the narrow - ridged finless porpoises examined in this study were collected during the period from 2010 to 2015 from the following areas : the omura bay , ariake 
 sound , tachibana bay , seto inland sea , ise bay , mikawa bay and the pacific coast of chiba and ibaraki prefectures .
the host animals were autopsied at the 
 stranding site or transported to the laboratory and frozen in 18c , to be autopsied on another day . in total ,
137 porpoises were used in this study ; the host 
 information is shown in table 1table 1.host animal sampling information : narrow - ridged finless porpoise ( neophocaena asiaeorientalis)location ( population)sampling periodnumber of animalscategory based on body length<90 cm90110
cm>110 cmunclearomura bay ( ob)201020155017726-ariake sound , tachibana bay ( at)2010201542610251seto inland sea ( sis)20122015245217-ise bay , mikawa bay ( im)2014191405-pacific coast of chiba and ibaraki prefecture ( ts)201320142101-total1374319741 . the parasitological examination followed the methodology described previously .
the surface of the host animal was 
 investigated before the autopsy to search for ectoparasites and epizoits .
the lungs and liver were cross - sectioned and observed macroscopically , then sliced into 
 1 to 2 cm thickness and washed in fresh water with slight pressure by hands . the stomach and intestine were opened entirely and washed in fresh water .
all of the washes obtained were left to stand , and then , the precipitation was observed under a stereomicroscope to 
 collect any helminths . in some cases ,
this method could not be applied to all organs and tissues owing to post - mortem change , including organ loss or degradation . 

only cross - sectioning and macroscopic observations were applied to highly degraded organs , and extremely degraded organs were not examined .
totally cranial sinus 
 of 118 , lungs of 114 , liver of 69 , stomach of 49 and intestine of 88 porpoises were examined . when parasites were found in other tissues or organs during the 
 autopsy , they were also collected .
nematodes were fixed in 70% or 99% ethanol , or 10% neutral buffered formalin , and cleared for observation using gater s solution .
in such cases , the total number of worms was estimated based on the 
 proportion of the weight of the worms .
one hundred entire worms were randomly selected from the collected worms and 
 identified ; then , the wet weights of the 100 worms and the total worms were measured .
the number of worms of each identified species was estimated from its 
 proportion in the sample , using the following equation : ( number of identified worms)(total worms weight)/(100 worms weight ) .
the identification of the female 
 lungworm was very difficult owing to morphological similarity between species ; therefore , only male lungworms were identified .
trematodes , cestodes and acanthocephalans were flattened between a pair of slide glasses and fixed in 70% ethanol .
after fixation , the worms were stained with 
 alum - carmine , semichon s carmine or heidenhain s iron hematoxylin , and mounted in canada balsam .
all specimens are deposited in the marine mammal research laboratory , nagasaki 
 university ( nu mmrl parasite collection nematoda 221 , 24 , 2831 , 3344 , 46 , 47 , 5091 , 96100 , 104114 , 118125 , 128 , 129 , 132 , 133 , 136193 , 195207 , 213 ; 
 digenea 413 , 1820 , 2224 , 2840 , 4253 , 5658 , 7283 , 88 , 89 , 91 , 92 , 95124 , 127132 , 135149 , 151 , 152 ; cestoda 1 , 9 , 69 , 71 ; acanthocephala 3 ) .
the prevalence of infection was calculated based only on the organs / tissues that had been washed and microscopically observed .
many helminths with indirect life 
 cycles are transmitted to their definitive host through dietary ingestion , including ingestion of infective larvae . therefore , whether the host has started to eat 
 prey or not must be considered for the prevalence calculation .
based on logistic models , shirakihara et al .   estimated that narrow - ridged finless porpoises start to eat solid food and wean at 98 cm and 101 cm in body length , respectively . in their models ,
a 
 porpoise is very unlikely ( 5% ) to be consuming solid food when it is 92 cm long , and to be consuming milk when it is 109 cm long .
therefore , in this study , < 90 
 cm long porpoises were regarded as nursing ( do not eat prey ) and > 110 cm long porpoises were considered weaned ( only eat prey ) .
porpoises of 90110 cm body 
 length were regarded as weaning ( in transition between nursing and weaned ) .
the differences in prevalence among host populations or between growth categories were tested using fisher s exact test .
pairwise fisher s exact test with the 
 benjamini  hochberg procedure was used for multiple comparisons between host populations .
all statistical analysies were performed using r , version 3.3.1 , and the statistical package fmsb   was used in multiple comparisons .
the identified species and sites of infection are shown in table 2table 2.list of the helminth species obtained in the japanese finless porpoises in this studyclasspredominant siteof infectionother sitesreferences for identificationspeciesnematodapharurus sunamericranial sinustrachea , lung p. asiaeorientaliscranial sinustrachea stenurus nanjingensiscranial sinus halocercus pingilungtrachea , uterus [ 26 , 36]h .
sunamerilung h. tauricalung trematodanasitrema sunamericranial sinus n. spathulatumcranial sinus campula oblongaliver / bile ductstomach , intestine synthesium elongatumintestinestomach , duodenum cestodadiphyllobothrium fuhrmanniintestine tetrabothrius sp.intestine acanthocephalacorynosoma sp.intestine a ) only male worms were identified .. in total , 13 species of helminths , 6 of nematodes , 4 of trematodes , 2 of cestodes and 1 of acanthocephalans were found .
most of the helminths were consistently found in a specific infection site ; however , some worms were in other sites .
pharurus 
 sunameri and pharurusasiaeorientalis were mainly found in the cranial sinus , with a few worms occurring in the 
 respiratory organs .
halocercus pingi was very long ; in cases of severe infection , the worms reached from the bronchiole to the trachea . 

campula oblonga and synthesium elongatum were 
 found in the liver / bile ducts and the intestine , respectively .
these trematodes were also found in the stomach , but no other helminths occurred in the stomach . 
 the detailed investigation and identification of tetrabothrius sp .
the general 
 appearance of corynosoma sp . was consistent with the description of unidentified specimens by kuramochi et al . .
table 3table 3.number of infected hosts and intensity of helminths in five japanese populations of the finless porpoise ( ob : omura bay , at : ariake sound - tachibana 
 bay , sis : seto inland sea , i m : ise - mikawa bays , ts :
taurica2 - 111 - 1 - 1 - 1 - 0 - --0 - --0 - --liver / examined host69241512162c .
fuhrmanni3- - 1231 - - -0 - --1 - - -0 - --1 - 123-tetrabothrius sp.1 - 1 - 0 - --1 - 1 - 0 - --0 - --0 - --corynosoma sp.1 - 426 - 0 - --0 - --0 - --0 - --1 - 426-a ) intensity was calculated using the number of identified worms and the ratio of the wet weight of the identified worms to the wet weight of total worms . 

shows each helminth species with the number of infected hosts , and the estimated mean , minimum and maximum intensity ( number of worms of positive 
 hosts ) for each population and the total of the populations . in total
, cranial sinus nematodes were found in 80 porpoises , including 4 hosts in which the worms 
 were highly degraded and unidentifiable .
stenurus 
 nanjingensis was found together with p. sunameri and p. asiaeorientalis in 2 hosts .
the species of the genus 
 nasitrema were found in 62 hosts , including 3 hosts in which the worms were highly degraded and unidentifiable . both species of this genus were 
 found together in 40 hosts , and coinfection of any trematode and any nematode species occurred in 61 hosts .
lungworms were found in 64 hosts , but were 
 unidentifiable in 29 of these owing to degradation or fragmentation .
h. pingi and h. sunameri were not found in the same host , 
 but h. taurica was found with h. sunameri in 2 hosts .
a ) intensity was calculated using the number of identified worms and the ratio of the wet weight of the identified worms to the wet weight of total worms . 

prevalence is shown as percentages in table 4table 4.prevalence of helminths in five japanese populations of the finless porpoise ( ob : omura bay , at : ariake sound - tachibana bay , sis : seto inland sea , i m : 
 ise - mikawa bays , ts :
- 600 - 100 s. nanjingensis004.000130000000 - 00 - 0 n. sunameri0107002567006700640 - 1000 - 100 n. spathulatum01072008000800100860 - 00 - 0lung / examined host34105011417441442131405101 h. pingi35202.04525025257.1250036 - 00 - 0 h. sunameri0102002524001400230 - 200 - 0 h. taurica0000000000000 - 00 - 0liver / examined host1883263142480151005000 c. oblonga01347007.102563 - 0800 - 100 - --intestine / examined host2413488615451412131005101 s. elongatum4.27.7290171300141000540 - 600 - 0 d. fuhrmanni004.2000000007.70 - 00 - 100 tetrabothrius sp.002.1000007.10000 - 00 - 0 corynosoma sp.002.10000000000 - 00 - 100a ) only hosts which were washed and microscopically observed are included .. the prevalence of all cranial sinus helminths was higher in > 110 cm hosts than in < 90 cm hosts in all populations .
the prevalence of 
 p. sunameri , p. asiaeorientalis and n. sunameri in > 110 cm hosts was not significantly different among 
 populations ( fisher s exact test , p>0.05 ) .
the prevalence of n. spathulatum was significantly different between i m and ob , at 
 and sis populations ( pairwise fisher s exact test with benjamini 
h. pingi was 
 frequently found in < 90 cm hosts in four of the populations , and its prevalence was not significantly different among populations ( fisher s exact test , 
 p>0.05 ) . in the total of the populations ,
the prevalence of h. pingi in < 90 cm hosts was significantly higher than in 
 > 110 cm hosts ( fisher s exact test , p<0.01 ) .
h. sunameri was only found in 90110 cm and > 110 cm hosts in four 
 populations .
its prevalence in > 110 cm hosts was not significantly different among populations ( fisher s exact test , p>0.05 ) , but it was 
 significantly higher than in < 90 cm hosts in the total of the populations ( fisher s exact test , p<0.01 ) . c. oblonga was 
 found only in > 110 cm hosts , and its prevalence in ob was significantly lower than in at , sis and i m ( pairwise fisher s exact test with benjamini 
s. elongatum was frequently found in > 110 cm hosts , and its prevalence was not significantly 
 different among populations ( fisher s exact test , p>0.05 ) .
pharurus sunameri and pharurus asiaeorientalis were originally 
 described as pseudostenurus sunameri yamaguti , 1951   and pharurus asiaeorientalis 
 petter , 1982 , respectively .
the former was included in the genus pharurus ; however , the name pseudostenurus sunameri has been used in several later studies [ 14 , 21 , 26 ] .
the latter was renamed and transferred to genus 
 otophocaenurus . both genera ( pseudostenurus and otophocaenurus ) 

s. elongatum was originally described as orthosplanchnus elongatus ozaki , 1935 ; 
 subsequently , it was transferred to the genus odhneriella   and then to hadwenius 
 .
therefore , in this study , a new combination synthesium elongatum n. comb . is used . a new location record and a new host record
s. nanjingensis was originally described based on specimens obtained 
 from a finless porpoise in nanjing , china .
the detection of this species in the present study is the first record in 
 japanese waters , although the worms were very rare and were only found in two hosts of the ob population .
this study is the first report about the helminths found in finless porpoises in the ob , at and i m populations .
it also adds further information on the sis and 
 ts populations , which have been studied previously .
eight species of helminths : p. sunameri , 
 p. asiaeorientalis , n. spathulatum , n. sunameri , h. pingi , h. sunameri , 
 c. oblonga and s. elongatum were commonly found and are considered common parasitic helminths of finless porpoises in japan . 
 in marine mammal helminths ,
anisakid nematodes in the stomach have been reported in several cetaceans [ 12 , 16 , 17 ] .
this is in line with previous 
 findings : another study on the finless porpoises in the sis and ts populations also found no anisakids .
this might be 
 due to the highly coastal habitat of the finless porpoise [ 14 , 19 ] .
on the other 
 hand , pilleri   reported anisakis typica in two hosts of the finless porpoise in pakistan .
jefferson 
 and wang   proposed that the genus neophocaena includes at least two species : n. 
 asiaeorientalis in east asia and n. phocaenoides in the indian ocean ; both species show extensive geographical variation . although the 
 prevalence of anisakid infection in n. phocaenoides is not reported , pilleri s report might indicate an inter - species or regional difference in 
 anisakid infection in the genus neophocaena , which probably reflects a difference in local habitat in the genus . comparing the helminthic fauna in the different host populations ,
the species composition or prevalence of helminths was more or less different between each 
 adjacent population .
although the number of examined hosts from ts was limited , a 
 previous study also found no n. spathulatum in the four hosts examined .
n. 
 spathulatum is considered absent or less prevalent in i m and ts populations than in the other populations .
kuramochi et al .   examined the intestines of three adult finless porpoises 
 from ts and obtained some specimens of the genus corynosoma from all of them .
the prevalence of c. oblonga in the ob population was lower than in the other 
 populations , with the exception of ts .
the only available information on c. oblonga infection in the ts population is from kuramochi et 
 al . .
in their study , one of three examined adult porpoises was infected . if the data from the present study 
 and kuramochi et al .  
are combined , the best estimate of the prevalence of c. oblonga 
 in the ts population increases to 50% ( 2/4 ) , higher than the ob population . not only for the finless porpoise ,
c. oblonga has been found 
 in the harbor porpoise ( phocoena phocoena ) [ 7 , 12 , 32 , 33 ] , dall s porpoise ( phocaenoides dalli )   and the common dolphin ( delphinus delphis )   from a wide range of habitats in the 
 northern hemisphere .
an atypical - host infection has also been reported in the common thresher shark ( alopias vulpinus ) .
based on the feeding habits of these hosts , the transmission of c. oblonga is suspected to involve the consumption of fish . 
 on the other hand ,
n. spathulatum has only been found in finless porpoises in japan , and there is no information available about its lifecycle . 
 in this study ,
n. spathulatum was found exclusively in porpoises that were longer than 90 cm , which were designated weaning or weaned ( see 
 materials and methods ) .
therefore , one or multiple prey species might serve as intermediate / paratenic hosts . from stomach content analyses ,
the finless porpoise 
 is known to be an opportunistic feeder , which feeds on several fishes , cephalopods , crustaceans and other marine organisms [ 20 , 30 , 31 ] .
the differences in the presence and prevalence of helminths 
 found in this study might reflect differences in the available prey species in each population .
the prevalence of h. pingi was higher in < 90 cm porpoises , which were considered nursing ( see materials and methods ) , than in > 110 cm 
 porpoises , considered weaned .
the preferential infection by h. pingi of neonate finless porpoises ( the exact species are unknown ) has been 
 reported in hong kong , and prenatal or transmammary infection has also been suspected [ 25 , 26 ] .
such vertical transmission has also been suspected during h. lagenorhynchi 
 infection of the bottlenose dolphin ( tursiops truncatus ) [ 8 , 10 ] and 
 the common dolphin ( d. delphis ) .
if the vertical transmission hypothesis is true , it is strange that 
 h. pingi is almost not found in > 110 cm porpoises . if the worms are transmitted from the mother to the fetus or the neonate , the prevalence 
 of infected adults should be equal to or higher than that of calves .
the pathogenicity of h. pingi could increase the fatality of severely 
 infected neonates , which would result in high numbers of young animals infected by h. pingi being stranded .
thus , the prevalence of h. 
 pingi in < 90 cm porpoises might appear higher than in > 110 cm porpoises . on the other hand ,
a congeneric species h. sunameri was 
 found frequently in > 110 cm porpoises , but not found in < 90 cm porpoises , indicating that h. sunameri infects porpoises via infected prey . 
 in lungworms found in the harbor porpoise , a correlation between the host age and
the number of species of lungworms has been observed , and a heteroxenous lifecycle , i.e. , the existence of intermediate host species , has been suggested .
although the species reported in the harbor porpoise are different from those in the finless porpoise [ 4 , 
 9 , 12 ] , a heteroxenous lifecycle of finless porpoise lungworms is plausible .
the 
 presence of completely different infection routes between the two congenerics ( the vertical transmission of h. pingi and the trans - prey infection 
 of h. sunameri ) is very interesting .
such shifts in lungworm species with the host s growth have not been reported in other cetaceans . to verify 
 the infection routes , detection of h. pingi in fetuses or milk , and the identification of infective h. sunameri larvae from prey 
 species are needed .
 a survey of helminths and ectoparasites , including epizoits , was conducted in narrow - ridged finless porpoises ( neophocaena asiaeorientalis ) 
 from japanese five populations using dead stranded or incidentally caught animals . in total , 13 helminth species were found ( 6 nematodes , 4 trematodes , 2 
 cestodes and 1 acanthocephalan ) in 137 porpoises . a new location record of stenurus nanjingensis and a new host record of 
 tetrabothrius sp . 
 were obtained . 
 eight species of helminth were considered common in the japanese populations of the finless porpoise : 
 pharurus sunameri , pharurus asiaeorientalis , nasitrema spathulatum , nasitrema sunameri , 
 halocercus pingi , halocercus sunameri , campula oblonga and synthesium elongatum . 
 no 
 anisakid nematodes were found . 
 n. spathulatum was found only in the western waters of the seto inland sea . 
 low prevalence of c. 
 oblonga in the omura bay was demonstrated . 
 h. pingi was mostly found in very young porpoises before starting to eat prey , indicating 
 prenatal or transmammary infection . 
 however , a congeneric species , h. sunameri , mainly infected weaned porpoises , indicating that these two 
 species possess different transmission pathways . 
 this study provides information on the geographical distribution and prevalence of helminth parasites in 
 finless porpoises off the japanese coast . 
MATERIALS AND METHODS
RESULTS
DISCUSSION