Lipid Droplet metabolism dependent microbial defense in pre-immune zebrafish embryos

Microbes present survival challenge to pre-immune embryos. Our study provides evidence for antimicrobial-secretion-based strategy of zebrafish embryos against microbes during pre-immune stages. Chorion prevents physical contact between embryos and microbes, yet microbes compromise embryonic survival through their secretions. Development of embryos in microbe-free medium involves secretion of pro-microbial compounds that bacteria utilize to accelarete growth. Embryo senses presence of microbes through microbial secretions. They respond by altering their secretions to include antimicrobial compounds along with regular pro-microbial ones. Upon sensing embryonic anti-microbial secretions, microbes too alter their secretions to include more potent toxins for embryos. In response to this embryos alter their secretions to include more potent antimicrobial compounds. Ability of embryos to secrete antimicrobial compounds is positively correlated with amount of lipid droplets (LDs) in them. Inhibition of LD metabolism prevents antimicrobial secretions by embryos. Thus, LDs protect zebrafish embryos from microbes. This manuscript establishes that pre-immune embryos employ dynamically evolving biochemical warfare to protect themselves from harmful microbes.


21
The immune systems of fish and mammals share significant similarities. For example, both the 22 vertebrates host comparable sets of lymphocytes (similar T-cell subsets) and hence, generate 23 analogous immune responses 1-3 . However, unlike mammalian embryos that develop inside the 24 body of the female, embryonic development of fish (oviparous) occurs in the open aquatic 25 environment. Therefore, right after fertilization these oviparous embryos require a functional 26 immune system to safeguard them from pathogenic microorganisms present in their natural 27 habitat. Studies on zebrafish embryos report that adaptive immune response develops four to six 28 weeks after fertilization of the egg 4-6 . Further, 'primitive macrophages' appear only after 16 hours 29 post fertilization (hpf) 7-9 . These macrophages and the newly generated neutrophils constitute the 30 innate immune system in one day old zebrafish embryos 2,10 . Hence, these embryos are exposed 31 to a large number of pathogens prior to the development of either the innate or the adaptive 32 immune system. However, it is not known how the zebrafish embryos protect themselves from 33 pathogens before 1 day post fertilization (dpf) when even the innate immune system is not value (LDD at 30mpf) associated with each parent set (Fig. 1B, Supp. S1). Interestingly, the rate-67 of-increase of LDD (slope) with embryonic development, for embryos from a particular parent 68 set, exhibits a strong positive correlation (correlation coefficient=0.75) with the corresponding 69 LDD 30 values (Fig. 1C). Therefore, we propose that the rate of synthesis of the LDs depends 70 upon the initial LDD values (LDD 30 ), which in turn depends upon the respective parent set used 71 for breeding. Next, we explored whether the unique embryonic LDD 30 is determined 72 independently or jointly by the male and the female parents. We found that the LDD 30 of the 73 embryos obtained from independent females bred with different males do not alter LDD 30 74 significantly (Fig. 1D), while similar analysis with the same male fish bred with different 75 females show significantly varying LDD 30 values in the offspring (Fig. 1E). Hence, embryonic 76 LDD is uniquely determined by the female parent alone. This allowed us to obtain the embryos 77 with distinct LDD by selecting the appropriate females for breeding. with/without bacteria in E3, with/without exogenous LD injection. LDD 30 of the embryo clutch 86 is 2971/mm 2 in A, B, D. Bacterial concentration used: 10 6 /ml. All embryos incubated with 87 external food supply so that bacteria is the only challenge to embryos.

88
LDs help the embryos endure microbial challenges 89 In the natural habitats, the zebrafish embryos are exposed to different microbes prevalent in the      observe that the embryos in E3 secrete mainly during the initial 3 hpf. There is no significant 193 secretion 3hpf onwards (Fig. 4C). On the other hand, the embryos exposed to BS-E3 continue to 194 release secretions until 9 hpf at a much higher rate. Interestingly, beyond 9 hpf neither are the 195 embryos vulnerable to BS-E3 (Fig. 3C), nor do they secrete antimicrobial compounds (Fig. 4C). 223 Surprisingly, neither protein translation nor protein degradation seems to be the mechanism of 224 synthesis of the cytostatic secretions (e1-e7). Next we explored the mechanism by which the 225 microbes challenge the embryos.  coulmn2) (Fig. 5D) reveals that the bacteria secretes additional compound 'b1' only if they are 249 exposed to ES-E3 (e0). More interestingly, bacteria exposed to compounds e0-e4 (antimicrobial  Thus, normal development of zebrafish embryos involves secretion of e0 which aggravates the 260 microbial challenge for the embryo itself by accelarating the bacterial growth rate. Fig. 5F 261 depicts the growth curve for bacteria (E.coli) exposed to e0-e4 (10 6 /ml bacterial grown in 2 ml 262 ES-BS ) obtained from embryos having distinct LDD . We observe an inverse relation between 263 the bacterial growth rate and the LDD of the embryos from which the ES-BS was obtained. This 264 observation is in agreement with Fig. 4, since the amount of antimicrobial secretion (Fig. 4B) is 265 positively correlated with LDD 30 (Fig. 4A). We further validate this hypothesis by studying the the growth rate is no more dependent on LDD (Fig. 5G).  (Fig. 2). The link between the abundunce of LDs and embryonic ability to cope with the 295 microbes is surprising. Antimicrobial chorion of the zebrafish embryo ( Fig. 3A-ii) which is the 296 first line of defense against microbes, prevents any physical contact with microbes (Fig. 3A).

297
The microbes attack the embryos through permeable toxic secretion (Fig. 5A). The embryos are 298 susceptible to microbial secretions (b0, Fig. 5A) only until 9 hpf. The development of zebrafish 299 embryos involves secretion of compound e0 (Fig. 4D) which is nutrient for bacterial growth (Fig.   300 5F). The embryos respond to bacterial toxin 'b0' by secreting antimicrobial amine containing 301 compunds 'e1-e4' (Fig. 4E). Bacteria too sense the presence of embryos through embryonic 302 secretion e0, and respond by secreting more potent pathogenic secretion 'b1' (Fig. 5B and D). 303 Interstingly, 'e0' accelarates the bacterial growth rate (Fig. 5F). As a result more pathogenic 304 secretion 'b0', 'b1' are generated, which compromise the survival of embryos even further. To 305 counter the detrimental effects of pathogenic secretions 'b0', 'b1'; embryos secrete antimicrobial 306 compounds 'e1-e4' (Fig. 5F) that checks the bacterial growth rate. The amount of embryonic 307 secretion (e1-e4) depends on the LDD in the embryo (Fig. 4A), as a result we find a reciprocal 308 relation between the bacterial growth rate (in ES-BS) and LDD of the embryos from which the 309 ES-BS has been obtained (Fig. 4A). The secretion of e1-e4 by embryos is dependent on the 310 lipolysis of the embryonic LDs (Fig. 4, 5). 311 We propose that both the embryos and the microbes perceive the presence of one another

315
4E and last coulmn in Fig. 5D). This mechanism of embryonic defenses is specially functional 316 during its pre-immune stages (0-9 hpf). Fig. 6 is the schematic representation of the mechanism 317 of how the embryos survive in the microbe-laced environment of their aquatic habitat.

358
To stain the LDs, the embryos were fixed with 4% paraformaldehyde (Merck) followed by for nearly 20 minutes. The embryos were then washed two to three times with PBS followed by 362 thourough wash with water to remove excess dye which may otherwise cause unnecessary 363 background fluorescence. The embryos were then mounted in 0.6% low melting agar and imaged 364 under 10X magnification using an inverted fluorescence microscope (Zeiss Axio Observer.Z1).

365
The images were processed using the '3D-deconvolve' plugin of ImageJ 1.47t to minimize any 366 unwanted background fluorescence and obtain a 3D-rendered image.  368 To determine whether the bacteria come in physical contact with the embryos/LDs, freshly laid          MgCl 2 , pH 6.8) was added to the blastodisc fraction followed by homogenization by syringe 478 plunging and pestle based homogenization. This was followed by ultracentrifugation in 479 decreasing sucrose gradient of 70%, 55%, 50% and 15% in T-buffer. LDs were collected from 480 the 15-50% layer and used for further experimentation. An overnight culture of E. coli (DH5) was spread over agar plates using a sterilized L-spreader.

483
The plate was allowed to dry and then inverted and incubated for around 30 min at 37°C till the