Dimorphic ejaculates and sperm release strategies associated with alternative mating behaviors in the squid

Abstract

Sperm competition is a powerful postcopulatory selective force influencing male adaptations associated with increasing fertilization success, and it is usually related to the evolution of different strategies of ejaculate expenditure between individuals. Ejaculates may also be influenced by additional selective pressures associated with sperm competition, such as timing between insemination and fertilization, female reproductive tract morphology, and fertilization environment. Also, males that adopt alternative mating tactics may face distinct sperm competition pressures, which may lead to the evolution of intraspecific diversity in ejaculates. In loliginid squids, males with alternative reproductive tactics (sneakers and consorts) differ not only in mating behavior, but also transfer spermatophores into two distinct sites within the female. Here, we compared structure and functioning of spermatophores between sneakers and consorts in the squid Doryteuthis plei applying microscopy techniques and in vitro experiments. Sneakers and consorts exhibit differences in spermatophore structure that lead to distinct spermatophoric reactions and spermatangium morphologies. Moreover, in sneakers, sperm release lasts longer and their sperm show an aggregative behavior not detected in consorts. Slow sperm release may be a strategy to guarantee longer sperm provision, given the wide interval between sneaker mating and egg release. For consorts, in turn, intense and quick sperm discharge may be advantageous, as timing between mating and egg-laying is relatively short. Within the complex squid mating system, factors such as (i) different fertilization sites and (ii) interval between mating and egg release may also influence sperm competition, and ultimately shape the evolution of divergent ejaculates between dimorphic males.

Behavior of “Intermediate” Males of the Dimorphic Squid Doryteuthis pleii Supports an Ontogenetic Expression of Alternative Phenotypes

“…Female status (as defined above) seems like a suitable candidate to explain the adoption of different tactics by intermediate males of D. pleii , as male fertilization success in loliginid squids is thought to be highly associated with sperm deposition site, due to temporal (i.e., timing between mating and fertilization) and spatial differences between both locations (i.e., near the oviduct vs. near the seminal receptacle) (e.g., Apostólico and Marian, 2017, 2018a). Due to the oocyte pathway during the egg-laying process, consort males are believed to be responsible for higher offspring paternity rates than do sneaker males, as sperm attached near the oviduct opening during MP is expected to contact the unfertilized eggs before those attached near the female’s buccal membrane by HH (Supplementary Figure S1G) (e.g., Buresch et al, 2009; Shashar and Hanlon, 2013).…”

“…As intermediate males are characterized by intermediate body size between those of sneaker and consort males, it was not possible to know a priori which males were indeed “intermediate males” based solely on ML measurements. As an accurate identification of these individuals requires the analysis of their spermatophores and spermatangia, they were first classified as either “sneaker males” or “consort males” based on their body size for the experiments, with male morph appropriate identification being confirmed later, only after anesthesia and analysis of their ejaculates’ morphology under stereomicroscope (Supplementary Figures S1H,I) (see Apostólico and Marian, 2017, 2018b).…”

“…In addition to contrasting behavioral strategies, different mating positions and spermatophore attachment sites (Supplementary Figure S1), male alternative phenotypes in loliginid squids also have singularities concerning morphological and physiological traits, particularly related to their body size ranges and to attributes of their ejaculates (e.g., Iwata and Sakurai, 2007; Iwata et al, 2015, 2018; Apostólico and Marian, 2017, 2018a). The overall morphology of spermatophores and spermatangia (i.e., everted spermatophores, implanted in the female during mating) of different male morphs is clearly distinct (Supplementary Figures S1C,F,H,I) (e.g., Iwata et al, 2015; Apostólico and Marian, 2017). They may also diverge, for example, in terms of spermatophore size and sperm size, volume, and swimming behavior (e.g., Iwata et al, 2011; Hirohashi and Iwata, 2014; Apostólico and Marian, 2017, 2018a).…”

“…The overall morphology of spermatophores and spermatangia (i.e., everted spermatophores, implanted in the female during mating) of different male morphs is clearly distinct (Supplementary Figures S1C,F,H,I) (e.g., Iwata et al, 2015; Apostólico and Marian, 2017). They may also diverge, for example, in terms of spermatophore size and sperm size, volume, and swimming behavior (e.g., Iwata et al, 2011; Hirohashi and Iwata, 2014; Apostólico and Marian, 2017, 2018a). Sneaker males, for instance, produce smaller spermatophores, with less but longer sperm, which also have the unique ability to swarm after release, a feature not observed so far in any consort sperm (e.g., Iwata and Sakurai, 2007; Hirohashi et al, 2013, 2016; Apostólico and Marian, 2017, 2018a).…”

Alternative male morphs solve sperm performance/longevity trade-off in opposite directions

“…Characteristics of nest male sperm correspond to the large time window between sperm activation and fertilization in these males, whereas sperm competition and the privileged position during spawning might be mainly responsible for sperm characteristics of dwarf males. This highlights the importance of the ecological context when considering sperm trait variations ( 23 , 60 ). We argue that the spatial and temporal dynamics of sperm in competition during the fertilization process merits consideration when studying functional properties of spermatozoa.…”