developmental stages and growth of pseudocaligus fugu yamaguti, 1936 (copepoda: siphonostomatoida:...

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Developmental stages and growth of Pseudocaligus fugu Yamaguti, 1936(Copepoda: Siphonostomatoida: Caligidae) host-specific to PufferSusumu Ohtsuka a; Ikuo Takami b; B.A. Venmathi Maran a; Kazuo Ogawa c; Takaki Shimono a; Yusuke Fujitad; Manabu Asakawa d; Geoffrey A. Boxshall e

a Takehara Marine Science Station, Setouchi Field Science Centre, Graduate School of Biosphere Science,Hiroshima University, Takehara, Hiroshima, Japan b Nagasaki Prefectural Institute of Fisheries, Nagasaki,Nagasaki, Japan c Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo,Japan d Graduate School of Biosphere Science, Higashi-Hiroshima, Hiroshima, Japan e Department ofZoology, The Natural History Museum, London, UK

Online Publication Date: 01 August 2009

To cite this Article Ohtsuka, Susumu, Takami, Ikuo, Venmathi Maran, B.A., Ogawa, Kazuo, Shimono, Takaki, Fujita, Yusuke, Asakawa,Manabu and Boxshall, Geoffrey A.(2009)'Developmental stages and growth of Pseudocaligus fugu Yamaguti, 1936 (Copepoda:Siphonostomatoida: Caligidae) host-specific to Puffer',Journal of Natural History,43:29,1779 — 1804

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Journal of Natural HistoryVol. 43, Nos. 29–30, August 2009, 1779–1804

ISSN 0022-2933 print/ISSN 1464-5262 online© 2009 Taylor & FrancisDOI: 10.1080/00222930902993757http://www.informaworld.com

TNAH0022-29331464-5262Journal of Natural History, Vol. 1, No. 1, May 2009: pp. 0–0Journal of Natural HistoryDevelopmental stages and growth of Pseudocaligus fugu Yamaguti, 1936 (Copepoda: Siphonostomatoida: Caligidae) host-specific to PufferJournal of Natural HistoryS. Ohtsuka et al.Susumu Ohtsukaa*, Ikuo Takamib, B.A. Venmathi Marana, Kazuo Ogawac, Takaki Shimonoa, Yusuke Fujitad, Manabu Asakawad and Geoffrey A. Boxshalle

aTakehara Marine Science Station, Setouchi Field Science Centre, Graduate School of Biosphere Science, Hiroshima University, 5-8-1 Minato-machi, Takehara, Hiroshima 725-0024, Japan; bNagasaki Prefectural Institute of Fisheries, 1551-4 Taira-machi, Nagasaki, Nagasaki 851-2213, Japan; cGraduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan; dGraduate School of Biosphere Science, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan; eDepartment of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK

(Received 21 January 2009; final version received 23 April 2009)

The post-embryonic development of the copepod Pseudocaligus fugu is described.This sea louse only parasitizes toxic pufferfish, and causes commercial loss ofcultured Takifugu rubripes in Japan. Two naupliar, one copepodid and fourchalimus stages preceding the adult are recognized in the species. The develop-ment pattern is similar to that of Caligus except for the suppression of leg 4 in theearly chalimus phase. Pseudocaligus fugu lacks any preadult stage, as in Caligus,but contrasts with Lepeophtheirus species in which two preadult stages arereported. The nature of the metamorphic changes in size and even in degree ofexpression of articulations can occur without moulting. The validity of somechalimus stages recognized in Lepeophtheirus species is questioned. Growth andegg production in P. fugu were obtained in the laboratory. Pseudocaligus fuguattained adulthood 9 days after the infective copepodid attached to the host.

Keywords: Caligidae; Copepoda; development; growth; Pseudocaligus fugu;pufferfish

Introduction

Members of the siphonostomatoid copepod family Caligidae are commonly known assea lice and cause significant commercial losses in aquaculture worldwide (Johnsonet al. 2004; Ho and Lin 2004). In Japan, five species are recognized as pests in marinefinfish aquaculture (Ho and Lin 2004; our unpublished data): Caligus lalandeiBarnard, 1948 on amberjacks, C. spinosus Yamaguti, 1939 on yellowtail and salmon,C. sclerotinosus Roubal, Armitage and Rohde, 1983 on red seabream, Lepeophtheiruslongiventralis Yü and Wu, 1932 on spotted halibut, and Pseudocaligus fuguYamaguti, 1936 on the tiger puffer Takifugu rubripes (Temminck and Schlegel, 1850).Caligus sclerotinosus is suspected to have been recently introduced to Japan from itsnatural range (Ho et al. 2004). Cultured tiger puffer have suffered serious infectionswith P. fugu in Nagasaki Prefecture, south-western Japan since 2003. Heavilyinfected puffer are irritated by skin lesions caused by the sea lice (Figure 1) andsecondary bacterial infection can result in significant mortality.

*Corresponding author. Email: [email protected]

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https://www.researchgate.net/publication/8905516_Two_species_of_parasitic_copepods_Caligidae_new_to_Japan?el=1_x_8&enrichId=rgreq-1d562d94-313e-424e-a74b-df9d15e3ed91&enrichSource=Y292ZXJQYWdlOzIzNDExNDI4MDtBUzoxMDE5MzQ0OTcyNzE4MjBAMTQwMTMxNDQ4NzE3OA==


1780 S. Ohtsuka et al.

Figure 1. Zero-year cultured tiger puffer Takifugu rubripes infected by Pseudocaligus fugu(indicated by small arrows). Scale = 50 mm.


Journal of Natural History 1781

The full life cycle is known in only 15 species of Caligidae representing just twogenera, Caligus Müller, 1785 (11 species) and Lepeophtheirus von Nordmann, 1832(four species) (Ho and Lin 2004), even though the family comprises more than 450species currently accommodated in 34 genera (Boxshall and Halsey 2004; Boxshalland Justine 2005; Boxshall 2008). All caligid species examined have two nauplii andone copepodid, the infective stage, in common. Subsequent development on the hostis more variable, with different numbers of stages reported in Caligus and in Lepeoph-theirus: four or six chalimi (chalimus stages), zero or two preadults, and one adultstage (Kabata 1972; Ho and Lin 2004). In this context it is especially interesting toobserve the complete cycle in a third genus, Pseudocaligus A. Scott, 1901. Thechalimus and preadult stages are juveniles between the copepodid and adult ratherthan larvae, unique to the family Caligidae and some other siphonostomatoid famil-ies (Ho and Lin 2004). The chalimus is a phase firmly attached to the host with afrontal filament, while the preadult is a subsequent phase which is initially attachedto the host by a temporary frontal filament but which becomes detached before thenext moult (Anstensrud 1990).

Pseudocaligus fugu is highly host-specific to pufferfish, including the grass pufferTakifugu niphobles (Jordan and Snyder, 1901), the panther puffer T. pardalis(Temminck and Schlegel, 1850) and T. rubripes, all of which are known to produce tet-rodotoxin (TTX) and related toxins (Yamaguti 1936; Kano 1988; Kim 1998; Itoh etal. 2006; Venmathi Maran et al. 2007). Our recent studies on this parasite of toxicpuffer have revealed that, through feeding on the mucus and skin of its host, theparasite accumulates TTX in most body tissues with the exception of the female repro-ductive organs and the epicuticle (Ikeda et al. 2006; Ito et al. 2006). In addition, epibi-ontic bacteria on P. fugu isolated from T. pardalis appear to produce TTX (VenmathiMaran et al. 2007). This parasite has become widespread in cultured puffer inNagasaki and other prefectures in western Japan so we need to understand the biologyof the species to better manage and control infestations in commercial aquaculturefacilities. This paper describes the post-embryonic stages of P. fugu except for theadults, which have already been well described by Yamaguti (1936) and Kim (1998).

Materials and methods

Description of developmental stages

Two ovigerous females of P. fugu were collected from the body surface of grasspuffer, T. niphobles, caught off Takehara City, Hiroshima Prefecture, western Japanin April 2008. Egg strings from these females were incubated at temperatures ofabout 15–17°C in sterilized seawater until hatching, to obtain nauplii and copepo-dids. Chalimus stages and adults were collected from the fins and body of the samehost species, collected off Takehara City also in April 2008.

Supplementary specimens of the copepodid, chalimus and adult stages were col-lected in May 2008 from about 70-day-old juveniles of tiger puffer T. rubripes kept inthe Nagasaki Prefectural Institute of Fisheries, Nagasaki City, Nagasaki Prefecture(see below). Only a few third chalimus stage females were collected from TakeharaCity so we used specimens of this stage selected from the Nagasaki material for themorphological description. There were no differences in morphology between thedevelopmental stages of P. fugu collected from Takehara and Nagasaki.


https://www.researchgate.net/publication/6854800_Detection_of_tetrodotoxin_TTX_from_two_copepods_infecting_the_grass_puffer_Takifugu_niphobles_TTX_attracting_the_parasites?el=1_x_8&enrichId=rgreq-1d562d94-313e-424e-a74b-df9d15e3ed91&enrichSource=Y292ZXJQYWdlOzIzNDExNDI4MDtBUzoxMDE5MzQ0OTcyNzE4MjBAMTQwMTMxNDQ4NzE3OA==




https://www.researchgate.net/publication/231788796_Moulting_and_mating_in_Lepeophtheirus_pectoralis_Copepoda_Caligidae?el=1_x_8&enrichId=rgreq-1d562d94-313e-424e-a74b-df9d15e3ed91&enrichSource=Y292ZXJQYWdlOzIzNDExNDI4MDtBUzoxMDE5MzQ0OTcyNzE4MjBAMTQwMTMxNDQ4NzE3OA==

https://www.researchgate.net/publication/237179107_Developmental_Stages_of_Caligus_clemensi_Copepoda_Caligidae?el=1_x_8&enrichId=rgreq-1d562d94-313e-424e-a74b-df9d15e3ed91&enrichSource=Y292ZXJQYWdlOzIzNDExNDI4MDtBUzoxMDE5MzQ0OTcyNzE4MjBAMTQwMTMxNDQ4NzE3OA==

https://www.researchgate.net/publication/7001572_Accumulation_of_tetrodotoxin_TTX_in_Pseudocaligus_fugu_a_parasitic_copepod_from_panther_puffer_Takifugu_pardalis_but_without_vertical_transmission-Using_an_immunoenzymatic_technique?el=1_x_8&enrichId=rgreq-1d562d94-313e-424e-a74b-df9d15e3ed91&enrichSource=Y292ZXJQYWdlOzIzNDExNDI4MDtBUzoxMDE5MzQ0OTcyNzE4MjBAMTQwMTMxNDQ4NzE3OA==


The copepod specimens cleared in lactophenol were observed following themethod of Humes and Gooding (1964). Drawings and measurements were made withthe aid of a camera lucida attached to a differential interference contrast microscope(Nikon Optiphoto).

Terminology basically follows Boxshall (1990a) and Huys and Boxshall (1991).

Attachment site and growth

The attachment sites and growth rates of the life history stages of P. fugu were observedin the laboratory. Adult females of P. fugu were collected on 8 May 2008 from 1-year-oldtiger puffer cultured in Kosasa Town, Sasebo City, Nagasaki Prefecture. Copepodidsproduced from these females on 10 May 2008 were transmitted to 97 juvenile tiger pufferheld in a 100-l tank (these fish were produced as seeds in Minamiarima Town,Minami-Shimabara City, Nagasaki Prefecture at the end of February 2008). Watertemperature in the tank ranged from 18.6°C (13 May) to 20.2°C (27 May). Five hostswere sampled from the tank and preserved in 10% neutralized seawater formalin from13 to 27 May 2008 to study the development of the parasite. Preference for attachmentsites of stages from the copepodid to the adult was analysed using a Friedman test.

Body lengths of the naupliar, copepodid and chalimus stages and young adults ofP. fugu were measured from the above material. Nauplii and copepodids cultured inthe laboratory were measured separately from those derived from ovigerous femalesof P. fugu collected from 0-year tiger puffer cultured in Kosasa Town, on 23 July 2008.

Number of eggs per string

The number of eggs per string was counted for 30 ovigerous females infecting 0-yearT. rubripes in a culture area off Kosasa Town, on 29 May 2008.

Results

Description

First nauplius (Figures 2, 3)

Body length (based on individuals originating from adult females collected from Take-hara). Range: 0.38–0.40 mm; average ± standard deviation: 0.39 ± 0.01 mm (n = 7);(Nagasaki): 0.32–0.37 mm; 0.34 ± 0.02 mm (n = 20) (Figure 2).

Description (based on individuals cultured from ovigerous females collected fromTakehara). Body (Figure 3A) oval, widest at midlength. Nauplius eye present.Labrum anteroventrally produced, mouth not open. Paired balancers located poster-olaterally, curved outwards, flattened distally.

Antennule (Figure 3B) two-segmented; proximal segment longer, with two subter-minal setae; distal segment separated from proximal by distinct ridged articulation;distal segment with three spiniform elements subterminally and two pinnate setae andshort aesthetasc terminally. Antenna (Figure 3C) biramous, with protopod indis-tinctly divided into coxa and basis; basis not separated from proximal segments oframi. Exopod five-segmented; free second to fifth segments each bearing setae withserrated outer and plumose inner margins; terminal segment also with short spiniform


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process. Endopod two-segmented; free second segment produced distally intorounded process, and armed with one short simple and two plumose setae terminally.Mandible (Figure 3D) biramous, with unsegmented protopod not separated fromproximalmost exopodal segment; exopodal segments 1 to 4 each bearing seta similarto that on antennary exopod. Endopod with single elongate free segment bearing twoterminal plumose setae and one short naked seta proximally on inner margin.

Second nauplius (Figures 2, 3)

Body length (based on individuals originating from adult females collected fromTakehara) 0.40–0.44 mm; 0.42 ± 0.02 mm (n = 8); (Nagasaki): 0.33–0.40 mm; 0.37 ±0.02 mm (n = 20) (Figure 2).

Description (based on individuals originating from ovigerous females collected fromTakehara). Body lengths (Figure 3E) more slender than preceding stage; posteriorend with tapering posterior process in mid-line and paired rounded knobs; balancerswith proximal part narrow and distal part flattened.

Antennule (Figure 3F) as in preceding stage except for two minute spinules onproximal segment and an additional rudimentary seta subterminally on distal

Figure 2. Body lengths of developmental stages of Pseudocaligus fugu collected from NagasakiPrefecture.

Max

Min

Sd

Av



segment. Antenna (Figure 3G) and mandible essentially as in preceding stage. Anlageof maxillule [= maxilliped sensu Piasecki (1996)] first appearing in this stage, consist-ing of pair of slender, posteriorly-directed processes (Figure 3 E).

Copepodid (Figures 2, 4)

Body length (based on individuals originating from adult females collected fromTakehara) 0.52–0.58 mm; 0.54 ± 0.02 mm (n = 7); (Nagasaki): 0.43–0.53 mm; 0.49 ±0.03 mm (n = 20) (Figure 2).

Description (based on individuals originating from ovigerous females collected fromTakehara). Body (Figure 4A) with dorsal surface highly pigmented from anterior part

Figure 3. Pseudocaligus fugu, naupliar stages. First (A–D) and second (E–G) stages. (A, E)Habitus, ventral view; (B, F) antennule; (C, G) antenna; (D) mandible. Scales in mm.

A

B

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of cephalothorax to caudal rami (pigmentation not illustrated). Cephalothorax incor-porating first pedigerous somite, about 1.6 times longer than free postcephalothoracicsomites and caudal rami combined: widest about at mid-length; rostrum (Figure 4B)weakly developed, rounded. Second pedigerous somite free, wider than long; thirdpedigerous somite with anlagen of leg 3 (Figure 4M) represented by paired, laterally-located papillae, each bearing one short spine and one plumose seta; third free somiteslightly smaller than preceding somite, unarmed; fourth somite (anal somite) with shortcaudal rami (Figure 4C) each having single flaccid element (seta IV – derived from nau-pliar balancer) and five setae, one of which carried on bi-articulated base.

Figure 4. Pseudocaligus fugu, copepodid stage. (A) Habitus, dorsal view; (B) rostrum; (C) leftcaudal ramus, dorsal surface; (D) antennule; (E) antenna; (F) mandible; (G) maxillule;(H) maxilla; (I) maxilliped; (J) postoral process; (K) leg 1, anterior surface; (L) leg 2, anteriorsurface; (M) leg 3, dorsal view. Scales in mm.

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Antennule (Figure 4D) with indistinctly subdivided proximal segment bearingthree setae anteriorly; distal segment bearing two aesthetascs and 11 setae, five ofwhich forked terminally. Antenna (Figure 4E) forming strongly sclerotized, raptorialorgan, three-segmented; first segment small, unarmed; second segment largest, withacutely pointed, inner process at proximal one-third; third segment smoothly curvedinward, with minute inner seta proximally. Mandible (Figure 4F) consisting of fiveparts; proximal part with vestige of naupliar palp; fourth part longest; distal partbearing about 10 teeth along inner margin. Maxillule (Figure 4G) comprising curvedposterior process plus anterior papilla armed with three unequal setae. Pair of short,curved postoral processes (Figure 4J) located between maxillule and maxilliped.Maxilla (Figure 4H) two-segmented; first segment unarmed; second segment as longas first, with calamus as long as canna, and with flabellum located distal to mid-pointof outer margin. Maxilliped (Figure 4I) subchelate; proximal protopodal segmentlongest, as long as distal subchela, armed with two minute acute prominences oninner margin; subchela comprising unarmed first endopodal segment and distal seg-ment carrying terminal claw and bifid setal element.

Legs 1 (Figure 4K) and 2 (Figure 4L) with distinct one-segmented rami; proto-pods indistinctly divided into coxa and basis. Seta and spine formula as follows(coxa/basis/exopod/endopod): leg 1, 0-0+1-0+III,I,4+7; and leg 2, 0-0+1-0+II,I,4+6.

First chalimus (Figures 2, 5)

Body length (based on individuals collected from the field off Takehara) 0.67–0.85mm; 0.73 ± 0.07 mm (n = 7); (individuals originating from adult females collectedfrom Nagasaki): 0.50–0.68 mm; 0.61 ± 0.05 mm (n = 20) (Figure 2).

Description (based on individuals collected from T. niphobles in Takehara). Body(Figure 5A) attached to host by short frontal filament. Cephalothorax about 1.84times longer than posterior somites combined; protruded anteriorly into triangularplate. First free (= second pedigerous) somite wider than long; second free (= thirdpedigerous) somite narrower than first; third free somite unarmed; anal somite bear-ing small caudal rami (Figure 5B) armed with six naked setae of unequal length.

Antennule (Figure 5C) two-segmented; proximal segment not subdivided by con-striction; distal segment with 11 setae and two aesthetascs. Antenna (Figure 5D)modified from that of preceding copepodid stage, consisting of broad base andweakly sclerotized tip carrying at least five small prominences terminally. Mandible(Figure 5E) different from that of preceding stage in loss of palp vestige. Maxillule(Figure 5F) similar to that of copepodid, but posterior process straight. Maxilla(Figure 5G) lacking flabellum on margin of distal segment. Maxilliped (Figure 5H)indistinctly three-segmented, protopodal segment robust; distal endopodal segmentof subchela bearing short inner seta and large curved claw.

Legs 1 (Figure 5I,J) and 2 (Figure 5K,L) with one-segmented rami; leg 1 bearing bul-bous process at inner proximal corner of coxa with minute prominence subterminally;leg 2 with similar but unornamented process. Leg 3 (Figure 5M) rudiment without seg-mentation, but with anlagen of rami distinct. Seta and spine formula as follows (coxa/basis/exopod/endopod): leg 1, 0-0+1-0+8+2; leg 2, 0-0+1-0+8+3/5*; leg 3, (1-0)+2+0.The symbol * indicates that the shape and number of rudiments of setal elements weresomewhat variable between individuals and even on both sides of same individual.



Second chalimus (Figures 2, 6)

Body length (based on individuals collected from the field off Takehara) 1.03–1.14 mm;1.09 ± 0.05 mm (n = 7); (individuals originating from adult females collected fromNagasaki): 082–1.38 mm; 0.95 ± 0.11 mm (n = 20) (Figure 2).

Description (based on individuals collected from T. niphobles in Takehara). Body(Figure 6A) with cephalothorax laterally expanded and incorporating both first andsecond pedigerous somites, but with suture visible dorsally marking plane of fusionbetween these somites; anterior margin with frontal filament (Figure 6B) bearing twobasal bulbs; anlagen of frontal plates visible; cephalothorax about 1.84 times longerthan free posterior somites combined. Third pedigerous somite free. Fourth pediger-ous somite free, bearing anlagen of leg 4 ventrolaterally (Figure 6L); caudal ramus(Figure 6C) broader than in preceding stage, with six unequal setae.

Figure 5. Pseudocaligus fugu, first chalimus stage. (A) Habitus, dorsal view; (B) right caudalramus, dorsal surface; (C) antennule; (D) antenna; (E) mandible; (F) maxillule; (G) maxilla;(H) maxilliped; (I) leg 1, anterior surface; (J) endopod of right leg 1, anterior surface; (K) leg 2, ante-rior surface; (L) endopod of right leg 2, anterior surface; (M) leg 3, anterior surface. Scales in mm.

A

C D F

M

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Antennule (Figure 6D) proximal segment bearing seven setae anteriorly; distalsegment with 11 setae plus two aesthetascs. Antenna (Figure 6D) unsegmented, withtwo short spiniform elements and two triangular processes terminally. Postantennaryprocess (Figure 6D) first appearing at this stage as papilla with one sensillum. Mandi-ble (Figure 6E) and maxillule (Figure 6F) as in preceding stage. Maxilla (Figure 6G)with minute spinules on distal quarter of outer margin. Maxilliped (Figure 6H) withsegments comprising subchela more completely fused than in preceding stage, i.e.only suture visible.

Legs 1 (Figure 6I) and 2 (Figure 6J) each lacking inner process on coxa found inpreceding first chalimus stage; protopod with indistinct segmentation; inner basalseta present in leg 1; inner coxal seta on leg 2. Leg 3 (Figure 6K) not expressingdistinct segments, but lobes representing rami larger. Setal formula as follows

Figure 6. Pseudocaligus fugu, second chalimus stage. (A) Habitus, dorsal view; (B) frontal fila-ment; (C) left caudal ramus, dorsal surface; (D) antennule, antenna and postantennary processin situ; (E) mandible; (F) maxillule; (G) maxilla; (H) maxilliped; (I) leg 1, anterior surface;(J) leg 2, anterior surface; (K).leg 3, anterior surface; (L) leg 4. Scales in mm.

A

B

C

D

E

F G

H

I JK

L



(coxa/basis/exopod/endopod): leg 1, 0-0+1-1+8+2; leg 2, 0-1+1-0+9+9; leg 3, (1-1)+8+4.Shape and number of rudiments of setal elements on rami somewhat variablebetween individuals and even on both sides of same individual. Leg 4 (Figure 6L)represented by lobe bearing two distal elements.

Third chalimus, male (Figures 2, 7)

Body length (based on individuals collected from the field off Takehara) 1.60–1.78 mm;1.67 ± 0.06 mm (n = 7); (individuals originating from adult females collected fromNagasaki): 1.31–1.55 mm; 1.41 ± 0.06 mm (n = 20) (Figure 2).

Description (based on individuals collected from T. niphobles in Takehara). Body(Figure 7A) showing full development of caligid cephalothorax with incorporation ofthird pedigerous somite and development of frontal plates. Cephalothorax subcircu-lar, about 2.7 times longer than posterior somites combined; frontal filament(Figure 7B) with three basal bulbs. Genital complex widest midway. Some of caudalsetae (Figure 7C) plumose.

Antennule (Figure 7D) proximal segment with 18 setae arrayed anteroventrally, fourof which ornamented with setules; distal segment bearing 12 setae and two aesthetascs.Antenna (Figure 7E) unsegmented, produced into rounded process distally, with twofine subterminal elements and small pointed tip. Postantennary process (Figure 7F)more or less irregular knob with single sensillum. Mandible (Figure 7G), maxillule(Figure 7H), maxilla (Figure 7I) and maxilliped (Figure 7J) as in preceding stage.

Leg 1 (Figure 7K,L) with exopod distinctly two-segmented; endopod vestigial,incompletely fused to basis, armed with two minute elements at tip. Leg 2 (Figure 7M)with two-segmented rami. Leg 3 (Figure 7N) with expanded protopod and one-segmented rami incompletely articulated at base. Setal formula as follows (coxa/basisendopod): leg 1, 0-0+1-1+1-0; 7+vestigial; leg 2, 0-1+1-0+1-1; 9+0-1; 8; leg 3,(1-1)+10+6.

Leg 4 (Figure 7O) represented by cylindrical process bearing two setae at tip. Leg5 (Figure 7P) comprising inner exopodal papilla bearing two setae plus isolated outerbasal seta on papilla.

Third chalimus, female (Figures 2, 7)

Body length (based on individuals collected from the field off Takehara) 1.61 and 1.65mm; 1.63 mm (n = 2); (individuals originating from adult females collected fromNagasaki): 1.26–1.55 mm; 1.44 ± 0.13 mm (n = 20) (Figure 2).

Description (based on specimens collected from T. rubripes in Nagasaki). Sexualdimorphism expressed only in antenna; body (Figure 7Q) and other appendages sim-ilar to male described above. Antenna (Figure 7R) acutely pointed with two fine ele-ments subterminally. Leg 5 (Figure 7S) as in male.

Fourth chalimus, female (Figures 2, 8)




Figure 7. Pseudocaligus fugu, third chalimus stage. Male (A–P), female (Q–S). (A, Q) Habitus,dorsal view; (B) frontal filament; (C) right caudal ramus, ventral view; (D) antennule;(E) antenna; (F) postantennary process; (G) mandible; (H) maxillule; (I) maxilla; (J) maxilliped;(K) leg 1, anterior surface; (L) endopod of right leg 1, anterior surface; (M) leg 2, anterior sur-face; (N) leg 3, anterior surface; (O) leg 4; (P, S) leg 5; (R) antenna and postantennary process.Scales in mm.

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QR

S



Figure 8. Pseudocaligus fugu, fourth chalimus stage. Female (A–N), male (O–R). (A, O) Habi-tus, dorsal view; (B) frontal filament; (C) left caudal ramus, ventral view; (D) antennule,antenna and postantennary process in situ; (E) distal segment of antennule; (F) maxillule;(G) maxilla; (H) maxilliped; (I) right leg 1, anterior surface; (J) endopod of right leg 1, anteriorsurface; (K) leg 2, anterior surface; (L) leg 3, anterior surface; (M, Q) left leg 4; (N, R) leg 5; (P)antenna and postantennary process. Scales in mm.

AB

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R



Description (based on individuals collected from T. niphobles in Takehara). Generalappearance as in preceding stage, but sexual dimorphism more clearly expressed ingenital complex, abdomen and antenna. Body (Figure 8A) with frontal plates dis-tinct; cephalothorax about 1.9 times longer than following somites and caudal ramicombined; frontal filament with four condensed basal bulbs (Figure 8B). Genitalcomplex swollen laterally; free abdomen one-segmented, rectangular; caudal ramus(Figure 8C) with six plumose setae.

Antennule (Figure 8D) proximal segment with 27 short plumose setae alonganteroventral margin; distal segment bearing 12 naked setae and two aesthetascsaround apex. Antenna (Figure 8D) indistinctly three-segmented: comprising twoshort, incompletely separated protopodal segments; basis with short, stout inner pro-cess; distal (= endopod) segment curved, with two short spines subterminally.Postantennary process (Figure 8D) tapering posteriorly, with two sensilla. Mandibleunchanged. Maxillule (Figure 8F) comprising posteriorly-directed process plus ante-rior papilla with three setae. Maxilla (Figure 8G) with distal segment more slenderthan in preceding stage. Maxilliped (Figure 8H) subchela with suture marking planeof fusion of original two endopodal segments. Rudiment of sternal furca of next stagevisible beneath cuticle of this stage.

Leg 1 (Figure 8I) with slightly tapering endopod (Figure 8J) incompletely fusedto basis; exopod two-segmented, proximal segment elongate, about twice as long asdistal segment. Leg 2 (Figure 8K) rami two-segmented; outer basal seta minute.Leg 3 (Figure 8L) with undivided protopod; endopod two-segmented; exopodtwo-segmented, outer spine on first exopodal segment stout and reflexed oversurface of segment. Setal formula as follows (coxa/basis/exopod/endopod): leg 1,0-0+1-1+I-0; III,1,3+vestigial; leg 2, 0-1+1-0+I-1; III,I,6+0-1; 8; leg 3, (1,1)+I-0; I-1;III,4+0-1; 6.

Leg 4 (Figure 8M) represented by trapezoidal process bearing one plumose setaand one spine at tip. Leg 5 (Figure 8N) located at posterovental corner of genitalcomplex, represented by outer and inner papillae tipped with one and two setae,respectively.

Fourth chalimus, male (Figures 2, 8)


Description (based on individuals collected from T. niphobles in Takehara). Generalappearance as in female, but differing in shape of genital complex, abdomen, antennaand legs from those of female. Body (Figure 8O) with genital complex somewhatwider than that of female and with different armature on leg 5; abdomen one-seg-mented, but constricted anteriorly.

Antenna (Figure 8P) indistinctly four-segmented; coxa incompletely separatedfrom basis; basis with short, stout process at inner angle; proximal endopodal segmentunarmed; distal endopodal segment with acutely pointed process midway along mar-gin and bearing two fine setae and minute prominence terminally. Leg 4 (Figure 8Q)as in female (setules on one seta seem have been detached). Leg 5 (Figure 8R)



represented by two papillae, outer bearing basal seta, inner representing exopod andbearing two setae.

Young adult female attached to host (Figure 9)

Description (based on material collected from T. niphobles in Takehara). Body length(Figure 9A) 4.15 mm (n = 1), with frontal filament (Figure 9B) attached to host. Gen-ital complex not as expanded as in adult females illustrated by Yamaguti (1936) andKim (1998); lateral margins with fine surface folds (Figure 9C). Appendages same asin previous descriptions; proximal segment of antennule with 28 setae as described inKim (1998). Paired, bulbous spermatophores (Figure 9C,D) attached to copulatorypores on ventromedial side of genital complex each via slender fertilization tube;spermatophores attached to female by secretion. Leg 4 (Figure 9E) minutely pointedat tip, armed with plumose seta and pinnate spine.

Adult female detached from host

Body length (based on cultured individuals originating from ovigerous femalescollected from Nagasaki) 3.09–4.14 mm; 3.65 ± 0.30 mm (n = 20) (Figure 2).

Adult male detached from host

Body length (based on cultured individuals originating from ovigerous females col-lected from Nagasaki) 2.69–3.46 mm; 3.05 ± 0.20 mm (n = 20) (Figure 2).

Figure 9. Pseudocaligus fugu, young adult female. (A) Habitus, dorsal view; (B) frontal fila-ment; (C) genital compound somite, ventral view, note sinuate lateral margins of somite andattachment of paired spermatophores; (D) spermatophore; (E) right leg 4. Scales in mm.

A

C

D

B

E



Attachment site and growth

More than 96% of all adults of P. fugu were attached to the body surface of the host,whereas all chalimus stages and young adults (immediately after moulting fromchalimus IV) were attached exclusively to the pectoral, dorsal, anal and caudal fins(Table 1). We observed adults moving rapidly over the body of the host (see Figure 1).Most copepodids (77%) were found attached to the host by their grasping antennaeand maxillipeds (Table 1). Chalimus stages were rarely detached from the fin evenafter fixation of the host in seawater formalin. Where detachment had occurred, itseems to have been as the result of their feeding activity on the fins having reducedthe stability of the frontal filament attachment to the host, or to rough movements ofthe host during fixation. In contrast, adults easily became detached from the hostsafter fixation.

In the laboratory, preference for attachment site of copepodids to adults was ana-lysed using the Friedman test (Table 1). Preference was calculated as average order:caudal fin > right pectoral fin > left pectoral fin > dorsal fin > anal fin. However, thepreferred sites differed between stages (p < 0.01).

Development from the copepodid through the chalimus phase of P. fugu was ana-lysed graphically using the percentages of the different stages commencing at day 1with an original inoculum of copepodids (Figure 10). On days 1 and 2 only the infec-tive copepodid stage was found, and copepodids were no longer present after day 6.Chalimus I was first found on day 3 and could be found up to day 10 at a low level(5.7%). Chalimus II occurred from day 3 until day 11, and on day 8, 72.1% of the par-asites were at this stage. Chalimus III, both male and female, first appeared on day 5.Chalimus IV, both male and female, first appeared on day 7, and adults firstappeared on days 9 and 10. Additional hosts were introduced to the experimentalaquarium on days 10 and 11 but did not become infected because no copepodids werepresent after day 6. The experiment was truncated as the result of the death of thehosts.

Using these data, weighted average duration of each post-naupliar stage was esti-mated as follows: copepodid = 2.1 days; chalimus I = 2.2 days; chalimus II = 4.6 days;chalimus III� = 3.6 days; chalimus III� = 4.6 days; chalimus IV� = 3.7 days; chalimusIV� = 3.0 days. The estimates for the duration of chalimus IV should be treated withcaution as they may have been affected by the truncation of the experiment.

Number of eggs per egg string

The number of eggs per string was significantly correlated with string length (r2 = 0.79,p < 0.01) (Figure 11A). Egg numbers ranged from 33 (2.25 mm) to 94 (5.63 mm) witha mean of 69 eggs per string. So an adult female with paired egg strings carried a meantotal of 138.7 eggs (range 67–178). No significant difference was detected between thenumber of eggs in the right and left strings (t-test, p > 0.05) (Figure 11B).

Discussion

Frontal filaments

The possession of a frontal filament is a feature of several families within the large,fish-parasitic lineage of siphonostomatoid copepods. In addition to the Caligidae,



Tab

le 1

.N

umbe

r an

d pe

rcen

tage

of

juve

nile

and

adu

lt s

tage

s of

Pse

udoc

alig

us f

ugu

atta

ched

to

or d

etac

hed

from

the

fin

s of

Tak

ifug

u ru

prip

es.

Stag

es

Att

ache

dD

etac

hed

from

bo

dy (

%)

Tot

al (

%)

Pec

tora

l (R

) (%

)P

ecto

ral (

L)

(%)

Dor

sal (

%)

Ana

l (%

)C

auda

l (%

)Su

b-to

tal (

%)

Cop

epod

id17

6 (1

4.6)

165

(13.

7)14

7 (1

2.1)

140

(11.

6)29

6 (2

4.6)

924

(76.

6)28

3 (2

3.4)

1207

(10

0)C

halim

us I

158

(18.

6)12

9 (1

5.2)

148

(17.

5)11

1 (1

3.0)

298

(35.

2)84

4 (9

9.5)

4 (0

.5)

848

(100

)C

halim

us I

I18

0 (2

2.7)

143

(18.

0)13

4 (1

6.9)

140

(17.

6)19

5 (2

4.5)

792

(99.

7)2

(0.3

)79

4 (1

00)

Cha

limus

III

�39

(32

.8)

29 (

24.4

)8

(6.7

)17

(14.

3)22

(18

.4)

115

(96.

6)4

(3.4

)11

9 (1

00)

Cha

limus

III

�14

(18

.9)

9 (1

2.2)

19 (

25.7

)8

(10.

8)21

(28

.3)

71 (

95.9

)3

(4.1

)74

(10

0)C

halim

us I

V �

3 (1

2.5)

4 (1

6.7)

2 (8

.3)

3 (1

2.5)

8 (3

3.3)

20 (

83.3

)4

(16.

7)24

(10

0)C

halim

us I

V �

7 (2

1.8)

5 (1

5.6)

3 (9

.4)

2 (6

.3)

11 (

34.4

)28

(87

.5)

4 (1

2.5)

32 (

100)

Adu

lt �

00

00

3 (2

.8)

3 (2

.8)

104

(97.

2)10

7 (1

00)

Adu

lt �

00

01

(1.2

)2

(2.3

)3

(3.5

)83

(96

.5)

86 (

100)

Sub-

tota

l57

7 (1

7.5)

484

(14.

7)46

1 (1

4.1)

422

(12.

8)85

6 (2

6.0)

2800

(85

.1)

491

(14.

9)32

91 (

100)

R, r

ight

; L, l

eft.

Fin

s



frontal filaments have been reported from at least some members of the Pandaridae(Wilson 1907), Cecropidae (Grabda 1974), Pennellidae (Sproston 1942; Schram 1979)and Lernaeopodidae (Kabata and Cousens 1973). Its possession by the Hatschekiidaewas inferred by Schram and Aspholm (1997) who recorded the presence of the frontal

Figure 10. Temporal change in composition of developmental stages of Pseudocaligus fugu(only specimens attached to the host) after infection of copepodids on juveniles of Takifugurubripes in the Nagasaki Prefectural Institute of Fisheries. Figure above each column repre-sents the total number of individuals examined. Ch-I, Ch-II, Ch-III and Ch-IV indicate chalimiI, II, III and IV, respectively.

100(%)

0

198 521 392 443 356 207 269 183 110 88 198

Figure 11. Relationships between length of egg string and number of eggs per string (A) andbetween numbers of eggs per right and left egg strings (B) in females of Pseudocaligus fuguobtained at a culture area off Kosasa Town, Sasebo City on 29 May 2008.

B

Y = 0.9119X + 4.8729

Number of eggs in right egg string

Num

ber

of e

ggs

in le

ft eg

g st

ring

0 20 40 60 80 100

20

40

60

80

100

A

Y = 14.911X + 3.694

r 2

= 0.9123

Length of egg string (mm)0 1.0 2.0 3.0 4.0 5.0 6.0

Num

ber

of e

ggs

in e

gg s

trin

g

20

40

60

80

100



filament-secreting organ in Hatschekia hippoglossi (Guérin-Méneville, 1837). How-ever, there is variability within families; for example, Cressey (1968) noted that thedevelopmental stages of the pandarid Phyllothereus cornutus (Milne-Edwards, 1840)lack frontal filaments, as do the post-copepodid developmental stages of Alebionlobatus Cressey, 1970 described Benz (1989), a genus now included within the Caligi-dae (Boxshall and Halsey 2004). In other families, such as the Dissonidae (Andersonand Rossiter 1969) and the Lernanthropidae (Cabral et al. 1984), no frontal filamenthas yet been reported, or no developmental stages are known, as in the case of theHyponeoidae. The presence or absence of frontal filaments (and, by definition, achalimus phase) appears to vary within the main fish-parasitic siphonostomatoid lin-eage and even within particular families.

Within the Caligidae the formation of the frontal filament and its fate duringmoulting follows one of two different patterns. In P. fugu, as in many Caligus species,the pre-formed frontal filament carried within the frontal region of the infective cope-podid is everted and attached to the host before the moult to chalimus I. This fila-ment is a discrete structure and remains permanently attached to the host (Pike et al.1993). At the subsequent moult to chalimus I an additional bulb of material issecreted at the origin of the filament, around its base. At each of the next threemoults a further bulb of material (an extension lobe) is secreted at the origin of thefilament, so chalimus II has two lobes at the base of its filament, chalimus III hasthree lobes, and chalimus IV has four lobes. These observations are in accordancewith the model first elucidated for Caligus centrodonti Baird, 1850 by Gurney (1934)and confirmed in C. elongatus von Nordmann, 1832 by Piasecki and MacKinnon(1993), in which an extension lobe of filament secretion is released from a frontalgland in the larval cephalothorax at each moult and serves to attach the newlymoulted parasite to its original filament, which has remained securely embedded tothe host throughout the moulting process. In addition to C. centrodonti and C. elong-atus, this pattern has been observed in other Caligus species, including C. punctatusShiino, 1955 (Kim 1993), C. orientalis Gusev, 1951 (Hwa 1965), C. pageti Russell,1925 (Ben Hassine 1983) and C. rotundigenitalis Yü, 1933 (Lin et al. 1997, as C. mult-ispinosus). However, not all Caligus species conform to this pattern (see below).

In contrast, the frontal filament of a Lepeophtheirus chalimus is simpler in struc-ture and forms an integral part of the anterior cephalothorax of the chalimus, with itssurface sheath being continuous with the integument of the chalimus (Pike et al.1993). It does not gain a freshly secreted bulb of material at each moult through thechalimus phase. There is uncertainty whether the frontal filament in Lepeophtheirus isreplaced at each moult or whether the moulting chalimus remains adhered to the ori-ginal frontal filament, although the mechanism permitting this during actual ecdysisis not clear. Given that Lepeophtheirus species have two mobile preadult stages intheir life cycle which secure themselves to the host during their moults (from preadultI to preadult II, and from preadult II to adult) by secreting a novel but temporaryfrontal filament (Anstensrud 1990), it seems probable that at least L. pectoralis(Müller, 1776) and L. salmonis (Krøyer, 1837) secrete a new filament at each moultthrough the chalimus phase. Interestingly, some Caligus species also have an enlargedfrontal zone in the chalimus and appear to have a simple frontal filament lackingecdysial extension lobes, such as C. clemensi Parker and Margolis, 1964 (see Kabata1972). In C. epidemicus Hewitt, 1971, Ho and Lin (2004) inferred that a new frontalfilament was formed during each moult during the chalimus phase; however, they



questioned the accuracy of the identifications of the chalimus II and III stages by Linet al. (1996). The secretion of an ecdysial lobe at the moult to chalimus IV suggests tous that C. epidemicus may conform to the multi-lobed filament type as found in C.centrodonti.

Caligids therefore, employ at least two different mechanisms that allow thechalimus to remain firmly secured to the host by the frontal filament during moult-ing. In the first, chalimus I secures itself to the original frontal filament by a newlysecreted ecdysial extension lobe; subsequent chalimus stages secure themselves to theextension lobe of the preceding stage in the same manner. In the second, no ecdysialextension lobes are formed and the filament is simple at every stage. It seems possiblethat a new filament is formed at each moult, with those of the preadult moults beingtemporary. The multi-lobed type occurs in some Caligus species and in P. fuguwhereas the simple type occurs in Lepeophtheirus and some Caligus species. Giventhis distribution of the filament types across generic boundaries it is unwise to gener-alize on patterns as life history data are currently available for so few taxa.

The structure of the frontal filament also varies between families. In pandaridsand cecropids for example, the filament has a double structure, consisting of twobroad bands (Wilson 1907; Grabda 1974). In pennellids, such as Lernaeenicus sprat-tae (Sowerby, 1806), the filament also comprises two strands, each with a distinct dis-tal expansion and each held by one of the paired antennae (Schram 1979). Inlernaeopodids the single filament is held by the paired maxillae (Kabata and Cousens1973). Benz (1989) inferred that the frontal filament was a primitive attribute of theentire siphonostomatoid lineage but considered that it “may have been independentlyrepressed within the order on several occasions”. In view of this variability, it wouldbe interesting to test this view rigorously. A frontal filament has also been reported inthe Nicothoidae (Hansen 1897; Ohtsuka et al. 2007), a siphonostomatoid familyusing crustaceans as hosts, but Huys et al. (2007) in a molecular-based analysisshowed that the Nicothoidae are not closely related to the fish-parasitic lineage andinferred that the frontal filament had originated convergently in the two groups.

Comparative life cycles among caligid genera

Of the 34 currently recognized genera in the family Caligidae, Pseudocaligus is onlythe third caligid genus to have its life cycle fully described. Previously, complete lifecycles have been elucidated for 15 species belonging to just two genera, Caligus andLepeophtheirus (Ho and Lin 2004). The description of development of the caligidAlebion lobatus by Benz (1989) is probably incomplete because Benz himself con-cluded that “it appears likely that several copepodid stages remain to be discovered”(see Table 2). The number of stages during the free-living phase is common to Pseu-docaligus, Caligus and Lepeophtheirus: two nauplii and the infective copepodid stage,but the number of stages between copepodid and adult appears to vary (Ho and Lin2004). In Lepeophtheirus species four chalimus and two preadult stages have beenreported (Lewis 1963; Boxshall 1974; Johnson and Albright 1991), with preadultsbeing distinguished by their ability to detach from the temporary frontal filamentshortly after moulting (Anstensrud 1990) and move over the surface of the host. Incontrast, in Caligus only four chalimus stages and no preadults have typically beenreported (cf. Ho and Lin 2004), although in C. epidemicus a total of six chalimusstages was reported by Lin et al. (1996). Inspection of the figures of chalimus II and



III stages from Lin et al. indicates that these two stages do not differ in any setationcharacters from chalimus I (see discussion in Ho and Lin 2004) and we consider thatthey represent late chalimus I larvae. On the basis of this re-interpretation, we con-clude that the parasitic phase of the life cycle of C. epidemicus comprises the copepo-did larva, chalimus stages I, II, III and IV [referred to as chalimus I, IV, V and VIrespectively by Lin et al. (1996) and by Ho and Lin (2004)] and the adult.

Ho and Lin (2004) concluded that there is no moult between so-called preadultsand adults in Caligus and that, in this genus, preadults are just young adults. In sup-port of Ho and Lin’s argument we would re-interpret the observation of attachedspermatophores on the genital complex of females referred to as “second preadults”of C. spinosus by Izawa (1969: fig. 17A,C) and as “preadults” of Caligus clemensi byKabata (1972: fig. 95). In copepods, mating only takes place between adult males andadult females: Izawa’s “second preadult” and Kabata’s “preadult” must therefore befreshly moulted, young adult females. However, the reported “copulation” between achalimus IV female (as first preadult) and an adult male (see Izawa 1969, fig. 18C),presumably represents an example of precopulatory mate guarding (Boxshall 1990b).These re-interpretations are in accord with Ho and Lin’s (2004) conclusion thatso-called preadults in Caligus are freshly moulted, young adults.

Development in P. fugu follows the same general pattern as in Caligus with twonaupliar, one copepodid and four chalimus stages preceding the adult, without anypreadult stage. The presence of paired spermatophores at the gonopores of youngadult females of P. fugu (see Figure 8C) confirms that these are adults and will notundergo any subsequent moult. In addition to having the same number of stages, thedevelopment of body tagmosis and limb segmentation/armature in P. fugu is similarto the generalized pattern of Caligus except for the highly reduced leg 4. The genusPseudocaligus is characterized by reduction of leg 4. In Caligus species, leg 4 typically

Table 2. Developmental stage(s) possessing different numbers of setae on the proximalsegment of the antennule in various caligid species.

Number of setae 3 7 13/14 20 27 27

Lepeophtheirus salmonis1 Co ChI-II ChIII-IV PaI PaII AdultL. pectoralis2 Co ChI-II ChIII-IV PaI PaII AdultCaligus epidemicus3 Co ChI-III ChIV ChV ChVI AdultC. rotundigenitalis4 Co ChI ChII ChIII ChIV AdultAlebion lobatus5 early-Co mid-Co PaI ? PaII Adult

Number of setae 3 3 7 18 26/27/29 27/28/29

C. clemensi6 Co ChI ChII ChIII ChIV AdultC. elongatus7 Co ChI ChII ChIII ChIV AdultPseudocaligus fugu8 Co ChI ChII ChIII ChIV Adult

Data and stage terminology from: 1Johnson and Albright (1991) – actual setal count [3, 7, 13, 20,27, 27], 2Boxshall (1974) – [3, 7, 14, 20, 27, 27], 3Lin et al. (1996) – [3, 7, 14, 19, 27, 27], 4(Ho andLin 2004) – [3, 7, 11, 20, 27, 27], 5Benz (1989) – [4, 7, 13, ?, 26, 29], 6Kabata (1972) - [3, 3, 8, 18,26, 26], 7Piasecki (1996) – [3, 3, 7, 18, 29, 29], 8Present account – [3, 3, 7, 18, 27, 28]. Setal num-bers in column headings indicate approximate categories: we consider that the numbers given initalics of actual setal counts require verification as some of them are clearly erroneous.Ch, chalimus; Co, copepodid; Pa, preadults



first appears at chalimus I as an anlage, and develops from a one- or two-segmentedcondition in chalimus II to attain the near-adult condition by chalimus IV (cf. Kim1993; Ho and Lin 2004). In contrast, the development of leg 4 of P. fugu is suppressedat the chalimus I stage and this leg is represented by a lobe-like vestige armed withone or two setal elements from chalimus II to the adult. This suppression generatesthe diagnostic apomorphy of the genus.

Now that the life cycle of a third caligid genus has been elucidated it provides anopportunity to revisit the anomalous case of Lepeophtheirus, which is the onlyexample in the entire Copepoda where the number of stages in copepodid phase devel-opment exceeds five before the adult (which is sometimes referred to as the sixth cope-podid stage). We consider the key question to be, are all the identified stages in the lifecycle of Lepeophtheirus separated by moults? Contrary to the erroneous statement inFerrari and Dahms (2007: 55), moulting between, for example, the second preadultand adult has been directly observed in Lepeophtheirus pectoralis by Anstensrud(1990), who described the production of the temporary frontal filament during thepreadult II to adult moult. The nature of development between first copepodid andadult caligids is perhaps the cause of the difficulty in determining the number of trueinstars (stages separated by moults). As noted by Boxshall (1974), during this phasethe copepod undergoes a metamorphosis from free-living copepodid to ectoparasiticadult. This metamorphosis comprises an initial regression (i.e. loss of, or weakerexpression of, limb and body segmentation, reduction in size of setal elements) fol-lowed by progressive development (stronger expression of limb and body segmenta-tion, increased setation). On the swimming legs, for example, segmental articulationsare more poorly expressed in chalimus I than in the preceding copepodid stage, andpreviously well-formed plumose setae become rudimentary. This regression renderscomparisons with free-living copepods, in which leg segmentation patterns can beindicative of developmental stage, more problematic. In addition, it is apparent thatchange can take place within an instar – lobes can swell, setae can lengthen, and wesuspect that even articulations can become more clearly expressed between moults. Inthe absence of continuous observations covering the entire moulting history of asingle individual, all available descriptions are based on analysis of a sampled popu-lation (whether from field or laboratory) and uncertainty about the number of trueinstars remains.

In our opinion, in L. salmonis and L. pectoralis there are two chalimus stageswhich require confirmation as true instars: chalimus II only differs from chalimus I insize and in the degree of development of certain limbs, and chalimus IV only differsfrom chalimus III in the same way, not in setal numbers (Johnson and Albright 1991;Boxshall 1974). It is conceivable that these paired stages (chalimus I–II and chalimusIII–IV) represent only intramoult growth stages and, given the commercial import-ance of sea lice, this should be tested. Reference to the number of setae on the proxi-mal segment of the antennule (Table 2) indicates that only five of the currentlyrecognized stages preceding the adult are distinguished by differing setal counts.There is no change in setal numbers at the final moult to adult, but this moult hasbeen directly observed in L. pectoralis by Anstensrud (1990). Similarly, in some spe-cies there is no change in setal numbers at the moult from infective copepodid tochalimus I, but the changes in endopod of leg 1 and the first appearance of the lobateleg 4 bearing two setae demonstrate changes in setal numbers, from which we inferthat there must have been a moult.



Specific features of P. fugu development include the presence of a vestige of themandibular palp in the copepodid, as also found in C. spinosus (see Izawa 1969, fig.9D). The number of setae on the endopodal segment of leg 1 in the copepodid differsbetween species: six setae are found in C. epidemicus, C. clemensi, C. rotundigenitalisand C. spinosus whereas seven are found in P. fugu, C. punctatus, L. dissimulatus C.B.Wilson, 1905, L. pectoralis and L. salmonis. In their meticulous comparison of adultmale antennules of monstrillids, Huys et al. (2007) highlighted the phylogenetic sig-nificance of the five branching setae on the distal segment of the caligid copepodidantennule. Pseudocaligus fugu copepodids also have five branched setae althoughthese are bifid rather than multi-branched.

Attachment, growth and egg production

The attachment sites of chalimus stages of caligids often differ from those of adults(cf. Bron et al. 1993; Johnson 1993). Johnson (1993) found copepodids and chalimuslarvae of L. salmonis on the gills, fins and body surface of the salmon host whileadults were restricted to the body surface. Pseudocaligus fugu showed a similar ten-dency, although the attachment site of the chalimus larvae is more strictly limited tothe fins than in L. salmonis. This site specificity may be linked to the mode of attach-ment because the basal plate of the frontal filament of any chalimus stage of P. fuguis always inserted through the epidermis onto a fin ray.

Durations of developmental stages of C. epidemicus at 24.5 ± 0.5°C given byLin and Ho (1993) are comparable to those of P. fugu. In C. epidemicus the nau-pliar phase lasts only 20.5 h. The non-feeding, infective copepodid finds a suit-able host within 3 or 4 days and the subsequent chalimus phase has a duration of8 days. In P. fugu, the total of weighted average durations from copepodid toadult was about 16 days in both sexes, although the shortest was only 9 days. Thegrowth rate is therefore generally similar to that of C. epidemicus. The bodylengths of all stages (nauplii to chalimi except for the third chalimus female) weresignificantly larger in Takehara specimens than in Nagasaki ones (t-test, p <0.01). This might have been caused by different water temperatures during incu-bation (ca. 15–17°C versus 18.6–20.2°C).

Little is known about egg production and fecundity in species of Caligidae. Thenumber of eggs per string ranges from 4 to 253 in Taiwanese and Japanese caligidsfor which such information is available (cf. Ho and Lin 2004; Ohtsuka et al. 2007;Venmathi Maran and Ohtsuka 2008). In P. fugu, an adult female carries a mean of 69eggs per string, placing it in the mid-range for the family.

Acknowledgements

The present study was partially supported by a grant-in-aid from the Japan Society for thePromotion of Science (nos. 20380110, 20580220).

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