Published: September 26th, 2018
Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) 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.
Intertidal copepods of the genus Tigriopus are widely distributed in rocky high intertidal pools across several continents1. These copepods exhibit mate-guarding behavior as a part of their reproduction, where an adult male captures a potential mate (juvenile or adult) utilizing its hooked first antennae prior to copulation (Figure 1 and Figure 2)2,3,4,5. Although this phenomenon has been a subject of ethological and biochemical studies for decades2,3,6,7, detailed procedures for studies of this behavior, including individual culture of virgin animals and criteria of behavioral events seen in a mate-guarding attempt, have not been well described. Thus, here we establish methods to enable studies of the behavior under a controlled experimental environment.
Individual culturing and staging of animals
Previous studies of reproduction of Tigriopus conventionally employed a pair detaching method to prepare juveniles (copepodids) and adult females for behavioral tests and breeding experiments3,8,9,10. However, this method allows animals to form pairs and potentially to copulate prior to tests (discussed in Ito 198811), which may alter behavioral properties of animals5. In addition, there is also a potential to misjudge developmental stages of copepods with the conventional protocols as they rely on apparent body size for staging. In this paper, we describe an individual culturing method used in our recent study with Tigriopus californicus5, which was designed to address these limitations by controlling pairing experience of animals and tracking their development from copepodid to adult stages.
Quantitative analysis of mate-guarding behavior
The mate-guarding behavior of Tigriopus species has been studied not only in the field of ethology2,6 but also in other fields such as ecotoxicology and evolutionary genetics3,4,7,8,9,10. However, the previous studies have explained the course of this behavior mainly in written forms without sufficient visual illustrations to outline both the behavior and the methods to study it, which creates technical obstacles for the replication and advancement of the studies. Here, we provide detailed descriptions of some of the key events in the mate-guarding behavior of Tigriopus copepods supported by visual materials. We also demonstrate equipment and methods for quantitative analysis of the behavior. These methods allow evaluation of behavioral properties of animals during mate-guarding attempts in precisely replicated experiments.
With these methods we aim to provide a methodological basis of controlled and reproducible studies on the mate-guarding behavior of the genus Tigriopus.
1. Preparation of Virgin Animals for Behavioral Observation
2. Behavioral Test and Video Recording of Mate-guarding Behavior
3. Manual Analysis of Behavioral Properties
4. Two-dimensional Tracking of Individuals and pairs
The individual culturing method described in step 1 allows preparation and staging of virgin animals with no prior experience of pairing.
The behavioral test described in step 2 allows video recording and observation of the mate-guarding behavior of Tigriopus copepods. The following examination of the recorded video with the methods described in steps 3 and 4 enables quantitative analysis of several aspects of the behavior shown in Figure 1.
Figure 11 shows a difference in average duration of guarding attempts between male-female pairs and male-male pairs of T. californicus. A manual analysis demonstrated that male-male pairs showed relatively shorter duration of pairing than male-female pairs.
Examples of trajectories tracked with the analysis method are presented in Figure 12 and a representative result of the velocity analysis is shown in Figure 13. Two-dimensional spatial tracking of newly formed guarding pairs of T. californicus revealed that male-male pairs tended to show higher velocity than male-female pairs in the first 3 s of guarding attempts.
Figure 1: Mate-guarding behavior in Tigriopus. (A) An adult male clasping a juvenile (copepodid) with the first antennae (indicated by blue arrows). Bar = 1 mm. (B) An outline of mate-guarding behavior. A male tries to capture a target individual (left) and forms a pair with it (middle). In male-male pairs and some male-female pairs, a guarding attempt terminates without copulation. This figure has been modified from Tsuboko-Ishii and Burton 20175. Please click here to view a larger version of this figure.
Figure 2: Developmental stages of Tigriopus. Tigriopus species generally undergo six nauplius stages (from NI to NVI), five copepodid stages (from CI to CV), and an adult stage (CVI)16. Males make guarding attempts to juveniles from an early stage of copepodids (CI in T. japonicus and T. fulvus6,17 and CII in T. californicus3), as well as to adults of both sexes5. This figure has been modified from Tsuboko-Ishii and Burton 20175. Please click here to view a larger version of this figure.
Figure 3: Morphology of adults (T. californicus). (A) Adult male. (B) Adult female. (C) Gravid adult female with an egg sac with fertilized and developed (clear orange) eggs. (D) Gravid adult female with an egg sac with unfertilized or undeveloped (dark green) eggs. Arrows indicate egg sacs. Bar = 1 mm. Please click here to view a larger version of this figure.
Figure 4: Outline of preparation for individual culture. Please click here to view a larger version of this figure.
Figure 5: Molted exuviae. (A) Exuviae from CI to CV stages of a single animal of T. californicus. Bar = 1 mm. (B) Exuviae from CI to CV stages in an individual culture well. White debris is excrement of an animal cultured in the well (not shown in the image). Please click here to view a larger version of this figure.
Figure 6: Search for exuviae under a stereomicroscope. Bars = 1 mm. (A) Focal change of a stereomicroscope for detection of exuviae at different depths. Magenta arrows indicate focused exuviae and gray arrows indicate unfocused exuviae. Top image focuses on the left exuviae, which is sunken at the bottom of the well. Bottom image focuses on the right exuviae, which is floating beneath the medium surface. (B) Top image shows an example of obstruction of examination by debris floating on the medium surface. A green arrow indicates an exuviae hidden beneath the debris. Bottom image shows a result of surface cleaning with a small piece of paper towel. An exuviae (indicated by a green arrow) is visible after the cleaning. Please click here to view a larger version of this figure.
Figure 7: Staging and sexing of animals. Examples of how to mark number of exuviae and sex of animals on a lid of a culture plate. (A) An example for a well containing five exuviae and an adult animal. The number of the lines in the tally mark on the lid represents the number of the exuviae found in the well. When the number of exuviae reaches five, the sex of the animal can be determined based on antennae morphology (see also Figure 3). (B) An example of a marked lid of a culture plate. Top rows contain older (CIV to adult) animals and bottom rows contain younger (i.e., newly collected) animals (CI to CIII). Please click here to view a larger version of this figure.
Figure 8: Behavioral test scheme. Setup for video recording of mate-guarding behavior (left) and an outline of behavioral test (right). This figure has been modified from Tsuboko-Ishii and Burton 20175. Please click here to view a larger version of this figure.
Figure 9: Rinse of animals prior to a behavioral test. Please click here to view a larger version of this figure.
Figure 10: Definition of events examined in the manual analysis. Illustrations of events observed in relation to mate-guarding attempt. Names of the events defined and analyzed in step 3 are highlighted. Events enclosed in a dotted line may not be observed in some mate-guarding attempts. Please click here to view a larger version of this figure.
Figure 11: Difference in guarding duration between male-female pairs and male-male pairs of T. californicus. Each triangle symbol represents data from one tested pair. Bars and whiskers represent medians and interquartile range respectively. Average duration of capture was greater for male-female pairs (male-female (n=22), male-male (n=29); **p < 0.01 by Mann-Whitney U test). Please click here to view a larger version of this figure.
Figure 12: Trajectories of pairs in the first three seconds of guarding attempts. Examples of tracked two-dimensional trajectories of male-female pairs (left) and male-male pairs (right) of T. californicus. Dots on the trajectories represent time points (30 frames per second). Bars = 10 mm. Please click here to view a larger version of this figure.
Figure 13: Difference in mean velocity after initiation of guarding between male-female pairs and male-male pairs of T. californicus. Each triangle symbol represents data from one tested pair. Bars and whiskers represent medians and interquartile range respectively. Average velocity of the pair in the first 3 s of guarding attempts was greater for male-male pairs (male-female (n=13), male-male (n=35). ***p < 0.001 by Mann-Whitney U test). Please click here to view a larger version of this figure.
Supplemental Figure 1: Effect of rinsing treatment on behavior of Tigriopus. Each circle or triangle symbol represents data from one tested individual or pair. Bars and whiskers represent medians and interquartile range respectively. Individuals in "rinsed" and "not rinsed" groups were handled in the same manner, except that the "not rinsed" group did not experience the rinsing treatment (step 2.1.3) before the 30-minute adjustment time (step 2.1.4). (A) Average velocity of rinsed males tended to be greater than that of not rinsed males (rinsed (n=6), not rinsed (n=6), n.s.: no significant difference was detected by Mann-Whitney U test). The velocity was measured for 30 s based on videos recorded after the adjustment time. (B) Average velocity of rinsed females tended to be greater than that of not rinsed females (rinsed (n=6), not rinsed (n=6), n.s.: no significant difference was detected by Mann-Whitney U test). The velocity was measured for 30 s based on videos recorded after the adjustment time, following step 4 (tracking interval = 0.5 s). (C) Frequency of guarding attempts was greater for pairs of rinsed individuals (rinsed (n=6), not rinsed (n=6). ** p < 0.01 by Mann-Whitney U test). (D) Duration of guarding attempts tended to be greater for pairs of rinsed individuals (rinsed (n=6), not rinsed (n=6), n.s.: no significant difference was detected by Mann-Whitney U test). Please click here to view a larger version of this figure.
Individual culturing and determination of stage and sex
Here we described the method used in our previous study5 to prepare virgin Tigriopus animals with their pairing experience controlled while tracking their development (Figure 4 and Figure 7). As Tigriopus species are utilized as model animals in various biological fields such as toxicology16,18, ecological physiology19,20,21, and evolutionary genetics13,22,23,24, this method has a potential to provide a valuable means to assess influence of environmental and genetic factors on the life cycle of these copepods.
To achieve successful staging, regular search and collection of CI copepodids from a mass culture (step 1.2) is critical, as collection at later stages may result in mis-staging of the animals. In addition, a thorough search for exuviae (step 1.3) is also essential for accurate staging. Increase the frequency of collection and staging if necessary, as the interval between molts varies from one to several days depending on species and rearing condition2,25,26. Differences in antennae morphology between copepodids and adult females are not visibly significant in some species and populations of Tigriopus16,25. Hence, staging prior to sexing is helpful to distinguish adult females from advanced copepodids of either sex.
Behavioral tests and manual analysis of behavioral properties
In general, one of the most critical parts of ethological studies is definition and description of events of interest. The methods introduced in this paper were first developed for our recent study5 and supplemented with the description of copulation and the visual aids (Figure 10). In addition to that, consistency in animal handling also plays an important role in behavioral experiments. For example, rinsing of copepods may potentially facilitate some aspects of their behavior (Supplemental Figure 1) and therefore is desirable to be performed in a consistent manner among samples, such as standardized in step 2.1.3. We expect the materials provided in this paper will assist controlled and reproducible studies on the mate-guarding behavior of Tigriopus, promoting reproductive and ecological studies of this abundant inhabitant of high tide pools.
One possible limitation with this method is low magnification of obtained images. Although movies recorded with our system allow identification of prominent body structures including urosomes and male first antennae, one may not be able to observe more subtle structures such as legs and genitalia with our method since it does not employ microscopic magnification for video recording. While Kelly et al. reported that they were able to observe spermatophore transfer from males to females of T. japonicus under a microscopic observation at 100X magnification (no video recorded)7, we have not been able to observe a spermatophore in our movies, perhaps due to the limitation of the image resolution.
Two-dimensional tracking of pairs
Although this method does not allow three-dimensional tracking of animals, it enables two-dimensional trajectory analysis of zooplankton without chemically labeling animals (cf. Lard et al. 201027) by utilizing programs distributed for free. If the size of a movie file is too large to be processed in ImageJ, one can reduce resolution of the file and convert it into a gray scale movie. While the described method was originally developed for adult pairs of T. californicus (Figure 12 and Figure 13), it is also available for adult-juvenile pairs and single individuals (Supplemental Figure 1) as well as other Tigriopus species in principle. We further expect the method to be applicable to short-term (from the millisecond to the second time scales) trajectory analysis of zooplankton of other taxa with appropriate adjustments.
The authors have nothing to disclose.
This work is supported by grants from the Sumitomo Foundation, Japan (Grant for Basic Science Research Projects, grant number: 150932) and Research Institute of Marine Invertebrates, Japan (2018 individual research grant) to STI and RSB, and a grant from the US National Science Foundation (DEB-1556466) to RSB. We thank Ms. Kiana Michelle Woodward for feedback on the culturing and staging method.
|Instant Ocean Sea Salt
|Spectrum Brands. Inc.
|For preparation of culture medium
|Food for copepods (used after being ground in a mortar)
|Flat bottom 6-well tissue culture plate with lid
|Corning Co., Ltd.
|Container for culture of gravid females and hatched nauplii
|Flat bottom 24-well cell culture plate with lid
|Corning Co., Ltd.
|Container for individual culturing
|Flat bottom 48-well cell culture plate with lid
|Nest Biotechnology Co., Ltd.
|Behavioral observation chambers
|LED light pad
|Shenzhen Huion Animation Technology Co., Ltd.
|Backlight for behavioral observation
|0591C003 (model: Rebel T6i)
|For recording of behavior
|For transfer of copepods
|P10 micropipette tips
|For transfer of C1 stage copepodids
|For semi-automatic analysis of movies
|ImageJ plugin for tracking developed by Dr. Erik Meijering (Biomedical Imaging Group Rotterdam, Erasmus University Medical Center, The Netherlands)
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