Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior.

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics

Prezygotic reproductive barriers in precopulatory behavior of tidepool copepod species.

Complexity in prezygotic mating behavior can contribute to the emergence of sexual incompatibility and reproductive isolation. In this study, we performed behavioral tests with two tidepool copepod species of the genus Tigriopus to explore the possibility of precopulatory behavioral isolation. We found that interspecific mating attempts failed prior to genital contact, and that this failure occurred at different behavioral steps between reciprocal pairings. Our results suggest that prezygotic barriers may exist at multiple points of the behavioral process on both male and female sides, possibly due to interspecific differences in mate-recognition cues used at those "checkpoints". While many copepod species are known to show unique precopulatory mate-guarding behavior, the potential contribution of prezygotic behavioral factors to their isolation is not widely recognized. The pattern of sequential mate-guarding behaviors may have allowed diversification of precopulatory communication and contributed to the evolutionary diversity of the Tigriopus copepods

Sex-specific rejection in mate-guarding pair formation in the intertidal copepod, Tigriopus californicus.

Securing a potential mate is one of the most important processes in sexual reproduction of animals. Intertidal copepods of the genus Tigriopus show mate-guarding behavior where a male captures a female and continues to clasp her for up to two weeks prior to copulation. Although these copepods form a mate-guarding pair between a male and a female with high accuracy, interactions between the sexes in pair formation have not been well described and the mechanism allowing successful male-female pair formation is not yet understood. In this study, we performed experiments with Tigriopus californicus to analyze the behavior of both a capturer (male) and a captured individual (female or male) in formation of a guarding pair. While capturer males were attracted by both females and males, capture of virgin males was terminated in a significantly shorter time than that of virgin females. However, when presented freshly killed females or males, regardless of the sex of the body, capturer males continued to clasp the body for a comparable time as in an attempt on a living female. Our results suggest that a sex-specific rejection signal actively sent by captured males prevents male-male pair formation. Experiments also suggest that mated females reject an attempt of pair formation. To our knowledge, this is the first study to suggest involvement of active rejection by a captured individual in facilitation of reproductively successful male-female guarding pair formation in the genus Tigriopus

Reduction of attraction and sex-specific capture interruption in dead targets.

<p>(A) Frequency of guarding attempts. Male to living female (n = 13); male to living male (n = 12); male to freeze-killed female (n = 10); Male to freeze-killed male (n = 13). Triangle symbols and whiskers represent means and SD respectively. ***p<0.001 on treatment (freeze-killed or not) by two-way ANOVA. No significant effect of sex and no significant interaction between treatment and sex were detected (p = 0.24 and p = 0.52). (B) Average duration of capture. Male to living female (n = 13); male to living male (n = 12); male to freeze-killed female (n = 10); male to freeze-killed male (n = 13). Each triangle symbol represents data from one tested pair. Bars and whiskers represent medians and IQR respectively. **p<0.01 by Mann-Whitney <i>U</i> test. No significant difference was detected between male to freeze-killed female pairs and male to freeze-killed male pairs by Mann-Whitney <i>U</i> test (p = 0.097). No significant effect of sex and no significant interaction between treatment and sex were detected (p = 0.35 and p = 0.24).</p

Outlines of mate-guarding behavior and the behavioral tests.

<p>(A) An adult male (capturer: indicated by white outlined arrowheads) clasping a juvenile (target: indicated by red arrowheads) with antennae (indicated by blue arrows). Bar = 1 mm. (B) Setup for video recording of guarding attempts. (C) An outline of behavioral tests. (D) Still image from a video recording. Arrowheads were added to indicate tested animals. The left well has unpaired individuals (two males) and the right well has paired individuals (a male and a female).</p

Difference in duration of capture depending on mating status of a target.

<p>Each symbol represents data from one tested pair. Bars and whiskers represent medians and IQR respectively. (A) Frequency of guarding attempts. Male to virgin female (n = 13); male to previously mated female (n = 9). No significant difference was detected by Mann-Whitney <i>U</i> test (p = 0.79). (B) Average duration of capture was greater for pairs with a virgin female target (male to virgin female (n = 13), male to previously mated female (n = 9). *p<0.05 by Mann-Whitney <i>U</i> test).</p

Difference in guarding attempts between male-female pairs and male-male pairs.

<p>Each triangle symbol represents data from one tested pair. Bars and whiskers represent medians and interquartile range (IQR) respectively. (A) Frequency of guarding attempts. Male to female (n = 32); male to male (n = 54). No significant difference was detected by Mann-Whitney <i>U</i> test (p = 0.13). (B) Average duration of capture was greater for male-female pairs (male to female (n = 32), male to male (n = 53). ***p<0.001 by Mann-Whitney <i>U</i> test).</p

Role of antennae.

<p>Each triangle symbol represents data from one tested pair. Bars and whiskers represent medians and IQR respectively. (A) Frequency of guarding attempts. Control surgery male to intact female (n = 14); antennae cut male to intact female (n = 16). ***p<0.001 by Mann-Whitney <i>U</i> test. (B) Average duration of capture. Control surgery male to intact female (n = 13); antennae cut male to intact female (n = 2). Statistical analysis was not performed since there were only two individuals that showed a capturing attempt in the antennae cut group (n = 16, also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183758#pone.0183758.s007" target="_blank">S1 Fig</a>).</p