A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida).

Gordius chiashanus sp. nov., a newly described horsehair worm that parasitizes the Spirobolus millipede, is one of the three described horsehair worm species in Taiwan. It is morphologically similar to G. helveticus Schmidt-Rhaesa, 2010 because of the progressively broadening distribution of bristles concentrated on the male tail lobes, but it is distinguishable from G. helveticus because of the stout bristles on the mid-body. In addition, a vertical white stripe on the anterior ventral side and areoles on the inside wall of the cloacal opening are rarely mentioned in other Gordius species. Free-living adults emerged and mated on wet soil under the forest canopy in the winter (late November to early February) at medium altitudes (1100-1700 m). Mucus-like structure covering on the body surface, which creates a rainbow-like reflection, might endow the worm with high tolerance to dehydration. Although Gordius chiashanus sp. nov. seems to be more adaptive to the terrestrial environment than other horsehair worm species, cysts putatively identified as belonging to this hairworm species found in the aquatic paratenic host, Ephemera orientalis McLachlan, 1875, suggest the life cycle of Gordius chiashanus sp. nov. could involve water and land. The free-living adults emerged from the definitive hosts might reproduce in the terrestrial environment or enter an aquatic habitat by moving or being washed away by heavy rain instead of manipulating the behavior of their terrestrial definitive hosts.

A rapid insect species identification system using mini-barcode pyrosequencing.

BACKGROUND: Rapid and accurate species identification is not only important for biodiversity studies and pest quarantine and management, but in some cases may also influence the results of international trade negotiations. In this study, we developed a rapid species identification system for insects. RESULTS: A universal DNA mini-barcode primer pair was designed to target ∼ 120 bp of the mitochondrial 16S rDNA gene. This primer set can amplify the targeted region from all 300 species of 26 insect orders tested as well as other classes of Arthropoda. Although we found no within-species variation in this region, it provided enough information to separate closely related species or species complexes, in particular Thrips spp. and Bemisia spp. By combining a quick DNA extraction method with pyrosequencing, we were able to generate DNA sequences and complete species identification within 5 h. CONCLUSION: Mini-barcode pyrosequencing of 16S rDNA coupled with the GenBank database provides a rapid, accurate, and efficient species identification system. This system is therefore useful for biodiversity discovery, forensic identification, and quarantine control and management. © 2019 Society of Chemical Industry.

The Effect of Insecticidal Stress on Reproductive Output of Susceptible and Field Strains of Aedes aegypti (Diptera: Culicidae).

The phenomenon of high egg reproduction when mortality risk rises is common in mosquitoes. However, the phenomenon may vary between insecticide susceptible and field-collected strains, due to the latter's decreased energy allocation in reproduction in the presence of insecticide resistance. In this study, we evaluated the effect of chlorpyrifos (CP) and temephos (TP) exposure on the oviposition and survival of Aedes aegypti (Linnaeus) (Diptera: Culicidae) using a susceptible strain (KHsm) and two field strains (KHly and TNnorth). We also dissected the female mosquitoes of each strain on fifth day after the first blood meal to examine the total number of eggs produced. Neither CP nor TP exhibited oviposition deterrent against female mosquitoes of any of the three strains, as the females did not show decreased reproduction activity on the insecticide-treated sites. Of the two insecticides tested, only CP had an adulticidal effect on Ae. aegypti. High mortality was recorded in KHsm after contacting the CP-treated oviposition sites on day 4. Before death, KHsm mosquitoes oviposited significantly more eggs compared to the two field strains. However, the difference of total egg production between susceptible and field-collected strains was subtle. Thus, the decreased reproductive output in field-collected strains might not be directly linked to energy and resource allocation. In this respect, we should consider the possible involvement of biogenic amines in the egg retention in field-collected strains when mortality risk rises. The phenomenon was not observed in nonadulticidal TP treatment.

A new orthopteran-parasitizing horsehair worm, Acutogordius taiwanensis sp. n., with a redescription of Chordodes formosanus and novel host records from Taiwan (Nematomorpha, Gordiida).

A description of a new species of horsehair worm, Acutogordius taiwanensissp. n., a redescription of Chordodes formosanus, and novel host records for the latter are provided. Acutogordius taiwanensissp. n. is morphologically similar to A. protectus with moderately flat areoles on its tail tips, but is distinguishable by small mid-body ornamentations. Despite the distinct differences in the post-cloacal crescents between 14 male samples, their conspecific status, along with that of nine female samples, was upheld by a phylogenetic comparison of partial cytochrome oxidase subunit I (COI) sequences. Chordodes formosanus is another common horsehair worm species in Taiwan, which was previously believed to specifically parasitize Hierodula mantids. However, in this study, five C. formosanus were observed emerging from an Acromantis mantid, and two long-horned grasshopper hosts (Leptoteratura sp. and Holochlora japonica). These five worms showed high degrees of similarity in COI sequences and morphology, but one of these individuals bore abnormal crowned areoles, which has never been observed in C. formosanus, and may be attributed to the incomplete development of this particular individual.

Annual Survey of Horsehair Worm Cysts in Northern Taiwan, with Notes on a Single Seasonal Infection Peak in Chironomid Larvae (Diptera: Chironomidae).

The life cycle of the freshwater horsehair worm typically includes a free-living phase (adult, egg, larva) and a multiple-host parasitic phase (aquatic paratenic host, terrestrial definitive host). Such a life cycle involving water and land can improve energy flow in riparian ecosystems; however, its temporal dynamics in nature have rarely been investigated. This study examined seasonal infection with cysts in larval Chironominae (Diptera: Chironomidae) in northern Taiwan. In the larval chironomids, cysts of 3 horsehair worm species were identified. The cysts of the dominant species were morphologically similar to those of Chordodes formosanus. Infection with these cysts increased suddenly and peaked 2 mo after the reproductive season of the adult horsehair worms. Although adult C. formosanus emerged several times in a year, only 1 distinct infection peak was detected in September in the chironomid larvae. Compared with the subfamily Chironominae, samples from the subfamilies Tanypodinae and Orthocladiinae were less parasitized. This indicates that the feeding behavior of the chironomid host likely affects horsehair worm cyst infections; however, bioconcentration in predatory chironomids was not detected.

Morphological allometry and intersexuality in horsehair-worm-infected mantids, Hierodula formosana (Mantodea: Mantidae).

Parasitic castration is a strategy used by parasites to minimize damage to the host by consuming its reproductive system, which results in the morphological alteration of the host. We determined that the forewing shape and density of the antennal sensilla of field-collected adult male mantids (Hierodula formosana), infected by horsehair worms (Chordodes formosanus) was partially feminized (intersexuality), and both male and female mantids infected by horsehair worms exhibited allometric changes in their wings and walking legs. In addition, the testes of most infected male adults disappeared or reduced in size, whereas the number of ovarioles in infected female adults was unaffected. The infection mainly influenced the structures related to host reproduction and locomotion, suggesting unbalanced energy exploitation and the reduction of parasitic virulence. In addition, the intersexuality of infected male adults indicated that sexual differentiation in insects, which researchers have considered to be an autonomous process, was influenced by the infection. The similarity of the antennae of infected male adults with those of last-instar female nymphs suggested that parasitic juvenilization may cause such feminization, but the mechanism of parasitic influence on insect sex characteristics should be studied further.

An improved bioassay facilitates the screening of repellents against cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).

BACKGROUND: Repellents are a common method for preventing flea bites, making an effective system for flea repellent screening advantageous. We describe an improved technique to facilitate repellent activity screening of numerous plant-based Ctenocephalides felis (cat flea) repellents. RESULTS: Two long strips of filter paper were impregnated with test compounds (dissolved in ethanol) and ethanol only, respectively. After drying, the two filter papers were glued together along the long side and inserted into a glass tube containing non-fed cat fleas. The distribution of cat fleas in each half of the filter paper was recorded after 30 min to calculate repellency. Results showed that the essential oil of Cinnamomum osmophloeum (from leaf), Taiwania cryptomerioides (from heartwood) and Plectranthus amboinicus (from leaf) exhibits repellent activity against cat fleas in a dose dependent manner. Moreover, the repellent activities against cat fleas of 2% trans-cinnamaldehyde (the main constituent of Ci. osmophloeum essential oil) and 0.5% thymol (the main constituent of P. amboinicus essential oil) are 97.6% and 90.6%, and can persist for up to 4 and 8 h, respectively. These results are comparable to those of 15% DEET. CONCLUSION: The proposed screening technique can facilitate the pre-screening of numerous flea repellents for further evaluation on animal or human subjects.

Genetic and physiological data suggest demographic and adaptive responses in complex interactions between populations of figs (Ficus pumila) and their pollinating wasps (Wiebesia pumilae).

To study interactions between host figs and their pollinating wasps and the influence of climatic change on their genetic structures, we sequenced cytoplasmic and nuclear genes and genotyped nuclear microsatellite loci from two varieties of Ficus pumila, the widespread creeping fig and endemic jelly fig, and from their pollinating wasps, Wiebesia pumilae, found in Taiwan and on nearby offshore islands. Great divergence in the mitochondrial cytochrome c oxidase subunit I (mtCOI) with no genetic admixture in nuclear markers indicated that creeping- and jelly-fig wasps are genetically distinct. Compared with creeping-fig wasps, jelly-fig wasps also showed better resistance under cold (20 °C) than warm (25 and 30 °C) conditions in a survival test, indicating their adaptation to a cold environment, which may have facilitated population expansion during the ice age as shown by a nuclear intron and 10 microsatellite loci. An excess of amino acid divergence and a pattern of too many rare mtCOI variants of jelly-fig wasps as revealed by computer simulations and neutrality tests implied the effect of positive selection, which we hypothesize was associated with the cold-adaptation process. Chloroplast DNA of the two fig plants was completely segregated, with signs of genetic admixture in nuclear markers. As creeping- and jelly-fig wasps can pollinate creeping figs, occasional gene flow between the two figs is thus possible. Therefore, it is suggested that pollinating wasps may be playing an active role in driving introgression between different types of host fig.

A gregarine from the gut of cat flea, Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae) in Taiwan: dynamic of infection patterns.

An understanding on host-parasite interaction is essential for control of disease causing organisms in domestic animals. The cat flea, Ctenocephalides felis (Bouché) is the predominant flea infesting dogs and cats in Taiwan. It was collected from 933 dogs and 197 cats from Taiwan. A total of 5878 C. felis adults were recovered; 14.6% fleas were observed to harbor Steinina ctenocephali. Female fleas were more susceptible to gregarine infection than males. Further, fleas were more likely to be infected with the gregarine at high temperatures, particularly during March-July with high parasite prevalence and intensity. Fleas harboring gregarines infection were higher in dogs than cats. Our study may help in development and application of appropriate flea control measures in Taiwan.

Identification of Rickettsia felis in fleas but not ticks on stray cats and dogs and the evidence of Rickettsia rhipicephali only in adult stage of Rhipicephalus sanguineus and Rhipicephalus haemaphysaloides.

Rickettsia spp. are zoonotic pathogens and mainly transmitted by various arthropod vectors, such as fleas, ticks, and lice. Previous epidemiological studies indicated that ectoparasites infested on dogs or cats may be infected by Rickettsia spp., and transmit them to human beings accidentally. In this study, the prevalence of Rickettsia infection was evaluated using fleas and ticks from stray dogs and cats in Taiwan. A total of 158 pools made by 451 cat fleas (Ctenocephalides felis) from 37 dogs and 4 cats were used for analysis. Besides, 386 Rhipicephalus ticks collected from the other 62 stray dogs were included in this study. Nymphal and adult ticks were individually analyzed but larvae were separated into 21 pools for molecular detection. Partial sequencing analysis of the gltA gene was applied for Rickettsia identification. The results showed that 44.3% (70/158) of the cat flea pools were harboring Rickettsia DNA. Although 6.9% (13/187) of adult ticks were infected with Rickettsia, neither larval pools nor nymphal ticks were found to contain Rickettsia DNA. According to the results of sequencing analyses, all Rickettsia PCR-positive cat flea pools were infected with R. felis, and all Rickettsia PCR-positive adult ticks were infected with R. rhipicephali. The results of this study demonstrated that C. felis but not Rhipicephlus sanguineus (the brown dog tick) and Rh. haemaphysaloides collected from stray animals in Taiwan could be infected the zoonotic pathogen R. felis. Moreover, R. rhipicephali was only identified in adult stage of Rhipicephalus sanguineus and Rh. haemaphysaloides.

Prevalence of Rickettsia felis and the first identification of Bartonella henselae Fizz/CAL-1 in cat fleas (Siphonaptera: Pulicidae) from Taiwan.

Cat fleas (Ctenocephalides felis [Bouché]) are the primary ectoparasites of dog and cat populations. In this study, we report the monthly population dynamics of Rickettsia felis and Bartonella spp. (two zoonotic pathogens that can cause human disease) in cat fleas collected from dogs and cats in Taipei, Taiwan, from December 2006 to December 2007. Natural R. felis infection in individual cat fleas was assessed by polymerase chain reaction (PCR) using pRF-, ompB-, and gltA-specific primer pairs. Samples positive by PCR were confirmed with DNA sequencing. R. felis was detected in cat fleas year round, and the average infection rate was 21.4% (90 of 420) in 2007. Cat fleas also play an important role in the transmission of Bartonella between reservoirs and other mammalian hosts. In this study, we used primer pairs specific for the Bartonella gltA and rpoB genes to detect Bartonella infections. Of the 420 cat fleas tested, 38 were positive by PCR for Bartonella. Sequence similarities to Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae were observed in 6.2% (26 of 420), 2.1% (9 of 420), and 0.7% (3 of 420) of the fleas, respectively. Based on the pap31 gene sequence, several amplicons of the B. henselae detected in the cat fleas could be subgrouped into three strains: Fizz/CAL-1 (n = 18), Marseille (n = 5), and Houston-1 (n = 3). These results demonstrate that cat fleas infected with R. felis are endemic to Taiwan, and highlight the role of C. felis in Bartonella transmission between reservoirs and other mammal hosts and demonstrate the genetic variability of B. henselae in Taiwan.

A new horsehair worm, Chordodes formosanus sp. n. (Nematomorpha, Gordiida) from Hierodula mantids of Taiwan and Japan with redescription of a closely related species, Chordodes japonensis.

A new species of horsehair worm, Chordodes formosanussp. n., is described and compared to a closely related species, Chordodes japonensis. Although both species possess the same six cuticular structures of areoles on the surface, the significantly longer filaments on the female crowned areoles can be used as diagnostic characters for the new species. The different taxonomic status of these two species was also confirmed after analyzing the partial cytochrome oxidase subunit I sequence, and the mantid hosts, which are respectively limited to the genus Tenodera for Chordodes japonensis and Hierodula for Chordodes formosanussp. n. In addition, the immature stages of eggs and larvae of the new species are also described and discussed in detail.

Bartonella infection in shelter cats and dogs and their ectoparasites.

Mainly through vector transmission, domestic cats and dogs are infected by several Bartonella spp. and represent a large reservoir for human infections. This study investigated the relationship of prevalences of Bartonella infection in shelter dogs and cats and various ectoparasite species infesting them (fleas, ticks, and lice). Moreover, relationships between Bartonella infection and animal gender and age and presence of ectoparasites were analyzed. Blood samples were collected from 120 dogs and 103 cats. There were 386 ticks and 36 fleas harvested on these dogs, and 141 fleas, 4 ticks, and 2 lice harvested on these cats. Isolation/detection of Bartonella sp. was performed by culture, polymerase chain reaction (PCR), and partial sequencing. Bartonella was isolated from 21 (20.4%) cats and detected by PCR from 20 (19.4%) cats, 2 (1.7%) dogs, 55 (39%) fleas collected from cats, 28 (10%) ticks DNA samples, and 1 (2.8%) flea collected from dogs. When combining culture and PCR data, 27 cats and 55 fleas collected on cats were positive for Bartonella henselae or Bartonella clarridgeiae, but none were coinfected. Approximately half of the B. henselae isolates from 21 cats were B. henselae type I. Moreover, B. henselae, Bartonella phoceensis, Bartonella queenslandensis, Bartonella rattimassiliensis, Bartonella elizabethae DNA was detected in ticks collected from dogs and one flea was B. clarridgeiae PCR positive. This is the first report of such a wide variety of Bartonella spp. detected in Rhipicephalus sanguineus. Further studies are required to understand the relative importance of these ectoparasites to transmit Bartonella spp. in dogs and cats.

A genome-sequence survey for Ascogregarina taiwanensis supports evolutionary affiliation but metabolic diversity between a Gregarine and Cryptosporidium.

We have performed a whole-genome-sequence survey for the gregarine, Ascogregarina taiwanensis and herein describe both features unique to this early diverging apicomplexan and properties that unite it with Cryptosporidium, the Coccidia, and the Apicomplexa. Phylogenetic trees inferred from a concatenated protein sequence comprised of 10,750 amino acid positions, as well as the large subunit rRNA genes, robustly support phylogenetic affinity of Ascogregarina with Cryptosporidium at the base of the apicomplexan clade. Unlike Cryptosporidium, Ascogregarina possesses numerous mitochondrion-associated pathways and proteins, including enzymes within the Krebs cycle and a cytochrome-based respiratory chain. Ascogregarina further differs in the capacity for de novo synthesis of pyrimidines and amino acids. Ascogregarina shares with Cryptosporidium a Type I fatty acid synthase and likely a polyketide synthase. Cryptosporidium and Ascogregarina possess a large repertoire of multidomain surface proteins that align it with Toxoplasma and are proposed to be involved in coccidian-like functions. Four families of retrotransposable elements were identified, and thus, retroelements are present in Ascogregarina and Eimeria but not in other apicomplexans that have been analyzed. The sum observations suggest that Ascogregarina and Cryptosporidium share numerous molecular similarities, not only including coccidian-like features to the exclusion of Haemosporidia and Piroplasmida but also differ from each other significantly in their metabolic capacity.

Repellency of essential oils of Cryptomeria japonica (Pinaceae) against adults of the mosquitoes Aedes aegypti and Aedes albopictus (Diptera:Culicidae).

The purpose of this study was to investigate the repellent activities of essential oils from Cryptomeria japonica (sugi) against adults of mosquitoes Aedes aegypti and Aedes albopictus . Comparison of essential oils from four different plant parts of C. japonica revealed that essential oil from its leaf exhibited the best repellent activity against mosquitoes. To understand the relationship between volatile organic compounds and repellent activity, the solid-phase microextraction (SPME) method was employed to analyze volatile organic compounds of leaf essential oil. The SPME fiber was coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS). The major volatile organic compounds in the cage were 3-carene, alpha-terpinene, limonene, gamma-terpinene, and terpinolene at 0 min. Results demonstrated that (-)-terpinen-4-ol was the major volatile organic compound adsorbed by SPME fiber during repellent assays. Furthermore, the repellent activities of six compounds against adults of the mosquitoes were evaluated, and the results revealed that (-)-terpinen-4-ol exhibited the best repellent activity against A. aegypti and A. albopictus.

Mosquito larvicidal activities of extractives from black heartwood-type Cryptomeria japonica.

The purpose of this study is to determine the larvicidal activities of ethanolic extracts from leaves, wood, and bark of black heartwood-type Cryptomeria japonica against fourth-instar larvae of mosquitoes Aedes aegypti and Aedes albopictus. Among three ethanolic extracts from C. japonica, wood extract exhibited the best larvicidal activity against A. aegypti and A. albopictus with LC(50) values of 63.2 and 93.8 microg/ml, respectively. Four major compounds, including ferruginol, epi-cubebol, cubebol, and isopimarol, were isolated from wood extract, and it was demonstrated that cubebol exhibited the best activity against A. aegypti and A. albopictus with LC(50) values of 60.1 and 50.0 microg/ml, respectively. Our findings showed that the wood extract and cubebol from C. japonica have good potential as a source for natural larvicides.

Rapid identification of the Mediterranean fruit fly (Diptera: Tephritidae) by loop-mediated isothermal amplification.

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), ranks as one of the world's most destructive agricultural pests. This pest is also widespread and highly invasive; thus, it is a high priority for pest detection and quarantine programs. Although Mediterranean fruit fly adult and third-instar larvae can usually be identified and distinguished from other species by morphological keys, it is often difficult or impossible to identify or distinguish this species from other tephritids by using material from other stages of development. In such situations, use of a molecular technique known as loop-mediated isothermal amplification (LAMP) would be valuable as a rapid and robust alternative species diagnostic tool. This method uses isothermal conditions and requires only relatively inexpensive equipment. In this study we have developed a simple and rapid procedure that combines a Chelex-based DNA extraction procedure with LAMP to rapidly detect the presence of Mediterranean fruit fly DNA and discriminate it from other species, by using material from different stages of development. Amounts of DNA as little as that recovered from a single egg were shown to be adequate for the analysis, and LAMP itself required only 45 min to complete.

Rickettsia felis in cat fleas in Taiwan.

We describe the first detection of Rickettsia felis in cat fleas (Ctenocephalides felis) in Taiwan. Natural infections of R. felis in cat fleas were isolated and confirmed using polymerase chain reaction (PCR) and an immunofluorescence assay. The infection rate in individual fleas and the minimum infection rate in pooled fleas detected by the PCR method were found to be 18.8% (13/69) and 8.2% (8/97), respectively. Partial sequences of the plasmid pRF, 17-kDa antigen, and outer membrane protein A genes obtained from the samples are identical to those of R. felis URRWXCal2. Serological studies confirmed R. felis infection in two stray cats, as demonstrated by the presence of serum IgG antibodies against R. felis with an immunofluorescence assay titer of 1:320.

Variations in insecticidal activity and chemical compositions of leaf essential oils from Cryptomeria japonica at different ages.

The larvicidal effects of the essential oils extracted from the leaves of Cryptomeria japonica at different ages (58, 42, and 26 years old) against 2 mosquito species, Aedes aegypti and Aedes albopictus, were studied. The analysis of major constituents of these essential oils was also investigated. Results obtained from the larvicidal tests, using essential oil from the leaves of 58-year-old C. japonica was found to be most effective against both A. aegypti and A. albopictus larvae, indicating tree age has significant influence on mosquito larvicidal activity. In addition, the eleven pure constituents from C. japonica leaf essential oil were also tested individually against the two mosquito larvae. Among them, alpha-terpinene, gamma-terpinene, p-cymene, 3-carene, terpinolene, and beta-myrcene shows strong larvicidal effect against the two mosquito larvae. Among these pure constituents, 3-carene exhibits the best larvicidal effect against A. aegypti and terpinolene shows an excellent inhibitory action against A. albopictus larvae. The results of this study show that the leaf essential oil and its effective constituents might be considered as a potent source for the production of fine natural larvicides.