Assessment of the cyst wall and surface microbiota in dormant embryos of the Antarctic calanoid copepod, Boeckella poppei

Abstract

Embryos of zooplankton from inland waters and estuaries can remain viable for years in an extreme state of metabolic suppression. How these embryos resist microbial attack with limited metabolic capacity for immune defence or repair is unknown. As a first step in evaluating resistance to microbial attack in dormant zooplankton, surface colonization of the Antarctic freshwater copepod, Boeckella poppei, was evaluated. Scanning electron micrographs demonstrate the outer two layers of a five-layered cyst wall in B. poppei fragment and create a complex environment for microbial colonization. By contrast, the third layer remains undamaged during years of embryo storage in native sediment. The absence of damage to the third layer indicates that it is resistant to degradation by microbial enzymes. Scanning electron microscopy and microbiome analysis using the 16S ribosomal subunit gene and internal transcribed spacer (ITS) region demonstrate the presence of a diverse microbial community on the embryo surface. Coverage of the embryos with microbial life varies from a sparse population with individual microbes to complete coverage by a thick biofilm. Extracellular polymeric substance binds debris and provides a structural element for the microbial community. Frequent observation of bacterial fission indicates that the biofilm is viable in stored sediments.

FIGURE 1

Variability in visible surface area covered by biofilm on encysted dormant embryos of B. poppei from stored Antarctic lake sediment, as observed with scanning electron microscopy: (A) 0%–10% biofilm, (B) 11%–50% biofilm, (C) 51%–89% biofilm and (D) 90%–100% biofilm; (E–H) identical to image above with biofilm identified by gold highlight.

FIGURE 2

Prokaryotic diversity in biofilm on encysted dormant embryos of the copepod, B. poppei, as observed with scanning electron microscopy. Putative identification based on morphology: Spirochete‐shaped (Sc; panels A, B, E and F), rod‐shaped (R; panels B, C, E, G and H), stalked/prosthecate rod‐shaped (SR; panel C), stalked/prosthecate vibrio‐shaped (SV; panels C and F), spirilla‐shaped (Sp; panels C and H), cocci‐shaped (C; panels C and E), filamentous‐shaped (F; panels D and H) and vibrio‐shaped (V; panel D).

FIGURE 3

Extracellular polymeric substance (solid white arrows) of putative microbial origin observed in biofilm on the surface of encysted dormant embryos of the copepod, B. poppei, using scanning electron microscopy. Extracellular polymeric substance was observed on (A) possible rod‐shaped prokaryote, (B) diatom, (C) cocci‐shaped prokaryote and (D–F) combination of debris and prokaryotes. D, diatom; P, prokaryotes.

FIGURE 4

Prokaryotic fission (black arrows) observed in biofilm on the surface of encysted dormant embryos of the copepod, B. poppei, using scanning electron microscopy: (A) rod‐shaped prokaryotes, (B) cocci‐shaped prokaryotes, (C) rod‐shaped prokaryotes in complex biofilm, (D) rod‐shaped prokaryote with minor extracellular polymeric substance attachment to embryonic surface, (E) stalked/prosthecate prokaryote with vibrio‐like morphology and (F) rod‐shaped prokaryotes. White arrows indicate extracellular polymeric substance on surface of microbes.

FIGURE 5

Putative fungal coenocytic hyphae observed in biofilm on the surface of encysted dormant embryos of the copepod, B. poppei, using scanning electron microscopy: H, spiral hyphae; S, asymmetrical ovoid spores; *, unidentified microbes.

FIGURE 6

Diatom diversity in biofilm on surface of encysted dormant embryos of the copepod, B. poppei, as observed with scanning electron microscopy: (A) araphid pennate diatom; (B–E) raphid pennate diatoms; (F) diatom with visible girdle band (gb). White numbered arrows identify seven potentially unique diatom species or morphotypes.

FIGURE 7

Representative images of amoeboid‐like organisms of varied size in biofilm on surface of encysted dormant embryos of the copepod, B. poppei, as observed with scanning electron microscopy. Measured diameter of organisms: (A) 8.2 μm, (B) 7.8 μm and (C) 4.4 μm. Arrows used to identify examples when more than one organism is visible in image.

FIGURE 8

Two previously unidentified layers to the cyst wall in the copepod, B. poppei, create a complex environment for microbes on the surface of the embryo. (A) Cross‐section of tri‐layered cyst wall (CW) previously identified by Reed et al. (2021) with thin ‘outer layer’ (OL), a thick lamellar layer (LL), and inner membrane (IM). An additional thin flexible layer (FL) and thicker spongey layer (SL) overlay the previously characterized cyst wall (B–D). Images captured with scanning electron microscopy.