First molecular detection of Hemolivia and Hepatozoon parasites in reptile-associated ticks on Iriomote Island, Japan.

Hepatozoon and Hemolivia are members of the haemogregarines and are reported in reptiles and reptile-associated ticks. However, no studies have reported on Hepatozoon and Hemolivia in Japanese reptile-associated ticks. This study aimed to molecularly identify and to characterize Hepatozoon and Hemolivia in Japanese reptile-associated ticks, Amblyomma geoemydae (Cantor, 1847) and Amblyomma nitidum (Hirst & Hirst, 1910). A total of 41 and 75 DNA samples from A. geoemydae and A. nitidum ticks, respectively, were used for screening of Hepatozoon and Hemolivia with polymerase chain reaction targeting 18S rDNA. As a result, Hemolivia and Hepatozoon were detected in two A. geoemydae and one A. nitidum, respectively. The sequences of Hemolivia spp. showed a 99.5% (1,050/1,055 bp) identity with Hemolivia parvula (KR069083), and the Hemolivia spp. were located in the same clade as H. parvula in the phylogenetic tree. The sequences of Hepatozoon sp. showed a 98.4% (1,521/1,545 bp) identity with Hepatozoon colubri (MN723844), and the Hepatozoon sp. was distinct from validated Hepatozoon species in the tree. Our findings highlight the first molecular record of Hemolivia in Japan and present the first detection of Hepatozoon in A. nitidum. Further investigations on these tick-borne protozoa are required to understand their life cycle and pathogenicity.

Morphology, Diet, and Reproduction of Coastal Hydrophis Sea Snakes (Elapidae: Hydrophiinae) at Their Northern Distribution Limit.

The Ryukyu Archipelago represents the northern distribution limit for hydrophiine sea snakes, the largest group of marine reptiles. Ryukyuan sea snakes may have developed distinct local adaptations in morphology and ecology, but they have been poorly studied. We examined preserved specimens of 111 Hydrophismelanocephalusand 61 Hydrophis ornatusfrom the Ryukyu Archipelago to obtain data on morphology, diet, and reproduction. Sexual size dimorphism was detected in H. melanocephalus (mean ± standard deviation of adult snout-vent length: SVL, females 1062 ± 141 mm vs. males 959 ± 96 mm) but not in H. ornatus. Female H. melanocephalus had larger head widths and shorter tail lengths relative to SVL compared to males. Relative girth was low in neonates of both species (1.0-1.3), but increased in adults to about 1.7-2.6 in H. melanocephalus and 1.3-1.8 in female H. ornatus. Stomach contents of H. melanocephalus consisted of ophichthid and congrid eels, a sand diver, and gobies, whereas in H. ornatus, gobies and a goat fish were found. Litter size of three reproductive H. melanocephalus ranged from five to seven, and parturition seems to occur from August to October. Litter size of six H. ornatus ranged from two to seven, and was correlated with maternal SVL. Parturition in H. ornatus probably occurs around November. Different selective forces related to locomotion, feeding and predation risk, which influence the pregnant mother and neonates, may have resulted in having few, long but slender offspring that show positive allometric growth in hind-body girth.

Rickettsia spp. and Ehrlichia spp. in Amblyomma ticks parasitizing wild amphibious sea kraits and yellow-margined box turtles in Okinawa, Japan.

Recently, several tick-borne pathogens were detected in reptile-associated ticks. However, studies on the microorganisms in reptile-associated ticks in Japan are limited. This molecular survey thus aimed to identify and characterize tick-borne pathogens (Rickettsiaceae and Anaplasmataceae) in reptile-associated ticks in Japan. In total, 77 Amblyomma nitidum and 104 Amblyomma geoemydae were collected from wild amphibious sea kraits (Laticauda semifasciata, Laticauda colubrina, and Laticauda laticaudata) and from yellow-margined box turtles (Cuora flavomarginata evelynae), respectively. Conventional polymerase chain reaction was performed using the DNA extracted from the ticks to detect the selected pathogens. Sequencing analysis of four Rickettsia genes (gltA, ompA, ompB, and sca4) led to the identification of a putative novel Rickettsia sp. and Rickettsia aeschlimannii-like rickettsia in A. nitidum and A. geoemydae, respectively. Sequencing analysis of gltA and groEL of Anaplasmataceae revealed that the Ehrlichia spp. in these ticks were novel and related to Candidatus Ehrlichia occidentalis. This is the first study on the microorganisms in A. nitidium and the first record of Rickettsia and Ehrlichia in A. geoemydae. Further studies are required to understand their pathogenicity to humans and animals and their life cycle in the wild.