Mai Sakai

Studies on the Conditions Required for Structural and Functional Maturation of Rabies Virus Glycoprotein (G) in G cDNA‐Transfected Cells

When the rabies virus G cDNA was expressed with the help of T7 RNA polymerase provided by a recombinant vaccinia virus (RVV‐T7), functional G proteins were produced in terms of their ability to induce low pH‐dependent syncytium formation and the formation of conformational epitopes, including the acid‐sensitive epitope recognized by mAb #1‐30‐44. Such an ability and the 1‐30‐44 epitope formation, however, were not associated with the G gene products when G cDNA was expressed without the help of RVV‐T7 using a tetracycline‐regulated expression vector (pTet‐G), although they were normally transported to the surface of established G protein‐producing BHK‐21 (G‐BHK) cells. But, when the G‐BHK cells were treated with 2.5 MM sodium butyrate (NaB) after the removal of tetracycline, we could observe not only a much increased frequency of G protein‐producing cells, but also the greatly enhanced maturation of the protein. Another short acylate, sodium propionate (NaP), similarly induced increased G protein synthesis at a concentration of 2.5 MM as NaB; however, such proteins were mostly not endowed with the fusion activity nor the 1‐30‐44 epitope, while NaP at a higher concentration as 5.0 MM did induce similarly the increased production and enhanced maturation of G protein, including the 1‐30‐44 epitope formation. From these results, we conclude that functional maturation of G protein to acquire fusogenic activity is correlated with 1‐30‐44 epitope formation, and 2.5 MM NaB not only stimulates G protein production, but also provides such cellular conditions as are required for the structural and functional maturation of the protein.

Social Touch in Apes and Dolphins

Social touch, or physical contact among two or more individuals in a nonaggressive context, seems to play important roles among both primates and cetaceans. However, with exception of social grooming among primates, it has rarely been studied in detail. Thus, in this chapter we review the descriptions of social touch in great apes and dolphins from the literature and from our own observations. After reviewing the social grooming among various mammalian taxa, we considered various types of social touch in apes and dolphins in more detail by dividing them into following seven categories: (1) social touch between mother and infant; (2) touch in play; (3) tactile gestures; (4) social grooming; (5) touch in greeting, reassurance, and appeasement; (6) touch to/with genital areas; and (7) simple body contact. Information from scattered descriptions in the literature suggests that social touch is widespread in apes and dolphins, yet frequencies may vary greatly among species. Although there has been no single theory to explain these diverse types of social touch, we briefly review theories that might be of relevance to explaining social touch.

A wild Indo-Pacific bottlenose dolphin adopts a socially and genetically distant neonate.

Alloparental behaviour and adoption have been reported in many mammals and birds. Such behaviours are energetically costly, and their causes and functions remain unclear. We observed the adoption behaviour of a wild Indo-Pacific bottlenose dolphin (Tursiops aduncus) near Mikura Island, Japan. A calf was seen with its mother on six observation days. Following the mother’s death, the calf was observed with a sub-adult female on all 18 observation days from May to September 2012. On three days, the calf was observed swimming with this female in the suckling position and milk was seen leaking from the female’s mammary slit. A five-year dataset revealed no significant social or kin relationships between the biological mother and allomother, indicating that kinship and social relationships did not play an important role in the observed adoption.

Sound variation and function in captive Commerson's dolphins (Cephalorhynchus commersonii).

Commersons dolphin (Cephalorhynchus commersonii), one of the smallest dolphin species, has been reported to produce only narrow-band high-frequency (NBHF) clicks and no whistles. To clarify their sound repertoire and examine the function of each type, we analysed the sounds and behaviour of captive Commersons dolphins in Toba Aquarium, Japan. All recorded sounds were NBHF clicks with peak frequency >110kHz. The recorded click-trains were categorised into four types based on the changing pattern of their Inter-click intervals (ICI): Decreasing type, with continuously decreasing ICI during the last part of the train; Increasing type, with continuously increasing ICI during the last part; Fluctuating type, with fluctuating ICI; and Burst-pulse type, with very short and constant ICI. The frequency of the Decreasing type increased when approaching an object newly introduced to the tank, suggesting that the sound is used for echolocation on approach. The Burst-pulse type suddenly increased in front of the object and was often oriented towards it, suggesting that it was used for echolocation in close proximity to the object. In contrast, the Increasing type was rarely recorded during approach, but increased when a dolphin approached another dolphin. The Increasing and Burst-pulse types also increased when dolphins began social behaviours. These results suggest that some NBHF clicks have functions other than echolocation, such as communication.

Further studies on the soluble form (gs) of rabies virus glycoprotein (g): molecular structure of gs protein and possible mechanism of the shedding.

In this study, we investigated the antigenic structures and maturation of some C‐terminal‐deficient derivatives of rabies virus glycoprotein (G). The Gs protein, a soluble form of G protein shed from infected cells, displayed antigenicity to most of our conformational epitope‐specific anti‐G mAbs, but took the 1‐30‐44 epitope‐deficient conformation (termed GC form). (The 1‐30‐44 epitope was acid‐sensitive and dependent on two separate regions, the Lys‐202‐containing and Asn‐336‐containing regions; Kankanamge et al., Microbiol. Immunol., 47: 507–519). Intact G proteins took the 1‐30‐44 epitope‐positive form (referred to as GB form) on the cell surface, but not inside the cell. A deletion mutant G(1–429) (termed GATC), lacking the transmembrane (TM) and cytoplasmic domains, was shown to be accumulated in the rough endoplasmic reticulum (rER) with BiP and did not seem to be shed. Another C‐terminal‐deficient mutant G(1–462) (termed CT1) was deprived of the whole cytoplasmic domain except for a basic amino acid left at the C‐terminus, but was transported to the cell surface, where it showed pH‐dependent cell fusion activity and almost full antigenicity to most of the anti‐G mAbs with the exception of very weak antigenicity to mAb #1‐30‐44. No Gs protein could be detected in the CT1‐producing cultures. Based on these results, we think that the cytoplasmic domain was not necessary for the G protein to be transported to the cell surface, but was necessary to keep its 1‐30‐44 epitope‐positive GB conformation. Gs proteins might have lost the C‐terminal regions during the maturation process after being exported from the rER.

Spontaneous Ejaculation in a Wild Indo-Pacific Bottlenose Dolphin (Tursiops aduncus).

Spontaneous ejaculation, which is defined as the release of seminal fluids without apparent sexual stimulation, has been documented in boreoeutherian mammals. Here we report spontaneous ejaculation in a wild Indo-Pacific bottlenose dolphin (Tursiops aduncus), and present a video of this rare behavior. This is the first report of spontaneous ejaculation by an aquatic mammal, and the first video of this behavior in animals to be published in a scientific journal.

Acoustic time synchronization among tags on porpoises to observe their social relationships

Observing and monitoring underwater social interactions of cetaceans is challenging. Because cetaceans spend most of their time underwater, it is important to monitor their underwater behavior individually. The finless porpoise is small and has no available natural identification marks that causes little knowledge of its sociality. Here we used acoustic datalogger to synchronize individual depth profile among individuals within a second. Acoustic and behavior tags were deployed on six free-ranging finless porpoises simultaneously and released in open water. Echolocation sounds were used as the trigger signal to synchronize the clock of all logging systems. Synchronous dives characterized by similar time-depth profile were used as an index of association. Two pairs tended to participate in long periods of synchronized diving more frequently than 13 other possible pairs, indicating that these four porpoises chose their social partners. Initiator and follower could be identified by precisely time synchronize...