| Termites are worldwide economic insects. To date 473 termite species that belong to 44 genera and 4 families have been recorded in China, among which there are 111 Reticulitermes species widely distributed in different parts of China. It is difficult to collect and observe the termite eggs and there are few reports on embryonic development because of the cryptic life style of termites. Swaming season of Reticulitermes is usually from February to June, but there are some overlappings for different species in the same distributed area. Whether there is hybridization between different species has been controversial for a long time. In this paper, Reticulitermes aculabialis, R. chinensis, R. labralis, R. flaviceps and R. bitumulus were used for studies on embryo development and interspecific hybridization. The results are as follows. (1) Based on paraffin section technics, the embryonic development of R. chinensis was observed, and the methods of eggshell sorting for fertilized eggs and unfertilized eggs were developed. The best condition is 1 minute, with 10 ml HOAc and 10% KOH for the fertilized egg, and 1.5 minutes, with 15 ml HOAc and 10% KOH for the unfertilizied egg. (2) Typical sections of different periods of Reticulitermes termite embryo development were obtained. The results showed that the double caudal flexure structure of R. chinensis embryo appeared during 10-12 days, which is occurred in the embryonic development of Termitidae, and parts of germ bands sank into the yolk. (3) The fertilized eggs (female-male paired eggs, FM eggs) and unfertilized eggs (female-female paired eggs, FF eggs) were used for observing the cleavage of two termite species, Reticulitermes chinensis and R. aculabialis. We used laser scanning confocal microscope for cleavage observation and digital microscope for external shape of eggs and embryos. The results indicated that the fertilized and the unfertilized eggs from queens of R. chinensis could both cleave in the nest. Comparison between the nuclei of FF egg and FM egg in 24 h and 48 h revealed no significant difference for FF egg, but significant difference for FM egg. On the 15th day of egg development, no significant changes occurred for the volume of FF egg, whereas the volume of FM egg significantly increased. The FF egg died and disappeared on the 15th-20th day, while the FM egg was in normal development. There was no significant difference in the number of nuclei in the same period between the fertilized and unfertilized eggs of R. aculabialis, and the number of nuclear in 48 h was significantly more than that in 24 h. The length and width of the fertilized eggs were significantly changed on the 10th day, with the volume significantly increasing at the same time. But the increase of length, width and volume of the unfertilized eggs occurred on the 15th day. This shows that the fertilized eggs and unfertilized eggs of the facultative parthenogenetic termite R. aculabialis have the same cleavage rate. But the change in the shape of the fertilized eggs was earlier than that of the unfertilized eggs. The unfertilized eggs of R chinensis can cleave, but they developed abnormally, and cannot hatch in the end. The length and width of these two termite species change simultaneously in the same development stage. The cleavage characteristics of FF egg of R. chinensis may be a preadaptive stage from bisexual reproduction to facultative parthenogenesis in termite reproductive evolution. (4) Heterospecific hybridizations of R. aculabialis, R. chinensis, R. labralis, R. flaviceps and R. bitumulus were carried out, pairing as Rc×Ra, Rl×Rf, Rc×Rl, Rf×Rb, respectively. Genotypes of parents and offsprings of Rc(?)×Ra? and Ra?×Ra? were detected by DNA capillary electrophoresis in order to determine the presence of hybrid pre-zygotic inhibition or post-zygotic inhibition, and to explore the possibility of the establishment of hybrid colonies and new termite species in some areas. For Rc×Ra, the time needed for spawning was located between the two conspecific pairs, and the survival rate of delates was relatively low and tended to be stable after six months. Egg number at 30 days from the beginning of egg-laying, and offspring number 6 months later were both less than those of the conspecific pairs, with significant difference. The death rate of R. aculabialis delates was relatively high in the initial colony. In the other groups, the survival rate of RfxRb was the highest, while the survival rate of Rl×Rf was the lowest. Although distribution and swaming season are different, these Reticulitermes species can produce offsprings by heterospecific pairing under laboratory condations, and do not exsit hybrid pre-zygotic inhibitions. |
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