Trophic upgrading via the microbial food web may link terrestrial dissolved organic matter to Daphnia

36 Direct consumption of allochthonous resources generally yields poor growth and reproduction in 37 zooplankton, but it is still unclear how trophic upgrading of terrestrial dissolved organic matter 38 (tDOM) via microbial food web may support zooplankton. We compared survival, somatic growth, 39 and reproduction of Daphnia magna fed with heterotrophic flagellate Paraphysomonas vestita and 40 three algal diets. Paraphysomonas was fed lake bacteria that used tDOM as a substrate to simulate 41 an allochthonous diet that zooplankton encounter in lakes. The highest survival, growth, and 42 reproduction was achieved with a diet of Cryptomonas, while Daphnia performance was the worst 43 when fed Microcystis. Paraphysomonas and Scenedesmus diets lead to intermediate growth and 44 reproduction. Cryptomonas contained high amounts of essential polyunsaturated fatty acids (PUFA) 45 and phytosterols that supported high somatic growth and reproduction, whereas poor performance 46 of Daphnia on cyanobacterial diet was most likely due to lack of sterols. Paraphysomonas 47 contained some phytosterols, but not in sufficient amounts, and also essential PUFA 48 (eicosapentaenoic and arachidonic acid) that enhance zooplankton growth and reproduction. Our 49 results indicate that tDOM-based microbial food web supports Daphnia performance even as a sole 50 food source, and may be important in providing zooplankton with essential biochemical 51 components when phytoplankton quantity or quality is low. 52 Introduction 53 The recognition that consumers in aquatic ecosystems may be fueled by terrestrial food 54 sources in addition to in-lake phytoplankton production has sparked vast amount of research during 55 the past three decades. The extent and possible pathways of consumer allochthony have been 56 studied both under laboratory conditions and in the field (e.g. Grey et al. 2001; Pace et al., 2004; 57 Berggren et al., 2014; Taipale et al., 2014). Field studies utilizing stable isotope ratios (C, N, H) 58 have found that large fraction of consumer biomass can be traced to allochthonous sources (Pace et 59 al., 2004; Berggren et al., 2014; Tanentzap et al. 2017, and references therein). Zooplankton 60 allochthony may also vary seasonally following the relative availability of phytoplankton and 61 allochthonous food sources (Grey et al. 2001). However, laboratory feeding experiments have 62 questioned the feasibility of high zooplankton allochthony. Although zooplankton (mainly 63 Daphnia) can survive on purely allochthonous diets, their growth efficiency, somatic growth rate 64 and reproductive output is very low on allochthonous compared to phytoplankton diets (Brett et al., 65 2009; Wenzel et al., 2012; Taipale et al., 2014). Consequently, high inputs of terrestrial carbon and 66 high consumer allochthony have been linked to low production of wild zooplankton (Kelly et al., 67 2014) and fish (Rask et al., 2014; Karlsson et al., 2015). 68 Most laboratory feeding trials testing consumer allochthony have been conducted using 69 terrestrial particulate organic matter (tPOM) as the food source. More than 90% of terrestrial 70 organic matter in lakes is in the dissolved form (DOM) (Kortelainen et al., 1993; Mattsson et al., 71 2005), and tPOM entering the lake in the shoreline or via river flow may rapidly sediment out of the 72 water column. Thus, pelagic consumers especially in large lakes may have limited access to tPOM. 73 Terrestrial DOM (tDOM) can be used as a substrate by bacteria, which can be grazed by 74 heterotrophic protists including flagellates and ciliates (the microbial loop) or directly by 75 zooplankton (Tranvik, 1992; Weisse 2004). Daphnia have been shown to benefit from tDOM 76 directly or via tDOM-supported bacteria when algae is limiting (McMeans et al., 2015). Previous 77 studies (Wenzel et al., 2012; Taipale et al., 2014) have found that Daphnia performance is better 78 when feeding on mixtures of phytoplankton and bacteria than on mixtures of phytoplankton and 79 tPOM, suggesting that DOM may be the more probable pathway for allochthonous organic matter 80 to enter the grazer food web. According to feeding experiments, bacteria alone cannot support 81 Daphnia growth and some taxa may even be toxic to Daphnia as a sole food source (Taipale et al., 82 2012; Freese and Martin-Creuzburg 2012). Few studies have been conducted on Daphnia 83 performance on diets of heterotrophic flagellates, but results have been variable (Sanders et al., 84 1996; Bec et al., 2003; 2006). 85 One of the reasons proposed why Daphnia has poor growth on allochthonous diets is their 86 lack of essential biomolecules, especially polyunsaturated fatty acids (PUFA) and sterols (Brett et 87 al., 2009; Taipale et al., 2014). Compared to many algae, tPOM contains very little PUFA, while 88 bacteria contain none (Lechevalier and Lechevalier 1988; Taipale et al., 2014). Some studies have 89 found that the fatty acid composition of heterotrophic flagellates depends on whether they feed on 90 algae or bacteria (Zhukova and Kharlamenko, 1999; Véra et al., 2001) while others conclude that 91 biosynthesis of lipids produces a consistent fatty acid (and sterol) composition in flagellates 92 irrespective of diet (Bec et al., 2010; Parrish et al., 2012). Bacteria, including cyanobacteria, also 93 lack sterols while phytoplankton contain various sterols in composition that is species-specific 94 (Taipale et al., 2016). The sterol composition of flagellates is poorly studied, but so far studies have 95 indicated that heterotrophic flagellates are capable of sterol synthesis (Klein Breteler et al., 1999; 96 Bec et al., 2006). In addition to concentrating PUFA and sterols present in their food e.g. by 97 selective retention, heterotrophic flagellates may enhance low quality bacterial or cyanobacterial 98 food for Daphnia by either biosynthesizing PUFA and sterols de novo or modifying dietary short99 chain PUFA to eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3). 100 This so called ‘trophic upgrading’ by heterotrophic flagellates has been observed in several studies 101 (Klein Breteler et al., 1999; Veloza et al., 2006; Bec et al., 2006; 2010). Also, indirect evidence of 102 trophic upgrading was obtained when increased abundance of Paraphysomonas vestita in a 103 decaying Microcystis culture was associated with rising EPA and DHA concentrations with a 104 concurrent decrease in short-chain PUFA prominent in Microcystis (Park et al., 2003). 105 Previous studies on Daphnia performance on allochthonous diets have used tPOM, single 106 strains of bacteria grown in artificial growth media, or heterotrophic flagellates growing on these 107 bacteria as a diet source (but see McMeans et al., 2015). We conducted a feeding experiment where 108 we constructed a simple microbial food web of tDOM (peat extract)-natural lake bacteria109 Paraphysomonas vestita to better simulate the pathway for allochthonous carbon to enter 110 zooplankton diets in lakes. We compared Daphnia somatic growth and reproduction on this 111 allochthonous diet to diets of three phytoplankton taxa (Cryptomonas, Scenedesmus, Microcystis) 112 known to vary in their quality as food for Daphnia. Our hypothesis was that 1) Daphnia survival 113 would be better on tDOM-based microbial diet than on pure bacterial diets (as seen in other studies) 114 and 2) Daphnia somatic growth and reproduction would be lower than on the algal diets. 115 116 Method 117 Experimental set up 118 We compared survival, growth and reproduction of Daphnia feeding on either diets of 119 algae or a diet of a heterotrophic flagellate that was grown on bacteria utilizing tDOM as a 120 substrate. For the experiment, we used Daphnia magna clone (DK-35-9), that originated from a 121 pond in North Germany and has been raised successfully in laboratory for several years. Prior to the 122 experiment Daphnia were reared several generations on Scenedesmus. Daphnia neonates (<24h 123 old) of multiple moms were pooled and randomly distributed among treatments (20 ind./treatment) 124 and some were used to determine Daphnia mean initial body weight. During the feeding 125 experiment, individual Daphnia were raised in 40mL vials in ADaM medium (Klüttgen et al., 126 1994). Daphnia were maintained on one of five different diets: no food, Cryptomonas marssonii, 127 Scenedesmus gracilis, Microcystis sp. (strain 130, unicellular, non-toxic) or the heterotrophic 128 flagellate Paraphysomonas vestita. The three algae were cultured in growth media optimal for each 129 of them (Table 1) in 14h:10h light:dark cycle at 20oC. The heterotrophic flagellate was grown in a 130 culture medium containing tDOM extracted from unfertilized garden peat (Kekkilä luonnonturve) 131 which was inoculated with lake bacteria (1 mL of 0.2 μm filtered lake water) a few days prior to 132 addition of the flagellate. Paraphysomonas was concentrated with gentle centrifugation, but the diet 133 given to Daphnia likely contained also bacteria. 134 The media was changed and the Daphnia fed every other day. We offered the food at non135 limiting concentration: 1.5 mg C L-1 on days 0-2, 2 mg C L-1 on day 4 and 5 mg C L-1 from day 6 136 onwards. Every day Daphnia were inspected and dead animals, and the number of offspring were 137 recorded. Sampling was conducted in the middle and in the end of the experiment (days 7 and 14), 138 and Daphnia (10 ind.) in each treatment were collected for measurements of length, weight and 139 subjected to fatty acid analysis. Due to difficulties in culturing the heterotrophic flagellate, we 140 ended the Paraphysomonas treatment already after 12 days. To facilitate the comparison with 141 Daphnia in algal diets that lasted 14 days, the eggs and embryos in Daphnia brood pouch in the 142 Paraphysomonas treatment on day 12 were included as “potential neonates” for day 14. 143 144 Fatty acid and sterol analysis 145 Prior to analy