Marine Chemical Ecology - Chapter 12 pptx

However, if small bacteria swim at high speeds, they would be able to detect a directional gradient before being rotated.26 Cell shape also has a large effect on the ability of a bacteri

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Chemokinesis and Chemotaxis

in Marine Bacteria and Algae

Charles D Amsler* and Katrin B Iken

CONTENTS

I Introduction 413

II Bacteria 414

A Chemokinetic Mechanisms 414

B Bacterial Chemokinesis in the Plankton: Models and Observations 415

C Chemoattractants of Marine Bacteria 418

III Algae 420

A Responses of Macroalgal Gametes to Sexual Chemoattractants 420

B Responses of Macroalgal Spores and Microalgae to Environmental Chemoattractants and Repellents 423

IV Concluding Remarks 425

Acknowledgments 425

References 425

I INTRODUCTION

Regardless of the size or phylogenetic placement of an organism, the capacity for motility confers

a potential ability to exploit spatial heterogeneity in the environment Motility, however, requires expenditures of organismal resources for the production, maintenance, and utilization of a propul-sion mechanism, regardless of whether the organism moves by use of pseudopodia, flagella, fins, legs, or other means There would appear to be little if any adaptive value to such investments unless an organism has a mechanism to sense environmental gradients in biologically significant resources (or hazards) and move towards more favorable areas The microscopic organisms and life history stages that are the focus of this chapter often have one or more such mechanisms that allow them to orient their movement These mechanisms include an ability to sense and respond

to light gradients,1,2 oxygen gradients,3 and differences in surface topography.4 They also include

an ability to sense and respond to chemical heterogeneity in the environment, as described below Strictly defined, chemotaxis refers to directed movement oriented by chemical gradients.5,6 This allows organisms to move directly towards (positive chemotaxis) or away from (negative chemo-taxis) a chemical source However, chemotaxis is sometimes defined more broadly to include mechanisms that allow organisms to have net movement towards or away from a chemical source

by indirect means such as varying their turning frequency and/or swimming speed These indirect mechanisms are more accurately defined as chemokinesis.5,6 Klinokinesis refers to the modulation

of turning frequency, and orthokinesis refers to modulation of swimming speed In some cases,

* Corresponding author.

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9064_ch12/fm Page 413 Tuesday, April 24, 2001 5:24 AM

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414 Marine Chemical Ecology

most notably with bacteria, such chemokinetic mechanisms are often referred to as chemotaxis for historical reasons For the purposes of this chapter, all chemokinetic and chemotactic mechanisms

by which marine bacteria and algae vary their movement in response to chemicals will be consid-ered Chemokinesis and chemotaxis will refer to these mechanistically distinct behaviors and will not be used as interchangeable terms

II BACTERIA

To our knowledge, all chemoattractive behaviors in marine bacteria are chemokinetic even though they are most commonly termed chemotactic responses in the literature Bacteria move through marine environments either by swimming, by swarming, or by gliding motility Gliding motility involves movements of a cell along a solid or semisolid surface without flagella.7 Swarming is movement along a solid or semisolid surface by means of flagella.8 Swimming occurs through more liquid environments, typically requiring the use of flagella, although some marine cyanobacteria swim without flagella or visible surface deformations.9 The mechanism for this is unknown but may involve propagation of submicroscopic waves along the cell surfaces.10 Bacterial flagella can

be restricted to only one or both cell poles, which is referred to as polar flagellation, or can be inserted at random locations throughout the cell surface, which is referred to as peritrichous flagellation Some marine species employ both forms of flagellation either simultaneously11 or under different environmental conditions.12

Chemokinetic behavior in bacteria is best understood in peritrichously flagellated enteric bac-teria.13 Peritrichously flagellated bacteria alternate between periods of forward swimming (called runs or smooth swimming) and brief stops when their orientation changes randomly, called tumbles

In the absence of chemokinetic stimuli, the cells tumble every few seconds, which causes them to move randomly about their environment.13,14 When the concentration of a chemoattractant is increas-ing (or that of a repellent is decreasincreas-ing), the cells tumble less frequently They do continue to tumble and, therefore, to undergo random changes in direction, but because the smooth swimming intervals are longer when attractant concentrations are increasing, the cells’ random walk is biased such that net movement is towards the attractant source.13–16 Although cells with polar flagellation are less commonly studied, they are certainly not uncommon17 and may well be even more important

in marine systems Cells with polar flagellation make high angle turns, nearly reversing their swimming direction rather than tumbling, but otherwise respond to chemical gradients much like peritrichously flagellated species.17 The chemokinetic signal transduction pathways of enteric bac-teria have been studied in great detail,13,18 and a good deal of information is also known about such pathways in other bacteria.17,18

Some bacteria only make an investment in the cellular machinery necessary for motility in suboptimal environments,19,20 presumably because there is little adaptive advantage in investing cellular resources in movement when in an optimal environment However, eventually, the ability

of cells in suboptimal environments to utilize their cellular machinery for motility diminishes as conditions become increasingly growth-limiting.19 Many marine environments, particularly in the plankton, can be severely growth-limiting to bacteria As an illustration of this, only 10% of cells

in a natural assemblage of coastal planktonic bacteria were motile before the addition of carbon and other nutrients, but over 80% had become motile 15 to 30 h after the addition.21 In combination, these observations suggest that bacterial motility represents a cellular expense that is not necessary

in an optimal growth environment and that is not affordable in a severely restrictive one However,

as discussed above, a cell must have the ability to receive and process relevant information about its environment for that expense to be of benefit

Because of their small body length, bacteria are assumed to have only limited ability to sense spatial chemical gradients, but they are known to integrate chemokinetic stimuli over time through

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Chemokinesis and Chemotaxis in Marine Bacteria and Algae 415

changes in the fractional amount of bound chemoreceptors.15,22–24 By movement through a spatial gradient, a cell experiences it as a temporal gradient.16 Dusenbery25 compared the efficiency of spatial vs temporal detection of stimuli in free-swimming bacteria using a numerical model and found that spatial detection could be superior to temporal detection for small cells under high concentrations and steep chemical gradients The problem of a temporal integration of a stimulus

is that organisms in a micrometer-scale size range are greatly affected by rotational diffusion due

to Brownian motion.25 A temporal integration does not work efficiently if a bacterium is moved into another random direction by Brownian motion during the time needed to react to the stimulus However, if small bacteria swim at high speeds, they would be able to detect a directional gradient before being rotated.26 Cell shape also has a large effect on the ability of a bacterial cell to detect chemical gradients.27 Cells with rod-like shapes have enhanced capabilities to sense chemical gradients, particularly by temporal detection, and they also have decreased drag while swimming relative to cells of other shapes.27 These adaptive benefits may help explain why rod-like shapes are more common than other shapes among motile bacteria.27

In marine and other aquatic environments, nutrients are not distributed homogeneously in the plankton but rather exist in patches and gradients Nutrients including sugars, amino acids, and hydrolyzed macromolecules such as dissolved proteins and polysaccharides are released by phy-toplankton into the surrounding water (leaking algae28–30) These may serve as nutrients for other microorganisms including heterotrophic bacteria.31 It is estimated that 10 to 50% of marine primary production is used for heterotrophic bacterial growth.32 Nutrients released from a cell will diffuse around the cell and, thus, result in an area of higher nutrient concentration compared to the background concentration.30,33 Nutrient concentration typically decreases with distance from an algal cell.29,30 Bacteria can only benefit from such nutrient patches if they are able to chemically detect these potential food sources and chemokineticly move and remain there.23,28 Considering the average abundance of phytoplankton and bacteria in coastal surface waters, bacteria will be within

a few hundred µm from a nutrient source,29 which reflects the distance bacteria will have to cover

to encounter a leaking phytoplankton cell

Over the last 15 years, considerable effort has been expended towards improving our under-standing of chemokinesis in marine bacteria through simulation models Jackson’s model34 simu-lated the factors influencing marine bacterial chemosensory ability based principally on behavioral and physiological mechanisms known from the enteric bacteria Escherichia coli and Salmonella

time The model is based on the simplified assumption that the only physical force active in the interaction between bacteria and leaking algae is diffusion From an idealized spherical alga, a substrate is released and builds a spherical concentration field around the alga Parameters consid-ered in the model to influence bacterial chemosensory response are substrate concentration gradient, substrate release rate, algal size, distance of bacteria to alga, binding capacity of bacterial receptors for the substrate, and bacterial swimming speed Chemokinetic responses in bacteria are especially high at high substrate concentrations or strong gradients This is directly related to substrate release rates by algae, since higher release rates enhance the substrate concentration surrounding the alga Bacteria in the model consistently exhibit a higher approach rate towards larger algal cells since those usually have higher specific release rates and build up stronger substrate concentration gradients Small algal size can be compensated for in the model by an increased release rate, but not infinitely so; there is a minimum size of chemosensitively detectable algae of about 2 µm The closer a bacterium is to an alga, the stronger the modeled response, and the distance at which an alga is still detectable depends on the size of the substrate cloud around the algal cell This sensing

of a substrate depends directly on the physiological binding capacity of bacterial receptors for that specific substrate The lower the half-saturation constant (KD) of a receptor, the higher the sensitivity

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The approach rate of bacteria to algae can be strongly increased by decreasing the KD This, however, does not work infinitely since there is a threshold at which the natural concentration of the substrate

in the surrounding water (background) will become too high to distinguish Also, lowering the KD, will increase the time bacteria need to react to the stimulus At a very low KD, this reaction time becomes too large to successfully bias orientation towards the alga cell The model organism E coli

is able to integrate temporal concentration comparisons in a time span of about 1 s, which is a nearly optimal time for reorientation for cells of that size.23

Another way to increase the detection of algae in the model is a decrease in bacterial swimming speed.34 Slower bacteria will stay longer in a substrate cloud and, therefore, increase the available response time and/or detectable algal size However, again there is a trade-off between increasing sensitivity towards small sources and needing to search a large volume for potential nutrient-rich sources Decreasing swimming speed increases detection of algae only when bacteria are moved

by physical forces (Brownian motion causing rotational diffusion) rather than by chemokinesis Smaller bacteria would be more easily subjected to rotational diffusion, and Jackson’s model34

suggests that the smallest marine bacteria may not even have any chemokinetic response This may

be compensated by a more efficient nutrient uptake rate from the surrounding water since small bacteria have a high surface-to-volume ratio This is in accordance with findings of Dusenbery35

that a minimum size limit exists for useful chemosensory motility Modeling the effect of size on locomotion in relation to different chemical, light, and temperature stimuli yielded bacterial size limits of around 0.6 µm diameter below which motility does not seem to be beneficial

In the marine planktonic environment, however, physical factors other than diffusion will also influence distribution of nutrient gradients and chemosensory interactions between bacteria and leaking algae.30 Turbulence, shear, and sinking of planktonic algae through the water column due

to gravitational and other forces cause a distortion in the symmetric distribution of substrate around the alga.30,36 Mitchell et al.30 estimated that these physical forces will prevent clustering of bacteria around exudating phytoplankton In an extended model, Jackson36 analyzed the chemosensory ability of bacteria to either stay in high substrate concentrations around sinking algae or to attach

to algae During sinking, substrate gradients become steeper upstream and tail off downstream Water velocity is lowest close to the algal surface because of the dragging force In order to benefit from algal substrate release, bacteria would need to stay close to these falling algae This model36

suggests that latency of the chemokinetic reaction is too long, and net movement in a biased random walk too slow, to keep up with sinking algae Increasing algal size, leakage rate, and bacterial swimming speed coupled with decreased algal falling velocity, however, could enhance the contact time between bacteria and algae and, thus, enhance bacterial exposure to elevated nutrient concen-trations.36 It has been suggested that significant enhancement of nutrient uptake due to chemokinetic response is only likely in eutrophic waters due to the specific relationships between algal size, algal abundance, nutrient availability, and slow sinking rates,36,37 and that planktonic interactions in eutrophic and oligotrophic waters may be of fundamentally different nature.36 Also, in low turbu-lence zones such as thermoclines, the lifetimes of nutrient gradients around phytoplankton cells can be long enough for bacteria to chemosensitively track and exploit them.30 Jackson’s model,36

however, indicated that chemosensory reactions are likely to be very important in the context of bacterial attachment to very large falling particles such as marine snow Independent from physical environmental conditions, attachment depends on the encounter of bacteria with a falling particle

As before, an increase or decrease in parameters such as particle size, algal stickiness, falling rate, bacterial swimming speed, binding capacity for substrate, etc can increase bacterial attachment, though there is a transition region where the attachment rate cannot be enhanced further

In contrast to these earlier models, Bowen et al.38 argue that shear, rather than sinking or Brownian motion, is the more important physical force influencing fluid motion for most natural phytoplankton assemblages in turbulent mixed ocean layers Nutrient concentrations around leaking algae are distorted by shear forces at irregular time intervals In modeling bacterial chemotaxis in turbulent waters, Bowen et al.38 referred to the same parameters as Jackson34,36 but applied a higher

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variation in bacterial swimming speed (12 to 80 µm s–1), based on observations of abundant marine

bacteria with polar flagellation Bacterial swimming, however, was still simulated according to the

run-and-tumble enteric bacterial pattern In agreement with the conclusions of Jackson,36 Bowen

et al.38 found that low water motion, short distance to the leaking phytoplankton, high exudation

rates, and a low KD will considerably enhance bacterial approach rate and exposure time to high

substrate concentrations High bacterial swimming speed strongly increases bacterial approach in

turbulent waters However, even with all factors optimized, the simulation indicated that bacterial

clusters will never occupy more than 10% of the substrate cloud volume Largely independent from

strength of shear, individual bacteria are transported into the vicinity of a leaking alga primarily

by water motion and random swimming rather than by chemokinetic behavior Once a bacterium

is close to an alga where fluid motion is weakest and concentration gradients are strongest,

chemokinesis can significantly increase residence time of bacteria near the alga However, physical

forces prevent a bacterium from remaining in a cluster for more than 1 min.38 Bacteria transported

away from one alga may be transported to the vicinity of another algal cell where chemokinesis

can once again be effective Overall, chemokinesis can enhance exposure time to nutrients by a

factor of about 10 over nonchemokinetic behavior, thus giving chemokinetic bacteria a competitive

advantage over nonchemokinetic cells, even in turbulent waters.38 Numerical simulation indicated

that bacterial nutrient uptake rates are unlikely to significantly reduce the high substrate

concen-trations close to algal cells Likewise, bacterial uptake of inorganic nitrogen does not seem to reduce

availability of nutrients for phytoplankton cells.38

Most analytical30 and numerical34,36,38 models of chemokinesis in marine bacteria to date are

based on movement and reaction parameters derived from the well-studied enteric bacteria These

models assume constant movement with changes in speed and direction in the presence of

concen-tration gradients They also assume mean swimming speeds of 10 to 30 µm per second23,34,36 or

80 µm per second,38 but recent analyses of natural assemblages of marine bacteria have yielded

much higher bacterial community swimming speeds of approximately 150 µm per second21,39,40

with individual peak speeds greater than 400 µm per second.21 This high speed mobility is possible

due to lateral and polar flagellation, where the polar flagellum is driven by a sodium-ion motor and

lateral flagella by a proton-motive force.41 Both motors are used simultaneously in marine bacteria,

and the sodium-motive force seems to account for about 60% of the swimming speed.11 Also,

marine bacteria are usually smaller (0.2 to 0.6 µm long26) than assumed in simulation models

(1 to 10 µm)

If small bacteria swim at very high speeds (>100 µm s–1), they are less affected by Brownian

motion,26,39 and chemokinetic reaction time is reduced by an order of magnitude.21 There may be

selection for fast moving cells and short reaction times because nutrient gradients in turbulent ocean

waters erode in a matter of tens of seconds,30 and in order for chemokinesis to function, bacterial

reaction time must be equally short or shorter.21,39,40 High speed, short chemotactic reaction time,

and very short turn times at tumbles enable natural assemblages of marine bacteria to cluster around

small sources of nutrients.40 The limiting factor for bacterial residence time close to high nutrient

patches seems to be more related to the lifetime of such patches rather than to bacterial chemokinetic

mechanisms However, the efficiency of bacterial clustering around nutrient sources has to be

considered within the observations of Mitchell et al.21 that only about 10% of a natural marine

bacteria community was motile when collected Motility was induced by the presence of nutrients

with a lag time of 7 to 10 h and the proportion of motile cells increased to more than 80% of the

population, but only after 15 to 30 h In this sense, on a community level, the significance of

bacterial chemokinesis can be limited by the fraction of motile individuals

Another essential assumption made in almost all previous studies of marine bacterial

chemok-inesis is the run-and-tumble mode of swimming The swimming behavior of marine bacteria near

air bubbles40 and in thin layers near sediments,42 however, suggests that marine bacteria change

direction by reversals much more than by tumbles This back-and-forth strategy was simulated in

a numerical model43 and explained clustering of bacteria around nutrient patches at high shear in

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a more effective way than the run-and-tumble mode Furthermore, rotational diffusion actually

seems to enhance the efficiency of this back-and-forth swimming mode The picture evolving is

that bacteria are brought into the vicinity of nutrient sources randomly by water flow and then

maximize their residence time in the nutrient patch by back-and-forth chemokinetic swimming.38,43

Chemokinetic behavior of marine bacteria has primarily been analyzed in isolation But what

role does bacterial chemokinesis play in the microbial food web? Blackburn et al.44 developed a

simulation model consisting of primary producers, nutrients, DOM, bacteria, and flagellate

preda-tors in order to evaluate the importance of bacterial chemokinesis in the microbial food web Release

of nutrients was supposed to be related to cell lysis or predation events, i.e., incomplete assimilation

or clearance of feeding vacuoles These nutrient sources around phytoplankton existed long enough

before they were physically dispersed that bacteria which clustered around the sources could

consume them.33,40 Simulation predicted that bacterial growth is enhanced by 50% due to

chemo-kinetic clustering in enhanced nutrient patches, emphasizing the adaptive value of chemokinesis in

marine bacteria.44 Results from experimental studies of microbial food webs confirmed the model

prediction that swimming speed is the more important factor influencing efficiency of chemosensory

behavior and foraging, with speed being directly correlated to nutrient patch size.45

There are a variety of compounds released by phytoplankton that could serve as chemokinetic

attractants for planktonic bacteria Bell and Mitchell28 showed that a suite of motile (but

uniden-tified) marine bacteria are attracted to filtrates of phytoplankton cultures but only in cultures old

enough to contain senescent or lysed cells They identified a number of amino acids, polyalcohols,

and sugars in the filtrates, many of which were chemoattractants for one or more of the bacterial

isolates (Table 12.1) Subsequently, amino acids were also shown to be chemoattractants for

planktonic isolates of an unidentified psychrophilic vibrio46 and a Pseudomonas sp., which is also

attracted to sugars47 (Table 12.1) Planktonic bacteria can also be chemokineticly repelled by

TABLE 12.1

Chemoattractants in Marine Bacteria

Heterotrophic Bacteria

Unidentified spp ala, arg, lys, met, val, mannitol, sucrose 28

Pseudomonas sp cys, glu, leu, met, pro, glucose, fructose, mannitol, glactose,

ribose, sucrose, lactose, maltose

47

Pseudomonas spp (pathogenic) glucose, cellobiose, cellotriose, cellulose 50

Vibrio alginolyticus acrylate, glycolate, dimethyl sulfide 53

Vibrio anguillarum glu, gln, gly, his, ile, leu, ser, thr, L -fucose, D -glucose, D -mannose,

D -xylose, taurocholic acid, taurochenodeoxycholic acid

55

Vibrio parahaemolyticus ala, gly, leu, ser 12

Vibrio furnissii ala, arg, asn, gln, gly, cys, his, ile, leu, lys, met, phe, pro, thr,

try, tyr, val, glucose, (GlcNAc)n with n = 1–6, sucrose,

trehalose, mannose, mannitol, galactose

57, 58, 59

Alcaligenes strain M3A dimethylsulfoniopropionate 60

Cyanobacteria

Synechococcus spp. NH4, NO3, urea, β-ala, gly 61

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nontoxic concentrations of copper, lead, tannic acid, benzoic acid, acrylamide, and several organic solvents,48,49 but the ecological relevance of the behavior is difficult to assess for some

of those compounds

Chemokinesis by pathogenic bacteria can play an important role in host–pathogen relationships

Two Pseudomonas spp., one isolated as a pathogen on the marine fungus Pythium debaryanum and the other isolated as a pathogen of the marine diatom Skeletonema costatum, are attracted to

exudates of their hosts.50 Each bacterial species is also attracted to exudates of the other eukaryote,

but to a lesser degree than its own host A nonpathogenic Pseudomonas sp is not attracted to either

host exudate even though it was attracted by nutrient broth at the same level as the pathogenic species.50 Both pathogens are attracted by cellulose and cellulose degradation products (Table 12.1)

with the response much greater in the fungus pathogen The nonpathogenic Pseudomonas sp is

not attracted by cellulose or its derivatives.50 In another pathogenic relationship, unidentified pseudomonads that attack environmentally stressed corals are chemokineticly attracted to mucus that is produced by the corals as part of their stress response.51,52

Vibrio alginolyticus, which can be a pathogen of marine algae, is attracted by the algal

extracellular products acrylate and glycolate as well as by dimethyl sulfide53 (Table 12.1)

V alginolyticus is also a common fish pathogen and is chemokineticly attracted to mucus from the

skin, gills, and intestines of host fish where it attaches to initiate colonization.54 Strains of V fischeri,

V harveyi, and V tubiashii are also attracted, often more strongly than V alginolyticus, by some

or all of the three mucus types but do not adhere.54 V anguillarum is strongly attracted by these

same mucus types as well as mucus from other fish and mammals, and it attaches to fish mucus

to initiate host invasion.54,55 Motility and a functional chemokinetic signal transduction pathway

are known to be required for invasion of a host by V anguillarum.56 Mutants deficient in either motility or chemokinesis have a 500-fold decrease in virulence compared to wild type cells but can cause infections following intraperitoneal injection into a fish.56 Specific chemoattractants include a variety of amino acids, sugars, and bile acids (Table 12.1) Other amino acids and sugars,

as well as other mucus components including a number of lipids, do not elicit significant chemokinetic effects.55 The marine bacterium and human pathogen V parahaemolyticus is also attracted by several

amino acids (Table 12.1) but not by aspartate, acetate, or sugars, and it is repelled by indole.12

The chitinivorous marine bacterium Vibrio furnissii apparently locates chitin sources initially

via chemokinesis to soluble attractants, such as the sugars glucose and trehalose, that would be released by dead animals and fungi57 as well as amino acids58 (Table 12.1) Attraction to these sugars is linked to sugar uptake mechanisms and is particularly strong after induction by individual sugars.57 Chitin is composed of subunits of N-acetyl-D-glucosamine (GlcNAc) which are strongly chemo-attractive as monomers or as oligosaccharides of 2 to 6 subunits58,59 (Table 12.1) Intact chitin is insoluble It has been hypothesized that new sources of chitin are initially colonized by chitinivorous bacteria that locate it by attraction to soluble cues other than GlcNAc, but that once chitin degradation begins, other bacteria are able to find the source via attraction to GlcNAc and its oligomers released during digestion by the initial colonizers.57

Induction of chemokinetic ability also occurs in Alcaligenes strain M3A.60 Dimethyl sulfonio-propionate (DMSP) is an attractant for bacteria that have been induced to produce DMSP lyase Such cells are attracted to DMSP at ecologically relevant concentrations, but uninduced cells do not respond to DMSP Since DMSP lyase produces dimethylsulfide, which is important to global climate regulation, Zimmer-Faust et al.60 suggest that bacterial chemokinesis may play a critical role in global sulfur cycles and climate

Chemokinetic responses have also been reported in marine cyanobacteria (Table 12.1)

Open-ocean isolates of Synechococcus spp., which move by swimming without means of flagella, are

attracted by ecologically relevant concentrations of ammonium, nitrate, urea, β-alanine, and glycine but not by numerous other amino acids, sugars, or vitamins.61 A mat-forming Oscillatoria sp.,

which moves by gliding motility, is attracted by carbon dioxide, bicarbonate, and oxygen.62

Phormidium corallyticum, which likewise moves by gliding motility and which is associated with

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black-band disease of corals, is also attracted by oxygen.63 Its movement is unaffected by ammo-nium, phosphate, acetate, glucose, and fructose, but it is strongly repelled by sulfide.63

III ALGAE

A RESPONSES OF MACROALGAL GAMETES TO SEXUAL CHEMOATTRACTANTS

Gamete chemoattractants (pheromones) are widespread in the brown algae (Class Phaeophyceae) Their structure, function, and biosynthesis have been the subject of a number of comprehensive reviews including those by Müller,64 Maier,65,66 and Boland.67 These chemoattractants have been identified in over 100 brown algal species and include 10 different C11 hydrocarbons and one C8 hydrocarbon67 (Figure 12.1) A total of over 50 different stereoisomers of these compounds have been identified.67 All are volatile and, consequently, form steep concentration gradients around female gametes with effective attractive radii that are probably limited to distances ranging from approxi-mately 500 µm in some species68 to over 1000 µm in others.69 They can often function as male gamete release stimulants in addition to male gamete attractants,64,65,67 and some of these compounds

or their degradation products may have additional roles in chemical defenses against herbivores.70

All of the known brown algal pheromones are involved in the attraction of male gametes to female gametes In most cases, the gametes are morphologically anisogamous or oogamous, but in

some cases, including in Ectocarpus siliculosus (Order Ectocarpales) where brown algal pheromones

were first identified, they may be morphologically isogamous but functionally oogamous because

of their behavior with respect to pheromones This differentiation in function between female and

male gametes in E siliculosus has been recognized since the work of Berthold and others in the

late nineteenth and early twentieth centuries (reviewed by Müller68 and Fritsch 71) but was not understood chemically until the pioneering work of Müller and colleagues in the late 1960s.72,73 The female gametes swim for 30 min or less before settling to the substrate Upon settlement, female

FIGURE 12.1 Pheromone chemoattractants for brown algal male gametes.

Viridiene

O Caudoxirene

Cystophorene

Desmarestene

Finavarrene

Fucoserratene

Hormosirene

O

Lamoxirene Multifidene

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gametes withdraw their flagella and begin to secrete the pheromone ectocarpene64 (Figure 12.1) This production continues at a rate of 105 molecules per second per cell for approximately 7 h.74

Male Ectocarpus siliculosus gametes respond to ectocarpene secreted by the settled females

via a chemokinetic response that has been described as chemothigmoklinokinesis.69,75 Upon detect-ing the pheromone, the male gametes exhibit a strong thigmotactic response which causes them to remain in close contact with the substrate (where the female gamete is presumably settled) and to move along it at reduced swimming speeds.76 Increasing concentrations of pheromone cause the rudder-like posterior flagellum to beat violently at a frequency directly proportional to pheromone concentration These violent beats cause the gametes to make sharp turns and, therefore, to swim

in circles Because of the concentration effect, the diameter of the circles becomes increasingly smaller as the male gametes approach the settled female, effectively trapping the males in the vicinity of females until contact is made.76 The posterior flagellum has also been reported to beat rapidly and unilaterally when pheromone concentration is decreasing, which results in the cells reversing direction.77 This reorientation or phobic response is functionally separate from the chemo-thigmoklinokinetic attraction response and has been called chemoklinotaxis.77 In culture, at least, female gametes can become surrounded by swimming male gametes.71 Initial but firm contact is made by the end of a male gamete’s anterior flagella anchoring to the female gamete’s plasma membrane A single male gamete pulls itself in to the female gamete by retracting its anterior flagellum and then fuses with the female The remaining male gametes release and swim away,71

presumably because the female gamete has ceased ectocarpene production Although ectocarpene

is the functional pheromone for several species of Ectocarpus as well as for members of two other

orders of brown algae,64–67 the initial attachment response is apparently species specific as it does

not occur even between reproductively isolated populations of E siliculosus.78

Because it relies on modulation of the frequency of swimming direction changes, the

chemo-thigmoklinokinetic response of Ectocarpus siliculosus male gametes is superficially similar to the

chemoklinokinetic behaviors described above which are used by marine and other bacteria

How-ever, the behaviors differ markedly since E siliculosus gametes increase their turning frequency

with increasing concentrations of attractant, while bacteria decrease turning frequency as chemoat-tractant concentrations increase This apparent incongruity seems likely to be a result of the very different objectives of the behaviors as well as of the different cell swimming behaviors in the absence of chemoattractants To be successful, a male gamete must locate the pheromone point source, i.e., the female gamete The male gametes swim in basically straight paths in the absence

of pheromones, and their increased turning frequency in response to increased pheromone concen-trations serves to retain them in the vicinity of the settled female gametes until contact is made Bacterial attraction to carbon or other nutrient sources that are taken-up by the cells does not require contact with an attractant point source Indeed, on the spatial scale of a bacterium, the source may well not be an isolated point The signal transduction pathway that controls the behavior of bacterial cells13,18 ensures that direction changes will occur randomly every few seconds and, consequently, that cells will move about in a random walk in the absence of chemoattractants By decreasing the frequency of turns in response to increasing chemoattractant concentrations, the random walk is biased such that net movement occurs in the direction of the source.13

Even though bacteria and Ectocarpus siliculosus male gametes use modulation of direction

changes in contrasting ways, the underlying adaptive advantage of chemoklinokinesis over chemo-taxis may be the same Koshland79 postulated that bacterial chemoklinokinesis is less efficient than the direct approach chemotactic mechanisms utilized by more advanced organisms, but this decreased efficiency is compensated for by the requirement for much less complex sensory and signal-processing mechanisms Although chemoklinokinesis may not be less efficient than chemo-taxis under some conditions,25 an analogous compromise between efficiency and complexity could favor chemokinetic mechanisms in brown algal gametes, particularly those from the Ectocarpales, which is typically considered the most primitive brown algal order.80 Male gametes in one of the

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most advanced brown algal orders, the Laminariales, have a chemotactic response to pheromones,

as described below

The C8 pheromone fucoserratene (Figure 12.1) is produced by female gametes of Fucus spp.67,69

(Order Fucales) The behavioral response of male gametes has been briefly described based on video recordings of male gametes swimming near an artificial pheromone source69 and differs from

the chemoresponsive mechanisms utilized either by Ectocarpus siliculosus male gametes or by bacteria The Fucus male gametes appear to swim randomly except when they are moving away

from the source Gametes that are moving away from the pheromone source execute near-180° turns at a relatively uniform distance from that source It appears, therefore, that when in a decreasing gradient of pheromone, the gametes make these reversals upon crossing some specific threshold concentration of fucoserratene This mechanism presumably traps the male gametes in the vicinity of the females until contact is made, a process called chemophobotaxis.69 A somewhat

similar mechanism has been reported for the response of male gametes of the brown alga Hormosira

pher-omone, the cells swim in helical patterns and turn only rarely Increasing concentrations of hor-mosirene further decrease the frequency of turns, and male gametes make sharp turns when moving away from a pheromone source.81

Lamoxirene (Figure 12.1) is known as the male gamete chemotactic attractant in a large number

of species in the more advanced families of kelps (brown algae in the Order Laminariales) and also stimulates male gamete release.82 The specific chemotactic mechanism has been described in detail based on video analysis.83 In addition to a thigmotactic response that causes the male gametes

to remain in contact with the substrate, the gametes exhibit directed movement towards the pheromone source (i.e., true chemotaxis) The specific mechanism is a phobic response When a male gamete begins to move away from the source, it beats its posterior flagellum such that it makes a greater than 90° turn which helps it reorient towards the general direction of the settled female gamete.83

Although a single pheromone is usually (but not always84) responsible for the attraction or release of gametes from specific brown algal species, additional brown algal pheromones are often produced by those species For example, ectocarpene, hormosirene, and dictyotene (Figure 12.1;

attractant in Dictyota spp., Order Dictyotales) are produced in combination with most of the other

brown algal pheromones.67 While these might only be phylogenetic remnants involved in the biosynthetic pathways of the active pheromones,67 their potential attractiveness to gametes of competitive (or biofouling) brown algae led Müller68 to speculate on possible allelopathic roles for them Though purely conjectural, pheromones produced by gametes could potentially “misguide” male gametes of competing or fouling brown algae away from conspecific female gametes and thereby decrease their reproductive success.68 Likewise, pheromones such as lamoxirene that stim-ulate the release of gametes in multiple species could have an allelopathic role if pheromone release

by female gametes of one species induced male gametophytes of a competing species to release their gametes before the female gametophytes of the competitor matured.68 Amsler et al.4 suggested

that such phenomena could explain the competitive dominance of the kelp Pterygophora californica over the kelp Macrocystis pyrifera in field experiments that investigated the consequences of

variable spore settlement on patterns of sporophyte recruitment.85

Chemotactic or chemokinetic responses are almost unknown in gametes of other marine

mac-roalgae Male gametes of the green macroalga Bryopsis plumosa are attracted by mature female

gametangia and female gametes.86 The presence of a chemical attractant has been confirmed with cell-free bioassays, but its chemical nature has yet to be elucidated.86 Similar gamete attraction has

been reported in the closely related species Derbesia tenuissima,87 but a chemical basis for this behavior has not been established Likewise, gamete attraction without known chemical basis has

been described in several species of the yellow-green alga Vaucheria.69 Gamete attraction, in some cases mediated by known pheromones, has been reported in a number of freshwater green microal-gae and macroalmicroal-gae.65

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