Suzanne M. Peyer

Zebra mussels anchor byssal threads faster and tighter than quagga mussels in flow

SUMMARY While the invasive zebra mussel Dreissena polymorpha has rapidly spread throughout the Great Lakes and inland waterways, it is being displaced by the quagga mussel Dreissena bugensis in shallow water habitats. However, zebra mussels remain dominant in areas with higher water velocity. We hypothesized that the persistence of zebra over quagga mussels in habitats with higher water velocity might result from greater rate and strength of byssal thread attachment. We examined whether zebra mussels relative to quagga mussels have: (1) higher byssal thread synthesis rate, (2) lower dislodgment in flow and (3) greater mechanical force required for detachment from substrate. Specifically, we examined byssal thread synthesis rate and dislodgment of both species in response to water velocities of 0, 50, 100 and 180 cm s–1. Byssal thread synthesis rate was significantly higher for zebra than for quagga mussels at all velocities. Dislodgment from the substrate increased for both species with increasing velocity but was significantly lower for zebra than for quagga mussels. We also tested the mechanical force to detach mussels after short (32 h) and long (two and three months) periods of attachment on hard substrate. Detachment force was significantly higher for zebra than for quagga mussels only after short-term attachment. Higher byssal thread synthesis rate in zebra mussels was a likely factor that minimized their dislodgment in flow and increased short-term attachment strength. Differences in byssal thread synthesis rate between the two species might partly account for the ability of zebra mussels to maintain dominance over quagga mussels in habitats with high velocities.

Bacterial Bioluminescence Regulates Expression of a Host Cryptochrome Gene in the Squid-Vibrio Symbiosis

ABSTRACT The symbiosis between the squid Euprymna scolopes and its luminous symbiont, Vibrio fischeri, is characterized by daily transcriptional rhythms in both partners and daily fluctuations in symbiont luminescence. In this study, we sought to determine whether symbionts affect host transcriptional rhythms. We identified two transcripts in host tissues (E. scolopes cry1 [escry1] and escry2) that encode cryptochromes, proteins that influence circadian rhythms in other systems. Both genes cycled daily in the head of the squid, with a pattern similar to that of other animals, in which expression of certain cry genes is entrained by environmental light. In contrast, escry1 expression cycled in the symbiont-colonized light organ with 8-fold upregulation coincident with the rhythms of bacterial luminescence, which are offset from the day/night light regime. Colonization of the juvenile light organ by symbionts was required for induction of escry1 cycling. Further, analysis with a mutant strain defective in light production showed that symbiont luminescence is essential for cycling of escry1; this defect could be complemented by presentation of exogenous blue light. However, blue-light exposure alone did not induce cycling in nonsymbiotic animals, but addition of molecules of the symbiont cell envelope to light-exposed animals did recover significant cycling activity, showing that light acts in synergy with other symbiont features to induce cycling. While symbiont luminescence may be a character specific to rhythms of the squid-vibrio association, resident microbial partners could similarly influence well-documented daily rhythms in other systems, such as the mammalian gut. IMPORTANCE In mammals, biological rhythms of the intestinal epithelium and the associated mucosal immune system regulate such diverse processes as lipid trafficking and the immune response to pathogens. While these same processes are affected by the diverse resident microbiota, the extent to which these microbial communities control or are controlled by these rhythms has not been addressed. This study provides evidence that the presentation of three bacterial products (lipid A, peptidoglycan monomer, and blue light) is required for cyclic expression of a cryptochrome gene in the symbiotic organ. The finding that bacteria can directly influence the transcription of a gene encoding a protein implicated in the entrainment of circadian rhythms provides the first evidence for the role of bacterial symbionts in influencing, and perhaps driving, peripheral circadian oscillators in the host. In mammals, biological rhythms of the intestinal epithelium and the associated mucosal immune system regulate such diverse processes as lipid trafficking and the immune response to pathogens. While these same processes are affected by the diverse resident microbiota, the extent to which these microbial communities control or are controlled by these rhythms has not been addressed. This study provides evidence that the presentation of three bacterial products (lipid A, peptidoglycan monomer, and blue light) is required for cyclic expression of a cryptochrome gene in the symbiotic organ. The finding that bacteria can directly influence the transcription of a gene encoding a protein implicated in the entrainment of circadian rhythms provides the first evidence for the role of bacterial symbionts in influencing, and perhaps driving, peripheral circadian oscillators in the host.

Developmental plasticity of shell morphology of quagga mussels from shallow and deep-water habitats of the Great Lakes.

SUMMARY The invasive zebra mussel (Dreissena polymorpha) has quickly colonized shallow-water habitats in the North American Great Lakes since the 1980s but the quagga mussel (Dreissena bugensis) is becoming dominant in both shallow and deep-water habitats. While quagga mussel shell morphology differs between shallow and deep habitats, functional causes and consequences of such difference are unknown. We examined whether quagga mussel shell morphology could be induced by three environmental variables through developmental plasticity. We predicted that shallow-water conditions (high temperature, food quantity, water motion) would yield a morphotype typical of wild quagga mussels from shallow habitats, while deep-water conditions (low temperature, food quantity, water motion) would yield a morphotype present in deep habitats. We tested this prediction by examining shell morphology and growth rate of quagga mussels collected from shallow and deep habitats and reared under common-garden treatments that manipulated the three variables. Shell morphology was quantified using the polar moment of inertia. Of the variables tested, temperature had the greatest effect on shell morphology. Higher temperature (∼18–20°C) yielded a morphotype typical of wild shallow mussels regardless of the levels of food quantity or water motion. In contrast, lower temperature (∼6–8°C) yielded a morphotype approaching that of wild deep mussels. If shell morphology has functional consequences in particular habitats, a plastic response might confer quagga mussels with a greater ability than zebra mussels to colonize a wider range of habitats within the Great Lakes.

Effects of shell morphology on mechanics of zebra and quagga mussel locomotion

SUMMARY Although zebra mussels (Dreissena polymorpha) initially colonized shallow habitats within the North American Great Lakes, quagga mussels (Dreissena bugensis) are becoming dominant in both shallow- and deep-water habitats. Shell morphology differs among zebra, shallow quagga and deep quagga mussels but functional consequences of such differences are unknown. We examined effects of shell morphology on locomotion for the three morphotypes on hard (typical of shallow habitats) and soft (characteristic of deep habitats) sedimentary substrates. We quantified morphology using the polar moment of inertia, a parameter used in calculating kinetic energy that describes shell area distribution and resistance to rotation. We quantified mussel locomotion by determining the ratio of rotational (Krot) to translational kinetic energy (Ktrans). On hard substrate, Krot:Ktrans of deep quagga mussels was fourfold greater than for the other morphotypes, indicating greater energy expenditure in rotation relative to translation. On soft substrate, Krot:Ktrans of deep quagga mussels was approximately one-third of that on hard substrate, indicating lower energy expenditure in rotation on soft substrate. Overall, our study demonstrates that shell morphology correlates with differences in locomotion (i.e. Krot:Ktrans) among morphotypes. Although deep quagga mussels were similar to zebra and shallow quagga mussels in terms of energy expenditure on sedimentary substrate, their morphology was energetically maladaptive for linear movement on hard substrate. As quagga mussels can possess two distinct morphotypes (i.e. shallow and deep morphs), they might more effectively utilize a broader range of substrates than zebra mussels, potentially enhancing their ability to colonize a wider range of habitats.

Characterization of the cell polarity gene crumbs during the early development and maintenance of the squid–vibrio light organ symbiosis

The protein Crumbs is a determinant of apical–basal cell polarity and plays a role in apoptosis of epithelial cells and their protection against photodamage. Using the squid–vibrio system, a model for development of symbiotic partnerships, we examined the modulation of the crumbs gene in host epithelial tissues during initiation and maintenance of the association. The extracellular luminous symbiont Vibrio fischeri colonizes the apical surfaces of polarized epithelia in deep crypts of the Euprymna scolopes light organ. During initial colonization each generation, symbiont harvesting is potentiated by the biochemical and biophysical activity of superficial ciliated epithelia, which are several cell layers from the crypt epithelia where the symbionts reside. Within hours of crypt colonization, the symbionts induce the cell death mediated regression of the remote superficial ciliated fields. However, the crypt cells directly interacting with the symbiont are protected from death. In the squid host, we characterized the gene and encoded protein during light organ morphogenesis and in response to symbiosis. Features of the protein sequence and structure, phylogenetic relationships, and localization patterns in the eye supported assignment of the squid protein to the Crumbs family. In situ hybridization revealed that the crumbs transcript shows opposite expression at the onset of symbiosis in the two different regions of the light organ: elevated levels in the superficial epithelia were attenuated whereas low levels in the crypt epithelia were turned up. Although a rhythmic association in which the host controls the symbiont population over the day–night cycle begins in the juvenile upon colonization, cycling of crumbs was evident only in the adult organ with peak expression coincident with maximum symbiont population and luminescence. Our results provide evidence that crumbs responds to symbiont cues that induce developmental apoptosis and to symbiont population dynamics correlating with luminescence-based stress throughout the duration of the host-microbe association.

Involvement of a host Cathepsin L in symbiont-induced cell death

The cathepsin L gene of the host squid, Euprymna scolopes, is upregulated during the first hours of colonization by the symbiont Vibrio fischeri. At this time, the symbiotic organ begins cell death‐mediated morphogenesis in tissues functional only at the onset of symbiosis. The goal of this study was to determine whether Cathepsin L, a cysteine protease associated with apoptosis in other animals, plays a critical role in symbiont‐induced cell death in the host squid. Sequence analysis and biochemical characterization demonstrated that the protein has key residues and domains essential for Cathepsin L function and that it is active within the pH range typical of these proteases. With in situ hybridization and immunocytochemistry, we localized the transcript and protein, respectively, to cells interacting with V. fischeri. Activity of the protein occurred along the path of symbiont colonization. A specific Cathepsin L, nonspecific cysteine protease, and caspase inhibitor each independently attenuated activity and cell death to varying degrees. In addition, a specific antibody decreased cell death by ~50%. Together these data provide evidence that Cathepsin L is a critical component in the symbiont‐induced cell death that transforms the host tissues from a colonization morphology to one that promotes the mature association.