The field information is lacked in most of the horsehair worms due to their complex life cycles and difficulty in survey. Thus, species frequently encountered becomes the valuable material in building the model life history of the horsehair worms in the field. Chordodes formosanus is the most common horsehair worm species in Taiwan. Before the systematic scientific research, C. formosanus is generally known to be the parasite of Hierodula mantids which usually emerge in early summer. This legend was first confirmed by the annual survey of the mantids in Taipei Zoo in 2007–2008 (Chiu and Wu, 2008), in which seven mantid species were identified and one Chordodes species parasitizing with H. formosana and H.
patellifera. The season of horsehair worm emergence is also confirmed since most of
the adult worms were found in the adult H. formosana during its reproductive season in the early summer (Chiu and Wu, 2008). These horsehair worms were described asC. formosanus in Chiu et al. (2011).
The infection of C. formosanus only in Hierodula mantids suggested the highly host specificity among the sympatric mantids (Chiu and Wu, 2008). It is also the first evidence suggesting the diverged species status from its morphological closed species,
C. japonensis (Chiu et al., 2011). Comparing with the annual infection rate of the
16.67% and 20% in H. formosana and H. patellifera, respectively, no worm was found in the 101 samples of the Chinese mantids, Tenodera sinensis. Actually, horsehair worms emerging from Tenodera have never been found in Taiwan to date.However, the mantids Tenodera and Hierodula were both recorded to be the definitive hosts of C. japonensis
(Inoue, 1952, 1955; Schmidt-Rhaesa, 2004). The further
examination of the detailed morphology and the barcoding sequences of the horsehair worms in Tenodera and Hierodula from Taiwan and Japan isolated the new species C.formosanus from C. japonensis, and thus, suggested the definitive host specific to the Hierodula mantids in C. formosanus, and the Tenodera mantids in C. japonensis (Chiu et al., 2011).
It has been a long time that I believe the definitive host range of C. formosanus is limited to the genus level, until five adult worms inside the mantid, Acromantis
japonica and two katydids, Leptoteratura sp. and Holochlora japonica, were
identified as C. formosanus by comparing the barcoding sequence (Appendix 4). The similar situation is also happened to C. japonensis, which has been once found to emerge from a katydid, Hexacentrus japonicus japonicus(Inoue, 1955). This result
extends the host range of C. formosanus to cross different orders of Insecta, but passes some closely related taxa. The newly found hosts of C. formosanus are likely to be novel hosts, which are not the main host supporting the parasite population. A parasites does not equally parasitize with all the hosts it potentially can infect (vanKlinken, 1999). The three main parasite metrics (prevalence, intensity, and abundance)
are commonly applied to quantify the host suitability to a parasite, and use to compare the host specificity level of different parasites (Poulin, 2007). However, in the case ofC. formosanus, these three parasite metrics among the known hosts are difficult to be
measured due to the difficulty in sampling. In addition, the contribution of each host to C. formosanus might be not completely represented by these three parameters. One reason is the low or no chance to mate is possible to be caused by the low infection rate. Most species of the horsehair worms prolong their lineage by sexual reproduction (with only one known exception of Paragordius obamai) (Hanelt et al.,2012), the horsehair worm cysts developing in the novel hosts might be finally not
able to find the mate and have to pay for the cost in the loss of chance to enter the"main hosts" through paratenesis (Hanelt and Janovy, 2004a). Another possible reason
is the size of the adult horsehair worms, which is highly determined by the host size and intensity, is the main factor to influence the fecundity of a female worm (Hanelt,
2009). The different among individuals can be higher than six folds (Hanelt, 2009) but
is not considered in any of the three main infection parameters.Other than the three parasite metrics commonly used, the seasonal infection of aquatic paratenic hosts provide an indicator in investigating the relative reproductive potential of the adult C. formosanus emerging from different hosts. The adult C.
formosanus from different species of definitive hosts were collected in different
seasons ((Chiu et al., 2016b): Hier. formosana (late June to early August, and late January), Hier. patellifera (September) (Chiu and Wu, 2008; Chiu et al., 2011, 2016a),Acromantis japonica (October, and February) (Appendix 4, personal observation), Holochlora japonica (November), and Leptoteratura sp. (March) (Appendix 4). Most
of the adult C. formosanus were collected from the adult definitive hosts, except a pair of adult worms collected in late January comes from a nymphal Hier. formosana. The phenomenon that adult worms generally emerge from adult definitive hosts meets our artificial infection of Hier. patellifera. In our experiment, nine of the ten Hier.patellifera released male adult C. formosanus around one month after the last molting
(32.8 ± 3.86 d (29–42 d)), despite some of the worm entered the host much earlier than others (two individuals developing for 103 and 143 days in the hosts which were infected earlier than the others 82.71 ± 7.52 d but they released the worms 32 and 35 days, respectively after the last molting). There is still one artificially infected host release an adult male worm one day after the last molting, but it is worth to note the possible developmental synchronization of the horsehair worms.The horsehair worms emerging from these five hosts reproduce in the water and theoretically create intensive infection risks for their aquatic paratenic hosts in the
different seasons. A regular survey was conducted in 2014 to early 2015 in Northern Taiwan
(Chiu et al., 2016a). The larval chironomids (Chironominae: Diptera) were
collected and examined the number of Chordodes cysts in each host. The short life span of the larval chironomids in aquatic environments provides a good indicator to monitor the amount of the larval horsehair worms in the water. In total, 806 cysts were found during the 25 times sampling and showed a single infection peak in mid-September(Chiu et al., 2016a). The horsehair worm larva is infectious for less
than two weeks in the water (Hanelt and Janovy, 2004b). The single infection peak indicates there is one mass reproduction of Chordodes, despite the adult worms emerge several times from different definitive hosts during a year.The seasonal infection in the paratenic host indicates the unequal contribution of the five definitive hosts, with a dominate one, to the parasite's reproduction. Egg of the horsehair worm goes through half to one month of embryogenesis before hatching
(Poinar and Doelman, 1974; Zanca et al., 2007; Bolek et al., 2015)
and spend 3-15 days to be encysted after enter paratenic hosts (Inoue, 1962; Hanelt and Janovy,2004b). In the case of C. formosanus, the egg spends around 25 days on
embryogenesis. In the artificial infection, 623.94 ± 102.12 eggs developing for different days under 28°C were feed to the newly hatched mosquito larvae (Aedesalbopictus) (Method 5-2). The infection rate, and the mortality rate at 24 hours of the
mosquito were almost zero in the eggs developing for less than 21 days, but increased rapidly in the eggs developing for 24 days which indicates the time when larval C.formosanus hatches and becomes infectious (Fig. 11). In summary, horsehair worms,
after being laid as the egg strings, spend approximately 30 free-living days in the aquatic environment. The closest time is when the adult C. formosanus emerges from the adult mantid hosts, H. formosana in July, which is around 2–2.5 months before theinfection peak (Chiu et al., 2016a).
Fig. 11. Mortality rate and infection rate of the 20 newly hatched mosquitoes, Aedes
albopictus, infected by the egg of Chordodes formosanus developing for
different days.According to both of our collecting experience and the seasonal infection of the paratenic hosts, H. formosana is the dominant definitive host of C. formosanus. The annual survey of paratenic hosts not only provide the information of geographic distribution
(Hanelt et al., 2001) and species composition (Bolek et al., 2013a), but
also the seasonality of the horsehair worms (Chiu et al., 2016a). The single mass reproduction of C. formosanus creates the high infection risk to the aquatic insects, and these infected aquatic insects will then bring the cysts to the definitive hosts. In Taiwan, H. formosana is the endemic species with one generation in each year. Its adults mate and reproduce in early summer and the eggs hatch in mid-August(Chiu and Wu, 2008). The newly hatched nymph will soon face to the highest infection risk
of a year in autumn. It is plausible that nymphal mantids are infected by preying on infected aquatic insects, and they release the adult worms in the following summer.However, the real situation seems to be more complicated because the adult worms mature and emerge from nymphal H. formosanain spring
(Chiu and Wu, 2008).
During our laboratory rearing, we found that some horsehair worms parasitizing early-instar mantids matured before the host emerged (unpublished data). This might suggest the existence of 2 generations of a horsehair worm population per year.
However, the scale of reproduction in spring is too small to cause the large number of larval worms produced in July. One possible scenario is that most of the cysts are not directly bring to the definitive hosts in autumn. They enter the food web of aquatic animals through paratenesis (Hanelt and Janovy, 2004b) and maintain their infectivity for several months until parasitizing with the definitive hosts
(Bolek et al., 2013b).
Another reason of the smaller scale of reproduction in spring is the smaller size of the adult horsehair worms emerging from the nymphal host. Length of adult worms emerging from the nymphal H. formosana in spring is less than half of that emerging
from the adult H. formosana in summer (Chiu et al., 2011), which makes the millions of difference in the egg number produced by each adult female (Hanelt, 2009).
The mass reproduction and high fecundity of the large adult worms might be the benefit to promote high host specificity of C. formosanus. The clear specificity is also noted in Gordius sp. by the free-living adults frequently collected in the winter and the cysts found in the aquatic insects collected in March. In the case of Acutogordius
formosanus n. sp., the seasonality is likely to be relatively unobvious. Adults of Acutogordius formosanus n. sp. are frequently found in several orthopteran insects
which can be found from mid May to late October. The high infection risk of the aquatic insects might be continued during the whole year. This phenomenon is unfortunately not confirmed in our annual survey in 2014 due to the temporal extinction of Acutogordius formosanus n. sp. after the stream remediation (Chiu et al.,2016a). Such temporal local extinction was also found after clear-cut logging in
artificial forests (Sato et al., 2014). While cysts of C. formosanus were still detected,Acutogordius formosanus n. sp. seems to be more sensitive to the human activity.
The life history model of C. formosanus is valuable to be the reference of other horsehair worms in Taiwan, and the investigation of the cyst stage provide an efficient tool and makes the large scale survey become possible. Although the identification of species is still relied on the adult stage, the seasonal dynamic of infection in aquatic insects provide the possible time period to collect the adult just emerging from the host. The application of the investigation in immature stage of the horsehair worms is likely to promote the understanding of the ecology of horsehair worms. Since the previous survey in Taiwan is mainly focused on the adult stage, the field information of more species with their horizontal and vertical distribution must to be structured soon by the newly developed method.
4 Horsehair worm-induced host developmental manipulation
Among more than 350 described horsehair worm species, our understanding of the horsehair worms mainly comes from the studies of a few "model species". The current knowledge of host behavior manipulation mainly comes from the studies in
Paragordius tricuspidatus and P. varius (Thomas et al., 2002; Biron et al., 2005b;
Sánchez et al., 2008a, b; Ponton et al., 2011; Barquin et al., 2015), host biochemical
physiological change in P. tricuspidatus and Spinochordodes tellinii(Thomas et al., 2003; Biron et al., 2005a, 2006; Ponton et al., 2006a), influence of the energy flow in
riparian ecosystems in Gordionus spp. (Sato et al., 2008, 2011), response to predator by P. tricuspidatus (Ponton et al., 2006b; Sánchez et al., 2008a), larval ecology in C.morgani, C. nobilii, G. robustus, P. varius, and P. obamai (Hanelt and Janovy, 2003, 2004a; de Villalobos et al., 2003; Zanca et al., 2007; Achiorno et al., 2009; Bolek et al., 2013b), and artificial infection in P. varius and Chordodes sp. (Inoue, 1962;
Hanelt and Janovy, 2004b). These horsehair worm species are relative easy to be
collected and reared in each area, and their influences on the hosts are obvious.Chordodes formosanus is one of the most common species in Taiwan. The large
population size and obvious seasonality makes it easy to be collected and provide a material to restructure the life history model in the field. In the study of the definitive host phase, despite the main definitive host, Hierodula mantids, is relative not easy to be cultured, the obvious morphological alternations frequently found on the infectedH. formosana suggests the possible developmental manipulation induced by C.
formosanus. In the very beginning of our studies from 2006, we already noted the
abnormal wing shape in the infected male H. formosana during surveying. The morphological change is obvious to be directly applied in distinguishing the infectedeven in another main host, H. patellifera. Although this phenomenon has also been found in the mantids, Tarachodella monticola