It was often possible to deduce the type of injury that had occurred in cryopreserved embryos from their appearance at recovery and during subsequent culture. This may help to improve cryopreservation protocols for embryos of many species, including man.

In the 1991-strain of Aquarius paludum in Kochi, both critical photoperiods for wing-form and diapause shifted to longer values by 1 h when specimens were grown under gradually decreasing photoperiods. However, the 1999-strain didn't respond at all to decreasing (14.5L to 13.5L) or increasing (12.5L to 13.5L) photoperiods. Photoperiodic response for wing-form determination by the 2002-strain was witnessed only in a small range between 60% and 95% as the proportion of long-winged form, and the critical photoperiod (14L) was a little bit longer than the critical value of 13.75L for the 1991-strain. The critical photoperiod for diapause induction in the 2002-strain was estimated to be 13

Seasonal records of nymphs of the water strider Aquarius paludum Fabricius (Heteroptera: Gerridae) at a waterway in Kochi prefecture, Japan (33 • 00 N to 33 • 30 N), in 2007, show that populations of new generation adults occur four or more times during the year, in mid and late May; late July to early August; September; and October to November. In laboratory experiments, more than 50% of adults are reproductive, irrespective of photoperiod, and this ratio increases to 80% even under a short-day LD 11.5 : 13.5 h photocycle, which corresponds to the winter season at Kochi (33 • N). There is a diminishing photoperiodic response in relation to the induction of diapause compared with measurements made in previous years (1995)(1996)(1997), suggesting that reproduction may be possible over a much longer period in the near future (and even in winter) if local climate warming is sustained. A high proportion of males sampled in 2008 have small testes and a high proportion of females do not have mature oöcytes even in summer. This contrasts with earlier data showing that the testis volume index for the same period is greater in 1995-1997, and that 75% of females have mature oöcytes. These changes suggest that the Kochi-Nankoku population of A. paludum is beginning to show partial aestivation. Overwintering adults of both sexes are found to have no mature flight muscles in October/November 2008. This is in marked contrast with earlier data from 1995 and 2004. Adults that are reared in the laboratory also show a lower flight propensity in response to shorter day lengths. The absence of mature flight muscles in the autumn and lower flight propensity under shorter days may indicate a cessation of dispersal between the summer habitats and overwintering sites on land far from the waterways. Taken together, these data suggest that populations of A. paludum in the Kochi-Nankoku region are continuing to show adaptive changes, apparently in relation to global warming.

No abstract

For the cryopreservation of embryos, vitrification has various advantages, but it also has disadvantages because embryos are vitrified with a considerable supercooling (i.e., in nonequilibrium). Here, we tried to develop a novel method in which embryos are vitrified in near-equilibrium. The extent of equilibrium was assessed by examining whether vitrified embryos survive after being kept at -80 degrees C. Two-cell embryos of ICR mice were vitrified with ethylene glycol (EG)-based solutions, either EFSa or EFSc solutions, which were mixtures of EG (30%-40%) and an FSa or FSc solution, respectively. The FSa and FSc solutions were PB1 medium containing 30% Ficoll plus 0.5 or 1.5 M sucrose, respectively. In vitro survival rate was high when embryos vitrified with 30%-40% EG (EFS30a, EFS40a, EFS30c, and EFS40c) were warmed rapidly. When embryos were vitrified and then kept at -80 degrees C for 4 days, large proportions survived with EFS30c and EFS40c. When embryos were vitrified with EFS35c or EFS40c, the survival rate was high even for those kept at -80 degrees C for 10 days. When embryos of ICR and C57BL/6J mice were vitrified with EFS35c or EFS40c and then kept at -80 degrees C for 4 days, the survival rate was high even after recooling in liquid nitrogen; a high proportion (75%) of C57BL/6J embryos vitrified with EFS35c developed to term after transfer. In conclusion, we have developed a novel method by which embryos are vitrified in near-equilibrium. This will be a supreme method for cryopreservation, retaining the advantages of both current vitrification and equilibrium slow freezing.