Angiostrongyliasis is a parasitic disease caused by nematodes of the genus Angiostrongylus. Distribution of this worm corresponds to the dispersal of its main intermediate host, the giant African land snail Achatina fulica. Genetic characterization can help identify parasitic pathogens and control the spreading of disease. The present study describes infection of A. fulica by Angiostrongylus, and provides a genetic outlook based on sequencing of specific regions. We collected 343 land snails from 22 provinces across six regions of Thailand between May 2017 and July 2018. Artificial digestion and Baermann’s technique were employed to isolate Angiostrongylus larvae. The worm and its intermediate host were identified by sequencing with specific nucleotide regions. Phylogenetic tree was constructed to evaluate the relationship with other isolates. A. fulica from Chaiyaphum province was infected with A. cantonensis, whereas snails collected from Phrae and Chiang Rai provinces were infected with A. malaysiensis. The maximum likelihood tree based on 74 A. fulica COI sequences revealed monophyletic groups and identified two haplotypes: AF1 and AF2. Only AF1, which is distributed in all regions of Thailand, harbored the larvae of A. cantonensis and A. malaysiensis. Two mitochondrial genes (COI and cytb) and two nuclear regions (ITS2 and SSU rRNA) were sequenced in 41 Angiostrongylus specimens. The COI gene indicated that A. cantonensis was closely related to the AC10 haplotype; whereas the cytb gene revealed two new haplotypes: AC19 and AC20. SSU rRNA was useful for the identification of A. cantonensis; whereas ITS2 was a good genetic marker for differentiating between A. cantonensis and A. malaysiensis. This study provides genetic information about the parasite Angiostrongylus and its snail intermediate host. The data in this work may be useful for further study on the identification of Angiostrongylus spp., the genetic relationship between intermediate host and parasite, and control of parasites.
Summary
Entomopathogenic nematodes (EPN) Steinernema and Heterorhabditis with symbionts with Xenorhabdus and Photorhabdus bacteria, respectively, are reported as biocontrol agents for insect control. The objectives of this study were to identify EPN and their symbiotic bacteria in national parks of Phitsanulok Province, Thailand, and to test how Xenorhabdus isolates attack Aedes aegypti larvae. We collected 810 soil samples from four national parks. The juvenile stage of EPN was isolated from soil samples using a baiting technique with Galleria mellonella followed by a White trap. Partial regions of 28S rDNA and internal transcript spacer were sequenced to identify EPN, and recA sequencing was used to discriminate between Xenorhabdus and Photorhabdus. We found that 74 of the 810 soil samples (9.1%) were positive for the EPN. The EPN were molecularly identified as S. surkhetense, S. longicaudum, H. indica and Heterorhabditis sp. SGmg3, and their symbiotic bacteria were identified as X. stockiae, X. griffiniae, X. indica, X. vietnamensis, P. luminescens subsp. akhurstii, and P. temperata subsp. temperata. Xenorhabdus griffiniae showed potential larvicidal activity against A. aegypti (91% mortality at 72 and 96 h after exposure). This study demonstrates the diversity of EPN and symbiotic bacteria in national parks of Thailand and the potential to use X. griffiniae as a biocontrol agent to kill A. aegypti larvae.
Cryptozona siamensis
, one of the most widespread land snails, is native to Thailand, and plays a key role as an agricultural pest and intermediate host for
Angiostrongylus
spp. However, its genetic diversity and population structure has not yet been investigated, and are poorly understood. Therefore, a genetic analysis of the
C
.
siamensis
population in Thailand was conducted, based mitochondrial 16S rRNA (402 bp) and COI (602 bp) gene fragment sequences.
Cryptozona siamensis
randomly collected from 17 locations in four populations across Thailand, between May 2017 and July 2018. Fifty-eight snails were used to examine the phylogeny, genetic diversity, and genetic structure. The maximum likelihood tree based on the 16S rRNA and COI fragment sequences revealed two main clades. A total of 14 haplotypes with 44 nucleotide variable sites were found in the 16S rRNA sequences, while 14 haplotypes with 57 nucleotide variable sites were found in the COI sequences. The genetic diversity of
C
.
siamensis
in term of the number of haplotypes and haplotype diversity, was found to be high but the nucleotide diversity showed low levels of genetic differentiation for the COI sequence as also noted with the 16S rRNA sequence. The population genetic structure of
C
.
siamensis
revealed genetic difference in most populations in Thailand. However, low genetic difference in some populations may be due to high gene flow. This study provides novel insights into the basic molecular genetics of
C
.
siamensis
.
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