanophages can also be broken down into multiple classes and sub-classes, differing in the types of cyanobacteria morphotypes and host ranges they are able to infect (Table 2) [36,37,38] (S. Where Class 1 cyanophages typically infect filamentous cyanobacteria that lack heterocysts, Class 2 cyanophages infect filamentous cyanobacteria, regardless of nitrogen fixation capabilities, and Class 3 cyanophages target unicellular or colonial cyanobacteria (Table 2). Furthermore, cyanophages can vary considerably in their host specificity, having both broad and narrow host ranges, where some cyanophages are unable to infect different strains even under the same host species, or conversely, may target multiple cyanobacteria genera [31,39,40]. For instance, the cyanophage known as Ma-LMM01 (M. aeruginosa—Lake Mikata Myoviridae 01) is only infectious to microcystin-producing M. aeruginosa strain NIES-298 [25]. Podovirus P-SSP7 is also strain-specific, infecting a single high-light-adapted Prochlorococcus strain out of 21 Prochlorococcus strains tested [41]. Additionally, Ma-LMM01, Ma-LMM02, Ma-LMM03, and Ma-HPM05 were found to specifically infect only microcystin-producing M. aeruginosa strains ([42] and references therein).

Similar to other viruses, cyanophages are considered an important regulator of both the abundance and composition of cyanobacteria in aquatic environments. It was found that genetic structure and diversity of cyanophages changed along water depth profiles, where maximum cyanophage diversity was correlated with maximum cyanobacterial abundances [43,44]. Furthermore, cyanophages only infect phage-sensitive cyanobacteria, which can result in the displacement of cyanophage-sensitive populations with cyanophage-insensitive populations [45]. For example, the Microcystis-specific phages that only infect microcystin-producing strains of M. aeruginosa have the potential to shift the composition of M. aeruginosa towards non-microcystin-producing populations, or vice versa [42].

Temperature, nutrients, and irradiance are important factors affecting the stability and infectivity of cyanophages and subsequent virulence against their host. For each parameter, there are three interconnected phases that directly impact cyanophage infectivity: (1) the tolerances of the host, (2) the tolerances of the free cyanophage and (3) the propagation of the cyanophage within the host.

Temperature has a profound effect on cyanobacteria propagation, which varies based on geographic location and taxa. Cyanobacteria tend to have heightened growth rates when water temperatures rise from 15 °C to 29 °C [46]. This is significant, as climate change scenarios predict that in the coming years, rivers, lakes and reservoirs will experience heightened conditions that favor cyanobacteria productivity [47]. Therefore, the ability of cyanobacteria to adapt to warming temperatures is an important consideration for future cyanophage-cyanobacteria control applications, as water temperature affects the survival rate of free cyanophages and therefore directly impacts their potential virulence.

As was observed with cyanobacteria, several studies found that cyanophage populations increased with a seasonal increase in water temperatures [48,49,50] and that their stability tended to be consistent with the stability of cyanobacteria at water temperatures up to 50 °C [37]. More specifically, at temperatures up to 40 °C, 85% of cyanophages remained virulent, while at 45 °C only 55% of cyanophages remained virulent, and at or above 50 °C, less than 0.001% of cyanophages remained virulent [36,51]. Thermotolerant cyanophage strains were able to survive at temperatures greater than 40 °C, whereas thermosensitive strains were unable to survive even at 35 °C [28]. For example, Safferman and Morris [36] and Safferman et al. [51,52] found that of the three cyanophage groups (LPP-1, SM-1, and AS-1), LPP-1 and SM-1 had the greatest temperature range, demonstrating stability between 4 °C and 40 °C. However, in LPP-1, mature particles were not formed within a host at temperatures above 31 °C. Furthermore, LPP-1 and SM-1 were inactivated at a lower temperature (55 °C) than the AS-1 group (60 °C), demonstrating various aspects of thermovariation in survivability across a diversity of cyanophages.

The infection rate of a cyanobacterium by a cyanophage is dependent upon (1) the contact rate and (2) how resistant the host cell is to the infection. Cheng et al. [53] found that cyanophages in warmer waters had more than a 50% increase in the efficiency of plaquing (EOP), which directly relates to the efficiency of cyanophage infectivity. For example, several studies have shown that an increase in water temperature led to a decrease in the water viscosity, which induced a 10.7% increase in cyanophage-host contact rate [53,54]. Furthermore, higher temperature can also lead to an increase in burst size and cyanophage adsorption on the host surface, and a decrease in the latent period [55]. Padan and Shilo [37] found that the lytic cycle could be induced under elevated temperatures, as increased temperature considerably affects cyanobacteria population homeostasis, making them more susceptible to lysis during infection. The association of the cyanophage lytic cycle with increasing temperatures is an important discovery, as global temperatures are predicted to rise owing to changing climates. Furthermore, seasonal changes could also be expected to induce cyanophages to enter the lytic cycle, which may be beneficial for operational cyanophage control scenarios. It should be noted that temperature is interrelated with pH and carbon dioxide (CO₂), which also affect virulence; however, the connection between these parameters is still unclear [53]. There is a direct relationship between water temperature and cyanophage infectivity, but more work is needed to establish correlations, specifically with regard to host tolerances. In general, cyanophage thermotolerance studies are lacking in freshwater strains.

Macronutrients, including phosphorus, nitrogen and CO₂ concentrations, are vital factors influencing both cyanobacteria growth and population dynamics ([56,57] and references therein). Phage proliferation strongly depends on host metabolism; host generation times affect phage latent periods and low nutrient availability results in longer latent periods and reduced burst size [58]. The metabolic status of the host is critical for viral infection and proliferation because it affects adsorption, replication, lytic activity, and survival of the phage [27]. Recently, it has been recognized that multiple nutrients may concurrently contribute to bloom occurrence [59]; however, the precise climatic and water quality conditions that trigger bloom events are still not well understood [12,60,61,62].

In the early stages of infection, cyanophages obtain the biomolecules needed to build progeny virions from the host cell and later shift to acquiring substrates that are extracellular in origin [63], which suggests that as an infection proceeds, ongoing host cell metabolism is an important factor for viral productivity [64]. It is important to note that cyanobacteria productivity is also heavily linked to extracellular nutrient concentrations, which may have even further implications for cyanophage success. For example, only 9.3% of cyanophage-infected cells lysed under limited phosphorous (P) conditions compared to 100% under replete conditions [65]. These results suggest that cyanophages became lysogenic in P-limited conditions. Continued studies have shown that, during low nutrient conditions, non-cyanophage bacteriophages enter the lysogenic phase due to unfavorable conditions for bacterial growth and production [66]. It is plausible to infer that cyanophages would function in a similar capacity given the overlap of comparable structure and function. This characteristic lifecycle shift has also been noted in cyanophages exposed to P-limited conditions, where cyanophages and their hosts can exist in an intermediate state between the lytic and lysogenic cycles, a phenomenon known as pseudolysogeny [67].

Furthermore, P-limitation in cyanobacterial host cells has also been shown to severely decrease cyanophage production rate and burst size [68,69,70]. For example, Wilson et al. [65] examined the effects of P-limitation on the cyanophage infection kinetics of S-PM2 cyanophages propagated on cultures of Synechococcus. Under P-limited conditions, lysis of Synechococcus was delayed by 18 h compared to a 9 h latent period in phosphate-replete conditions [65]. Additionally, there was an 80% reduction in burst size under P-limited conditions when Synechococcus was infected with cyanophage S-PM2 in comparison to replete conditions, which was also noted in a second study by Rihtman [67], who used a purified cyanophage for infection. Further, Cheng et al. [70] demonstrated significant decreases of 85% and 73% in cyanophage production rate and burst size, respectively, in P-limited Phormidium sp., demonstrating that P-related effects on infectivity are strongly tied to specific host–phage relationships. Cheng et al. [70] also documented increases in viral adsorption in P-limited samples from 21% to as high as 51%, further underscoring P-related effects on infectivity. Research on phage production in cyanobacteria has shown that there is a strong dependence on light and nutrient availability, but more research needs to be conducted on this topic [27].

The direct effects of nitrogen (N) on the virulence of cyanophages have not been studied extensively; however, during infection, cyanophages are known to utilize the host cell’s machinery to obtain nutrients from the extracellular medium for protein synthesis. For example, in Synechococcus sp. WH8102 infected with the cyanophage S-SM1, Waldbauer et al. [64] observed that proteins in progeny virion particles were composed of 41% extracellular N. Although more than half of the proteins in the phage particles were derived from the host, nutrients from the extracellular medium played an important part in viral replication. Furthermore, in a study by McKindles [71], viral replication did not occur when a strain of Microcystis Aeruginosa was infected with cyanophage Ma-LMM01 in N-limited media, further supporting the theory that N may be an important nutrient in phage absorption and viral replication. Lysogenic activity of cyanophages also appears to be affected by N. One study using samples of natural populations of Synechococcus spp. from Tampa Bay and the Gulf of Mexico showed that prophage induction is inversely correlated with the abundance of Synechococcus, suggesting that lysogeny may be a survival response to resource limitation [72]. This finding is further supported by another study that showed prophage production is favored over lytic behavior during periods of reduced population and vitality of Synechococcus spp. [66]. It should be noted that information regarding freshwater strains of Synechococcus spp. is lacking in this context.

The effects of CO₂ on cyanophage behavior are not as well-described in the literature as those of P or N, but it remains an important factor in phage infectivity nonetheless. Elevated dissolved CO₂ concentrations have been shown to increase adsorption ratios as well as burst size in at least one cyanophage, coinciding with increases in host growth rate; however, there were no significant changes in the latent periods or lytic cycles between the high (740 ppm) and low (370 ppm) CO₂ concentrations [73]. Additionally, Zhou et al. [73] documented a greater abundance of the host (Leptolyngbya sp.) population when cultured at the higher CO₂ concentration compared to the lower concentration. Furthermore, it is of note that an increase in CO₂ concentration may coincide with a decrease in environmental pH [74], and, at low pH levels, the release of cations from the culture can promote an increase in the host cell surface charge [73]; as a result, this may improve cyanophage stability and increase adsorption [75]. In a study whose findings support this, Cheng et al. [70] investigated the effects of elevated (800 µatm) CO₂ partial pressure (pCO₂) on cyanophages, and found a 96% increase in cyanophage production rate and a 57% increase in burst size compared to ambient (400 µatm) pCO₂ at various host growth rates. In addition, elevated pCO₂ resulted in a shortened latent period compared to ambient pCO₂. In another study, during viral infection of Synechococcus, elevated pCO₂ also resulted in a shortened latent period, although a decrease in burst size was observed [76]. These studies indicate that increases in CO₂ concentration may improve infection capabilities of cyanophages by increasing their adsorption ratio and burst size. However, due to the complexity in the mechanisms involved in the host–phage relationships, additional research is necessary to investigate CO₂ impacts on cyanophage infectivity. Furthermore, more information is needed on the combinatorial effect of changing CO₂ concentrations alongside other factors, such as temperature, nutrients, and light conditions, particularly as the climate shifts toward warmer temperatures and as anthropogenic pollution increases.

Solar irradiance levels have been shown to directly impact cyanophages and cyanobacteria productivity, and can influence the dominant strain in cyanobacteria communities. For example, several studies have shown that toxigenic cyanobacteria species generally dominate in high-light, high-temperature, and highly stratified environments [77,78]. Alternatively, non-toxic strains thrive in a mixed water column partly because of a generally higher affinity for light absorption and unique photopigment composition [79]. Furthermore, a study by Zilliges et al. [80] highlights that this selection for toxigenic strains can be directly traced to the greater levels of ultraviolet radiation tied to a shifting climate. Collectively, this suggests that toxigenic cyanobacteria are more likely to occur in the coming years, which further drives the need to develop species/genus-specific, environmentally benign control strategies to reduce environmental and human health impacts from cyanoHABs.

Solar irradiance can also directly impact the stability of free cyanophages in aquatic systems. High solar irradiances are believed to significantly contribute to the loss of cyanophages in the natural environment as a result of impairment to phage genetic material. Specifically, the formation of pyrimidine dimers when exposed to increased irradiance has been shown to impact phage replication and infectivity [28,81], although such damage from exposure to ultraviolet light may be reversed through common photo repair mechanisms [82]. Additionally, the impact of sunlight on the rate of cyanophage decay depends on the intensity of the germicidal wavelengths that reach the cyanophage, which is impacted by the ultraviolet absorbance of the water, as well as the location of cyanophages throughout the water column [81].

Unlike other bacteriophages, light is crucial for cyanophages in the infection of cyanobacteria [83], as the adsorption and replication of some cyanophages to their host cells is light-dependent [84]. Cyanophage adsorption and replication derives most of its energy and certain resources from photosynthetic metabolism of the host cells, and is often synchronized to the light–dark cycle [83]. It was also observed that the first sign of infection is invagination of the photosynthetic lamellae, with viral particles later appearing in the space between the folded lamellae and the plasma membrane [37]. Multiple studies have also shown a heavy reliance of certain cyanophages upon the photosynthetic activity of their host cyanobacterial cells, with total losses of infectivity observed under dark conditions [85,86] and at least one cyanophage harboring a genetic homolog capable of stemming photoinhibition [87,88]. This active role of cyanophages in securing photosynthetic byproducts from their hosts further underscores the integral nature of solar irradiance to their collective success.

Most cyanobacteria produce a protective boundary between themselves and the surrounding environment in the form of extracellular polymeric substances [89,90]. These substances are primarily made up of complex heteropolysaccharides, which enable cyanobacteria to dynamically regulate their extracellular glycan levels to alter mucilage complexity and function [91]. Exopolysaccharides (EPS) have many functional purposes related to their physio-chemical properties [92]. In cyanobacteria, EPS are polyanionic, forming hydrated gels that help form the scaffolding of the colony and enable metal sequestration [90]. EPS are also involved in colony formation, as they provide the “glue” that holds the individuals together into a colony.

EPS produced by cyanobacteria can act as a physical barrier to the adsorption of cyanophages, interrupting the infectivity and effectiveness of the phages [93]. EPS are known to cause lower phage mobility and even trap cyanophages [91]. Given the poor mobility of cyanophages in EPS, cyanobacteria near the outer edges of colonies and biofilms would be most susceptible to infection. As cyanobacteria colonies grow from the center outward, with the more mature cells in the center and the younger, more metabolically active cells on the edges, the majority of phage population growth in a biofilm could involve infection of bacteria that are more metabolically active, which would better support larger phage bursts [94].

Although EPS can be an effective defense strategy against bacteriophages, bacteriophages have developed mechanisms to combat them. For example, some bacteriophages can synthesize enzymes capable of degrading polymers on the cell surface of their host [95]. Some bacteriophages can also produce enzymes to depolymerize the scaffolding of the EPS and rapidly reduce the hindrance of diffusion by phages within the matrix [91]. Additionally, given the negative correlation between EPS production and cyanobacteria growth rate, EPS production is unlikely to interfere with cyanophage infectivity as it is likely to be low during active bloom events. However, more information is needed to better characterize EPS production during a HAB and its possible effects on cyanophage infectivity.

Several environmental factors significantly influence (1) cyanobacteria growth, (2) free cyanophage populations, and (3) cyanophage infectivity. Temperature, nutrients, and irradiance are the predominantly studied environmental parameters that have been shown to directly impact cyanophage success. Table 3 summarizes the aforementioned findings regarding the impacts of these parameters on various facets of cyanophage ecology: burst size, latent period, infectivity, adsorption, life cycle, and overall abundance. Broadly, increasing temperature coincides with an increase in all listed ecological aspects, with cyanophage life cycles being predominantly lytic in nature [48,49,50,53,55]. P-limitation resulted in decreased burst size and infectivity and an increase in latent period [65,67,68,69,70]: this limitation also drove cyanophage life cycles toward the lysogenic pathway [65,66]. It is of note that an increased P concentration has been shown to correlate with increased free cyanophage abundance, further underscoring the relationship between P and cyanophages [70]. N-limitation results were solely based on marine strains of Synechococcus spp. and should be explored further in freshwater systems. N-limitation drove cyanophages to lysogenic life stages and also potentially reduced adsorption and/or overall abundance [71,72]. The effects of CO₂ on cyanophage ecology are not as well-described as the other environmental parameters discussed here; however, studies have shown that an increase in the pCO₂ has resulted in both a decrease in latent period as well as an increase in cyanophage production [70,76]. Finally, solar irradiance is critical to the viability of host cyanobacteria cells and is therefore a major factor in cyanophage ecology. Facets such as infectivity and adsorption are strongly tied to the host cell’s photosynthetic metabolism and fluctuate alongside their host’s own optimal irradiance values [83,84]. However, it should be noted that free cyanophage abundance has an explicitly described relationship with solar irradiance, in which increased irradiance results in damage to phage genetic material [81]. The production of EPS by cyanobacteria is unlikely to provide an obstacle to the propagation of cyanophages [93], particularly as EPS production is negatively correlated with cyanobacteria growth rate [96] and growth rates are often high during bloom events, but there is limited information on EPS impacts on cyanophage infectivity, and this should be explored further. In short, understanding the effects of these critical environmental parameters on cyanophage ecology is critical to their potential operational use as a cyanoHAB control measure.