This is the first study to demonstrate a significant increase in Cryptosporidium numbers over time within a biofilm system, highlighting that biofilms can readily provide a suitable environment for not only the retention, but also the multiplication of Cryptosporidium parasites, in aquatic environments. Previous studies [32, 33] have shown that the number of oocysts retained within biofilms remained constant while oocysts were continually supplied to the biofilm system. However, the apparent discrepancy between these results and those presented here can be explained by the difference in the type of biofilm and methods used to detect Cryptosporidium. In studies by Wolyniak et al.[32, 33], natural biofilms were used with filter sterilised creek water used as the medium. Therefore, when compared to our artificial Pseudomonas biofilms, their biofilms would have a different community structure and nutrient levels. In addition, through the use of the qPCR technique, our analyses quantified not only the oocysts within the system but also other Cryptosporidium life stages that were produced through multiplication.
The significant increase in Cryptosporidium DNA in six day old Cryptosporidium exposed biofilm was further supported by confocal microscopy observation. Cell free culture studies by Hijjawi et al. [7, 8], Karanis et al. and Zhang et al.  support our observation that Cryptosporidium can multiply extracellularly, and that encapsulation within a host cell is not essential for multiplication to occur. Due to the difficulty of identifying Cryptosporidium from a large background of bacteria from biofilms, not all Cryptosporidium stages were identified. Nevertheless, several key developmental stages representing both asexual and sexual reproduction were observed, including sporozoites, trophozoites, and types I and II meronts. The observation of morphological changes of Cryptosporidium sporozoites agrees with previous in-vivo and in-vitro culture observations [34–36], including sporozoites becoming oval shaped during the trophozoite transformation process. Although previous studies by Petry et al.  and Matsubayashi et al.  suggested these changes were due to aged sporozoites that could not multiply in the nutrient-limited cell free culture environment, our observation of subsequent Cryptosporidium development stages of the life cycle, such as types I and II meronts (based upon the developmental stage descriptions by Hijawi et al. ) within six day old biofilm provides evidence that these sporozoites were not simply aged sporozoites. The misinterpretations by these authors [37, 38] have also been clearly defended and clarified by Karanis and Aldeyarbi . Furthermore, the environments that liberated sporozoites are exposed to in aquatic biofilms are nutrient rich micro-environments that could allow Cryptosporidium to salvage their metabolite needs to fuel their high rate of growth and multiplication. This ability to multiply either intracellularly or extracellularly [6, 8–12] suggests that Cryptosporidium i) is capable of extracting the nutrients required for growth and multiplication from the surrounding environment, ii) is not an obligate intracellular parasite, and iii) may be physiologically as well as genetically similar to the closely-related gregarines [40, 41]. Further high resolution imaging [6, 29, 42, 43] and flow cytometry-based studies [44–46] are now needed to fully characterise any additional life stages that may be produced within biofilms.
Interestingly, the presence of Cryptosporidium was also shown to significantly affect biofilm development and maturation. Cryptosporidium- exposed biofilms were found to form mature biofilms significantly faster than biofilms forming without exposure to Cryptosporidium. Consistent with this, Singleton et al.  also showed that biofilms that contain both prokaryotic and eukaryotic cells often formed extensive dense and thick mature biofilms. As the biofilm sloughs off after growing to a certain density, we are unable to conclusively determine if Cryptosporidium detected in the effluent was a result of cells sloughing off with the biofilm, or free floating cells. It is likely to be both. Although it is not possible to determine what proportion of the increase in the mature biofilm thickness was due to increases in the number of bacteria or of Cryptosporidium, our qPCR analyses demonstrated that even very immature biofilms were capable of capturing and accumulating Cryptosporidium oocysts. These may be incidentally incorporated into cell clusters during the biofilm aggregation process. Additionally, during transformation from an immature to a mature biofilm, matrix and water channels that form on the biofilm surface may have enhanced the adhesion of oocysts and also encased both parasites and bacteria, trapping those that were already retained within the biofilm [32, 48]. However, while the thickness and maturation rate of the biofilm was affected by Cryptosporidium, the two factors were not strongly correlated, thus biofilm thickness cannot be reliably used as an indicator of the number of Cryptosporidium residing within the biofilm, a finding also concluded by other studies [32, 33, 49, 50].