The non-flagellated male gamete is free and vigorously motile, propelled by pseudopodia. However, the cues if any in their search for compatible female gametes and the general search patterns to locate them are unknown. We tracked and compared male gamete movements in the presence and absence of receptive female gametes.
Path linearity of male movement was not affected by presence of female gametes. Male gametes did not move towards female gametes regardless of their proximity to each other, suggesting that the detection range for a compatible mate is very small compared to known algal examples mostly spermatozoids and that mate recognition requires near contact with a female gamete. We therefore investigated how male gametes move to bring insight into their search strategy and found that it was consistent with the predictions of a random-walk model with changes in direction coming from an even distribution.
We further investigated the type of random walk by determining the best-fit distribution on the tail of the move length distribution and found it to be consistent with a truncated power law distribution with an exponent of 2.
This is somewhat surprising because female gametes were often outnumbered by male gametes, thus contrary to the assumption that a Brownian search mode may be most optimal with an abundant target resource. This is also the first mathematically analysed search pattern of a non-flagellated protistan gamete, supporting the notion that principles of Brownian motion have wide application in biology.
In complex, heterogonous environments, mobile organisms are confronted by the daunting task of finding resources food, host, sexual partner, etc. To do so they use a wide variety of cues to locate target resources from a distance. However, these cues can typically only be detected within a specific range.
In the absence of cues, various types of random movement may be used to increase the probability of an encounter [1] , [2] , [3]. The optimal search strategy seems to be dependent on the abundance and distribution of the target resource [3] , [4] , [5]. When it is sparse, highly directed movement e. On the other hand, when the target is abundant and randomly distributed, a series of randomly oriented moves with little variation in move length, i.
Individuals may switch from one search strategy to another in response to a change in the environment [7] , [8] , [9]. Although sensing environmental cues e. To our knowledge only a few such studies have been published thus far, and exclusively for raphid pennates. One such work involved a motile, raphid pennate diatom [10] which demonstrated positive chemotaxis to specific sugars, similar to that observed in a number of green and other brown algae [11] , [12].
For fertilization to occur, gametes have to meet and also recognize each other as compatible partners. Among diatoms, flagellated sperm in centrics are credited with the capacity to move towards an immotile egg [13].
In raphid pennate species, sexualized motile gametangia find each other and pair, usually following sex-induction cues [14] and within a specific cell-size range [15]. Recently, the behavioural, directional response to identified chemical cues exuded by a sexual partner was documented for a species of Seminavis , a motile raphid pennate [16] , joining a relatively small volume of work of well-documented cases from other brown macroalgae [12] and various microalgae [17].
In contrast to other protists, including a few microalgae, the movement pattern of cells responding to chemical stimuli including compatible mates is yet to be analyzed mathematically for any diatom. Tabularia fasciculata C. Agardh D. Williams and Round is one of only a few araphid pennate species known to produce vigorously motile, free, non-flagellated male gametes [18] , [19] , [20]. Male gamete motility in two Tabularia species coincides with extrusion and retraction of pseudopodia, thereby making gametes capable of spinning, shuffling and making jump-like displacements over a distance much greater than the diameter of the gamete [18] , [19].
Therefore, they are a good candidate to investigate the male gamete search method for locating a receptive sex-partner. In largely immotile vegetative cells of araphids, parents-to-be are believed to be passively carried to the vicinity of each other by water turbulence. The absence of flagellated sperm and inherent immotility of parental gametangia of most of the araphid pennate species would seem to place the prospects for syngamy for araphid diatoms at a disadvantage relative to centrics and raphid pennates.
Therefore we compared the movement of male gametes in presence and absence of stationary female gametes to determine whether males can detect and consequently orient themselves towards potential mates.
After determining they could not, we evaluated what type of search strategy may be used by males to enhance the probability of their encounter with a compatible female gamete. The significance of our results is discussed in the context of diatom reproductive and evolutionary biology.
Tabularia fasciculata is a benthic diatom typically growing on filaments or blades of small intertidal macroalgae. It is a colonial species forming pin-cushion clusters of needle-shaped cells [21]. Its sexual reproduction is primarily heterothallic [22]. The entire process and cells involved are described in [18]. Male and female gametogeneses and syngamy are readily inducible [21] and were observed numerous times in this and other studies.
Two gametes are produced by each male and female gametangium [18] , each gamete with sex-specific morphology and behavior. Male gametes are released into the environment where they can move freely and often vigorously by means hitherto unknown among diatom gametes and possibly in any gamete in general [18] , [19].
Female gametes stay within maternal thecae that are attached to the substratum mating well bottom throughout auxosporulation. Fertilization results in a zygote, which elongates becoming an auxospore more or less parallel to the maternal theca, eventually producing a progeny initial cell with a large frustule. We routinely observed all these stages in the course of our experiments, confirming that gamete behavior and resulting zygotes observed in this study conform to the normal sexual process for this species.
Measurements involving female gametes in proximity to male gametes were more frequent in the latter half of the experiments, thus involving more slightly smaller gametes. The three clones of Tabularia fasciculata used in this study were isolated using micropipetting methods as described in [21].
All three clones are genetically and morphologically characterized in [25]. Stock cultures were transferred weekly to maintain exponential growth phase. Immediately prior to mating, the flasks with newly transferred cells of parental clones were first kept for one day in the same growth conditions as the stock described above. Parents were then mixed together in pair-wise fashion in 9 replicate wells and two control wells each with only one parent clone in well trays.
Terminology associated with sex cells and processes follow [22]. Reconnaissance mating experiments showed that the optimal time for gamete movement observation was after 48 hours incubation of mating parents in the growth chamber.
Male gamete movement paths were recorded as a time-lapse video captured using a QImaging MicroPublisher 3. Each video capture consisted of 50 frames, each 15 seconds apart, viewed through a 20x objective lens for the gametes that remained within the focal plane of this objective 5. Given the typical diameter of the observed gametes, this criterion essentially restricted data collection to gametes moving in two dimensions.
Occasional recordings where the male gamete moved out of the focal plane up or down in the well medium were disregarded. An image of a larger field of view using a 10x objective was taken immediately prior to video recording to record the cell layout to be used as a reference for the start positions of all sex cells involved. Only one video was captured per well to minimize photic exposure of the gametes. The most vigorously motile male gamete was selected in each recording to represent performance with the greatest potential for syngamy because the age of individual male gametes and possibly age-dependant vigour could not be measured.
Representative trajectories for each experimental condition are shown in Fig. ImageJ v. Steps and turning angles were calculated using these coordinates for every fourth frame one minute intervals in order to avoid oversampling [2]. Each step was calculated using the Pythagorean distance formula with the current frame and previous frame coordinates to obtain the distance traveled from the previous point Figure 2. The turning angle was measured as the difference between the direction the cell traveled during the previous time interval and the direction for the current time interval Figure 2.
If the turn was counter clockwise from the starting direction, the angle was within the range [0,]. A Trajectory of male gamete in the absence of receptive female gamete. B Trajectory of male gamete in the presence of receptive female gamete. Plots represent position of male gamete at each of the 15 second intervals of time-lapse imaging 50 frames over Velocity for the particular male gamete in A ranged between 0. The sign of the x-values in A and the y-values in B have been changed so that trajectories are in a similar orientation and directly comparable.
Dots represent position of the gamete at one-minute intervals, d t shows the move lengths, and t t shows turning angles. Velocities were logtransformed to correct for problems with the assumption of homogeneity of variances. We computed a linearity index defined as the ratio of net displacement distance between starting and end point and gross displacement sum of distances traveled within each minute step.
The index ranges from 0 to 1, with values close to 0 corresponding to very convoluted paths and values close to 1 corresponding to a directed movement. We also compared the linearity index known also as the McCutcheon Index [26] , in relation to presence of female gamete with a t-test. To determine whether male gamete movement was directed towards female gametes, we conducted an analysis of orientation.
We first determined the direction that would lead the male gamete to the closest female gamete, i. We then determined the net direction of the male gamete movement, i. We then conducted a Rayleigh test on the mean vector [27] to determine if the direction of male gamete movement, in relation to position of the female gamete, deviated from a random orientation.
Finally, to determine if proximity of a female gamete influences the direction of male gametes, we conducted a linear regression analysis with absolute deviation absolute value of the deviation described above as the dependent variable and distance between the male and female gametes at the start of a video as the independent variable.
We compared the realized paths with the predictions of random-walk models in which the direction of a step is independent of the direction of the previous step. This was done to evaluate the possibility that gametes use correlated random walks, i. This would result in superdiffusivity and would be detected by a poor match between predictions of a random walk model and observed displacements.
A model was fitted for each of the two treatments female gamete present or absent. The expected net square displacements were calculated by generating random paths.
A path consisted of 12 steps describing the position of gametes at one-minute intervals. The distance moved during each step was randomly selected from the empirical distributions obtained from the videos.
The direction of subsequent moves was determined by randomly drawing a turning angle coming from the same distribution. We then computed the net square displacement squared distance from the initial position after each step. These reproductive cells are produced through a type of cell division called meiosis. During meiosis, a diploid parent cell, which has two copies of each chromosome, undergoes one round of DNA replication followed by two separate cycles of nuclear division to produce four haploid cells.
These cells develop into sperm or ova. The ova mature in the ovaries of females, and the sperm develop in the testes of males. Each sperm cell, or spermatozoon, is small and motile. The spermatozoon has a flagellum, which is a tail-shaped structure that allows the cell to propel and move.
In contrast, each egg cell, or ovum, is relatively large and non-motile. Plants Latest Protocol Video. Author Map. Protocols in Current Issue. By Date By View. Lopez , Katayoun Mansouri , Jason S. Henry , Nicholas D. Flowers , Kevin C.
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