How female squid inseminate their eggs with stored sperm.

How sperm reach ova after mating is one of the central questions in reproductive biology. Many species copulate and store sperm in female reproductive organs until spawning [1]. The way females use stored sperm is closely associated with sperm competition and cryptic female choice. However, it is difficult to observe the process of fertilization in natural spawning, as fertilization usually occurs in some 'hidden place' within the female's body. Here, we report the fertilization process of a squid using a glass plate as a spawning substratum, enabling observation within the female arm crown where the sperm storage organ is located and where fertilization may occur. Additionally, we detail the distribution of sperm around newly spawned eggs. Our observations reveal that: the female places her sperm-storage organ (seminal receptacle) over an egg held within her arm crown and inseminates the eggs one-by-one during attachment to the spawning substratum; sperm pass through a pathway within the jelly layers surrounding an egg; and such direct insemination behavior and the pathway through the egg jelly enables a female squid to externally fertilize her eggs using relatively few sperm. This study is the first to reveal the fertilization process using stored sperm, under female control.

Impact of cryptic female choice on insemination success: Larger sized and longer copulating male squid ejaculate more, but females influence insemination success by removing spermatangia.

In polyandrous mating systems, sperm competition and cryptic female choice (CFC) are well recognized as postcopulatory evolutionary forces. However, it remains challenging to separate CFC from sperm competition and to estimate how much CFC influences insemination success because those processes usually occur inside the female's body. The Japanese pygmy squid, Idiosepius paradoxus, is an ideal species in which to separate CFC from sperm competition because sperm transfer by the male and sperm displacement by the female can be observed directly at an external location on the female's body. Here, we counted the number of spermatangia transferred to, removed from, and remaining on the female body during single copulation episodes. We measured behavioral and morphological characteristics of the male, such as duration of copulation and body size. Although males with larger body size and longer copulation time were capable of transferring larger amounts of sperm, females preferentially eliminated sperm from males with larger body size and shorter copulation time by spermatangia removal; thus, CFC could attenuate sperm precedence by larger males, whereas it reinforces sperm precedence by males with longer copulation time. Genetic paternity analysis revealed that fertilisation success for each male was correlated with remaining sperm volume that is adjusted by females after copulation.

Spermatangium formation and sperm discharge in the Japanese pygmy squid Idiosepius paradoxus.

In cephalopods, sperm discharge is an important event not only for sperm transfer but also influencing sperm storage capacity of attached spermatangia (everted spermatophores). To investigate sperm discharge from spermatangia and the condition of naturally attached spermatangia in Japanese pygmy squid (Idiosepius paradoxus) we (i) investigated the morphology of spermatophores and spermatangia, and the process of spermatophore evagination and sperm discharge from spermatangia obtained in vitro; (ii) observed spermatangia that were naturally attached to female squids at 6, 12, 18, 24 and 48 h after copulation to investigate alterations in naturally attached spermatangia with time. The spermatophore of I. paradoxus is slender and cylindrical and consists of a sperm mass, a cement body and an ejaculatory apparatus, which is similar to those of loliginid squids. The spermatangium is fishhook-shaped, its distal end being open and narrow. After the spermatangium is formed, the sperm mass gradually moves to the open end of the spermatangium, from where sperm are released. Sperm discharge is a rapid process immediately after the beginning of sperm release, but within 5 min changes to an intermittent release of sperm. Although the volume of residual spermatozoa differed among spermatangia that were naturally attached to a single individual, the probability that spermatangia would be empty increased with time. Most naturally attached spermatangia discharged almost all of their spermatozoa within 24h after copulation, and no spermatangia were attached to females 48 h after copulation. These results suggest that sperm transfer from the spermatangium to the seminal receptacle must occur within 24h, and that the spermatangium functions as a transient sperm storage organ in pygmy squids.

Evolution of the cephalopod head complex by assembly of multiple molluscan body parts: Evidence from Nautilus embryonic development.

Cephalopod head parts are among the most complex occurring in all invertebrates. Hypotheses for the evolutionary process require a drastic body-plan transition in relation to the life-style changes from benthos to active nekton. Determining these transitions, however, has been elusive because of scarcity of fossil records of soft tissues and lack of some of the early developmental stages of the basal species. Here we report the first embryological evidence in the nautiloid cephalopod Nautilus pompilius for the morphological development of the head complex by a unique assembly of multiple archetypical molluscan body parts. Using a specialized aquarium system, we successfully obtained a series of developmental stages that enabled us to test previous controversial scenarios. Our results demonstrate that the embryonic organs exhibit body plans that are primarily bilateral and antero-posteriorly elongated at stereotyped positions. The distinct cephalic compartment, foot, brain cords, mantle, and shell resemble the body plans of monoplacophorans and basal gastropods. The numerous digital tentacles of Nautilus develop from simple serial and spatially-patterned bud-like anlagen along the anterior-posterior axis, indicating that origins of digital tentacles or arms of all other cephalopods develop not from the head but from the foot. In middle and late embryos, the primary body plans largely change to those of juveniles or adults, and finally form a "head" complex assembled by anlagen of the foot, cephalic hood, collar, hyponome (funnel), and the foot-derived epidermal covers. We suggest that extensions of the collar-funnel compartment and free epidermal folds derived from multiple topological foot regions may play an important role in forming the head complex, which is thought to be an important feature during the body plan transition.

Shorter telomere length with age in the loggerhead turtle: a new hope for live sea turtle age estimation.

We verified whether telomere length shortens with age in the loggerhead sea turtle (Caretta caretta) by measuring telomere lengths (relative telomere to single copy gene [T/S] ratios) in whole blood and epidermis from 20 captive individuals with a real-time PCR method. There was no significant correlation between age and relative T/S ratios in blood. Although the correlation between age and relative T/S ratios in epidermis was not significant, older turtles had smaller relative T/S ratios in epidermis. It was thus demonstrated that telomere length in epidermis could be a useful age estimator for sea turtles. Relative age information obtained with this simple, rapid, non-invasive technique may help to advance our understanding of the ecology of endangered sea turtles. This is the first publication on age-related changes in telomere length among chelonians.