Mating Behavior in Littorina planaxis Philippi. Daniel G. Gibson, III Hopkins Marine Station of Stanford University Pacific Grove, California The habitat of the grey periwinkle, Littorina planaxis Philippi (1847), well above high water on rocky western North American shores, exposes this snail to desiccating sun and wind and high temperatures to a greater degree than most other intertidal organisms (Ricketts and Calvin, 1952). This degree of exposure presents several problems to the process of re- production. Fertilization is internal, which requires that males must have some means of locating females, recognizing them as such, and mating with them. This paper will attempt to explore the mating behavior of Littorina planaxis and ex- plain its method of locating other individuals, differences in the behavior of males and females, and the anatomy and mechanics of copulation. Studies were made at Hopkins Marine Station, Pacific Grove, Calif., during April and May, 1964. Egg-laying and copulating individuals were seen throughout this period. Ricketts and Calvint (1952, p.13) state that some individuals can be found copulating at any time of year, but spring and summer seem to be the times for heavy occurrences of mating. Locating of Mating Partners All movement and any subsequent locating of mating partners in Littorina planaxis occurs when the rocks on which these winkles are living are moist. This may be at night or at any time during the day, and may take place even in direct sunlight. Tide height and wave action are two of the main factors that govern rock wetness. Males and females follow mucus trails of other individuals over moist rock, with ap- parent disregard for the sex of the snail that laid down the trail they are following, using their cephalic tentacles to feel their way along (Peters, 1964). Such following of mucus trails has selective advantage, since it leads winkles to other winkles, which may be potential mating partners. Pairing Behavior Males differ from females in that they immediately climb upon the shell of any other Littorina planaxis individual they encounter, usually over the head end of the shell and often after touching tentacles with the other snail. Once on the shell, the male will migrate to the right side and insert his penis into the mantle cavity of the snail beneath him, assuming the the pairing position shown in Figure 1. This position is necessary for copulation, since the genitalia in both sexes are found on the right side, behind the head (for a complete illustration of Littorine anatomy, see Fretter and Graham, 1962, Chapter 2, pp. 14-48). When the animal below is a female, copulation may ensue. However, it appears that success or failure of the tip of the penis in contacting the bursa copulatrix of the female mantle cavity is the only way in which a male can tell whether he has encountered a female or another male. A male exhibits no defensive reaction to having his mantle cavity explored by the penis of another male, but the male above will soon climb off and seek another mate if his probings do not meet with success. Observations were made of 32 males placed on a moist rock in the laboratory and watched for an hour and a half. No females were present. During this time, 12 pairs formed, and the length of time that each remained in the pairing position was recorded. None of the male-male pairs observed persisted for more than 8 minutes, and more than half of them broke up in two minutes or less. Be- canselof theiryshort duration, male-male pairseare nõt prevalent indthetfield atcanyionentime.1 Paralleliobservations ofg 32 females inathe absence ofmales under the above conditions yielded no pairing or climbing on one another's shells. This apparent trial-and-error methodcof finding a mate could be of great selective advantage in the range where Lit torina planaxis lives. A male becomes aggressive if he comes into contact with another male on the shell he is exploring or intending to explore. Ten battles resulting from such circumstances have been observed, lasting between 30 seconds and 3 minutes, con- sisting of the males jerkily pushing each other with the head ends of their shells until one is dislodged.. Figure 2. shows such a battle in progress. In two cases, the snail the fighting males were on was also a male, but they apparently did not know it, for the victor in each case attempted copulation with the snail beneath him. Often a third snail will be found very close to or touching two paired individuals. In 50 checks of the sex of this third individual, 48 were males, 2 females. The proximity of the second male to the pair might be due to the fact that he has just been pushed off the female and has not yet left the area, or it might suggest a chemo-attraction of males to the vicinity of females. Normal (heterosexual) pairs persist for quite some time after formation, often spanning an entire moist-rock activity period of 10 hours or more. A field study of pairiformation and persistence over one activity period is shown in Figure 3. Pairs are seen to form over a wide range of dark and daylight — 12 Figure 2. Mating battle; Figure 1. Pairing position; males push each other with female from above, male from head ends of shells until left side. Part of male s one is dislodged. X2. foot visible. During pairing, male's foot is entirely on the other shell. X3. KEY Figure 3. Field study of formation -paired and persistence of pairs during a moist-rock ac- tivity period. Pairs formed broke up are seen to form over =========== Same 9 wirh - — a wide range of dark new d and daylight hours and to persist for greatly varying lengths of time. Some animals paired more than once (with dif- ferent partners) during the same activity period. aaaaaaaa. — — „----- == - 9AM 3AM 6 AM -DAYLIGHT hours, and are seen to persist for greatly varying lengths of time. A certain percentage of snails are to be found paired, with different partners, more than once during the same activity period, as Figure 3. also shows. The amount of pairing occurring at any time in the field seems to be affected by the level of the tide. Figure 4. shows two pairing frequency studies made over 25-hour tide cycles a week apart, in order to show that the time shift in the tide level causes a concomitant shift in the time of day that the greatest and least numbers of individuals are paired. The area observed was about 1 meter square in area, containing about 100 Littorina planaxis individuals. Each hour the num- ber of pairs and single winkles in the area was counted, the percentage pairing calculated, and plotted. The second week, as the tide came in later in the day, pairing percentage also peaked later. In both studies, pairing percentage rose as the tide came in, and then began to fall as the tide rose further. An increasing amount of splash, capable of dislodging paired snails, since in a pair only the female's foot is attached to the substrate, may account for this. MAA2 14 50 u 0 om a -7 6- 950 —— 254 OL Figure 4. Two 25-hour field studies of percentage pairing in relation to tide level, made one week apart in order to illustrate that a shift in the time of tide-level peaks is accompanied by a time-shift in pairing peaks. Overall drop in pairing the second week may have been due to ex- tremely calm conditions, with little splash. K 4 6 Fig. 5a. Male ex¬ 5b. Beginning of in- 5c. Penis deep in mantle tending penis prior sertion. Front edge cavity of female, as during to copulation. It is pushed forward and sperm transfer. Longest elongates downward and in first, tip of penis time penis observed remained backward, becoming en- trails behind. Fretter inserted: 15 mins. Female gorged with blood. Fe- & Graham (1962) state in Fig. 5c waving foot male shown while attach that mammaliform glands about in an attempt to ing foot to glass plate, on leading edge of penis right herself. illustrating hows may yield secretions spreading of foot blocks which help hold penis in view. place during copulation. Copulation The trial-and-error behavior displayed by male Littorina planaxis in finding females suggests that copulation is the first purpose of pairing, and probably occurs immediately upon finding the female's genital opening. Figures 5a, b, & c illustrate insertion of the penis. It is first extended downward and backward, becoming engorged with blood and elongating (5a.); as it continues to elongate, the front edge is pushed into the mantle cavity (5b.); the tip trails in after the front edge and is eventually inserted in the bursa copu- latrix (5c.). Involuntary cilia in the penis cause a very rapid flow of sperm and prostatic secretions down the seminal groove that can be observed with a dissecting microscope. The rate of flow here makes it very unlikely that copulation takes very long, and it has been observed that sperm transfer is not always occurring during the entire time the penis is inserted. Of ten cases of copulation observed without disruption, the longest time the penis remained inserted was 15 minutes, more usually 4 or 5. The technique of observation was thelcol- lection of dry, closed pairs stuck together with mucus which were then placed under seawater with the female on her back. Under these conditions the snails soon extended themselves, and the male usually attempted to insert the penis. The only drawback to this method of observation is occasional dis- ruption of copulation as the female swings her foot around. trying to find a substrate in order to right herself. An at- tempt was made to remedy this problem by allowing the female to contact a glass plate with her foot while upside down; however, contact with the plate was followed by a spreading and flattening of the foot which obscured the view of the penis and the female's mantle cavity. When the female is righted, the positionpofithe male shell during pairing completely obstructs the view of the penis. Since it is impossible to tell by male shell orientation whether or not the pair is copulating, field studies of duration or frequency of copulation are practically impossible. Disruption of 100 active (extended) pairs in the field revealed only 17 in which the male's penis was extended, so paired snails cannot be regarded as always copulating. Size Ratios in Male-Female Pairs. A question arises as to what size males will be found with pairediwithewhätssize females in the field, and whether any selection for size of mate occurs. Figure 6 is a scattergram of allothefpairseofttwoidifferent Littorina planaxis popula- tions taken from two different areas, in which the size of each female is plotted against the size of the male she was paired with. The method of measurement used for both Figures 10 6 and 7 is shown in Figure 6a. All symbols between the diverging slope lines in Figure 6 represent pairs in which the smaller member is at least 2/3 the size of the larger. This suggests itself to be the practical limit in size disproportion in pairs found in the field. An overall survey of populations in which only the most disproportionate pairs were selected yielded only 4 out of 84 pairs taken that failed to fall within the suggested limits. A few of these limit-defining pairs are also recorded on the scattergram. A breakdown of size frequency by sex of the two scattergram populations is presented in Figure 7. Here is suggested one reason why more disproportionate pairs are not found: very large males and females are not found in the same populations with very small individuals. Therefore, in addition to the me- chanical problems of copulation between Littorina planaxis in- dividuals with grossly disproportionate genitalia, there are undoubtedly many ecological pressures involved which keep large and small snails apartiin nature, such as size as a factor in withstanding wave shock, desiccation, etc. However, laboratory experiments in which large males were kept isolated with small females, and conversely, for two weeks yielded only one pair, out- side the 2/3 limit but not significantly so, while control in- dividuals under the same environmental conditions paired readily. Figure 6. Scatter- gram of all pairs of two different field papulations, plus 14 eral of the most disproportionate pairs found.any- where, all illustra- ting a practical limit of disproportion in pairs found in the field: All symbols 9 between the diverging slope lines represent pairs in which the smaller member is at least 2/3 as large as the larger member. - pop. 41 (61 pairs) x- pop. 2 (72 pairs) o- pairs selected for their disproportion 20- 54 210 6 4 8 m at mn 11 r „ * 14 16 Figure 6a. Method of measurement used in figs. 687. Across shell aperture, from center of lip to widest part of whorl. Figure 7. Breakdown of size frequency by sex for the two scattergram popu- lations, showing that the largest and smallest indi- viduals do not occur in the same areas. Every half-millimeter is plotted as a category, i.e., 7.0-7.5 mm contains 25 females. — pop. 1 2 (93) — — - pop. 1 (126) —— - pop. 2 ? (96) - - - - - pop. 2 d' (120 Sex Ratio As Figure 7 indicates, there are generally more males than females in the population. A survey of 1000 unpaired individuals yielded a ratioof two males to each female; however, when the population as a whole and the large number of snails that are pairing at any one time are considered, the balance between the sexes is such that there is probably little or no significance in the greater number of males present. Literature Cited Fretter, Vera, and Graham, Alastair. 1962. British Prosobranch Molluscs. London: The Ray Society. xvi755pp.; 310 figures. Peters, Ronald. 1964. Function of the Cephalic Tentacles in Littorina planaxis. Hopkins Marine Station of Stanford University, Pacific Grove, California. unpublished. Ricketts, Edward F., and Calvin, Jack. 1952. Between Pacific Tides; an account of the habits and habitats of some five hundred of the common, con- spicuous seashore invertebrates of the Pacific Coast between Sitka, Alaska, and northern Mexico. Stanford, California: Stanford University Press. xii-502 pp.; 134 figures.