First record of Caligusdussumieri Rangnekar, 1957 (Copepoda, Siphonostomatoida, Caligidae) from Malaysia, with notes on caligids found from Malaysia and on host-specificity of caligids on lutjanid fishes

Abstract Background In total, 14 species of Caligus have been reported from Malaysia. Amongst them, four species are reported from lutjanid fishes. New information Caligusdussumieri Rangnekar, 1957 is reported from Malabar snapper, Lutjanusmalabaricus, purchased from a local wet market in Terengganu, Peninsular Malaysia. This is the first record of this species in Malaysia and it is only the second species assigned to the bonito-group of the genus Caligus to be reported from Malaysia. A key to species of the bonito-group is presented herein. The list of caligids infecting lutjanid fishes and the geographical distributions plus the known hosts of members of the bonito-group of Caligus are discussed.


Introduction
Parasitic copepods of the genus Caligus Müller, 1785 (Siphonostomatoida, Caligidae) are well known as pests that cause serious economic losses in the marine aquaculture and fisheries industries globally (Ho and Lin 2004, Johnson et al. 2004, Nagasawa 2013, Arriagada et al. 2019, Hemmingsen et al. 2020).Heavy infestation by caligids, especially in fish kept in captivity, often results in secondary infection and disease outbreaks (Johnson et al. 2004).Therefore, the taxonomy, ecology and physiology of sea lice are particularly important and intensively studied.
Seven species groups have been established within the genus Caligus: the bonito-, confusus-, diaphanus-, macarovi-, productus-, pseudrorhombi-and undulatus-groups (Boxshall 2018, Ohtsuka and Boxshall 2019, Ohtsuka et al. 2020b).The bonito-group can be recognised on the basis of the following features: well-developed inner setae on the distal exopodal segment of leg 1 and the presence of a row of robust denticles along the outer margin of the second endopodal segment of leg 2.
The fish genus Lutjanus Bloch, 1790 (Lutjanidae) plays an important role in fisheries and aquaculture in Malaysia.The lutjanid catch contributed about 5.3% of the overall marine fish catch in 2021 (DOFM 2021).Bakar et al. (2018) identified 17 species of Lutjanus from Malaysia.Amongst them, Lutjanus malabaricus (Bloch and Schneider, 1801), commonly known as Malabar snapper, is one of the economically important species as they are recorded in 10 fish landing facilities across Malaysia: in Kedah, Penang, Perak, Selangor, Johor, Terengganu, Kelantan and Sabah (DOFM 2021).To date, only one species of parasitic copepod (C.malabaricus) has been described, associated with L. malabaricus from Malaysia (Leong 1984).
We have conducted taxonomic studies on metazoan parasites on commercially important fish obtained in the fish markets in Terengganu since 2019 (Ohtsuka et al. 2020b, Nitta et al. 2022) and, in this paper, we report a new locality record for Caligus dussumieri Rangnekar, 1957 from Malabar snapper, Lutjanus malabaricus.This species is redescribed herein and we present notes on Caligus species parasitic on Lutjanus around the world and on the host-specificity of members of the bonito-group.

Materials and methods
A single female specimen of Caligus dussumieri was found on the gills of L. malabaricus purchased from the fish landing facility of the Fisheries Development Authority of Malaysia (LKIM) Complex Pulau Kambing, located in Kuala Terengganu, Terengganu State, Malaysia on 19 October 2019.The fish host was purchased dead and brought back to be examined for parasites in the Aquatic Laboratory, Faculty of Bioresources and Food Industry, University Sultan Zainal Abidin Besut Campus.The body surface, head and gills of the host were examined for parasites.Parasite specimens were fixed in 70% ethanol until identification.The copepod specimen was immersed in lactophenol and examined using the Humes and Gooding (1964) slide method, on a differential interference microscope Olympus Co.,Ltd.),equipped with a drawing tube.The body length was measured from the frontal margin of the cephalothorax to the posterior margin of the caudal ramus excluding the caudal setae.The specimen is deposited at the South China Sea Repository and Reference Center, Universiti Malaysia Terengganu, Malaysia (UMTCrus 1645).A second specimen of C. dussumieri from the Natural History Museum, London (Reg. No. NHMUK 2022.189-197),fixed in 70% ethanol and collected from L. johnii from the Ord River, Camden Sound, Western Australia on 11 September 2013, was also examined.It was cleared in lactophenol on a glass cavity slide and examined with light microscopy using a Leitz Diaplan microscope, equipped with differential interference contrast and with confocal laser scanning microscopy (CLSM) as detailed below.Terminology of adults and developmental stages follows Huys and Boxshall (1991), Ho and Lin (2004) and Piasecki et al. (2023).

Confocal Laser Scanning Microscopy
One female of Caligus dussumieri (NHMUK 2022.189-197) was examined using CLSM.The specimen was stained overnight in a saturated solution of Congo Red in 100% ethanol, then rinsed in distilled water until no Congo Red could be seen diffusing and prepared as a temporary mount in a 50% solution of glycerine and distilled water on a glass slide under a coverslip.The specimen was examined using a Leica TCS SP5, equipped with a Leica DM5000 B upright microscope and the Leica Application Suite Advanced Fluorescence software LAS AF 2.2.1.(Leica, Wetzlar, Germany).We used a 561 nm excitation wavelength from a DPSS 10 mW 561 nm laser set at 80% power and collected the emitted fluorescence in two channels: 570-630 nm artificially coloured green and 630-715 nm artificially coloured red.A series of image stacks were collected and the final images were obtained by maximum projection of the overlaid channels using the same Leica software.For the full-body dorsal and ventral images, multiple fields of view were combined using Adobe Photoshop v.25.1.
Australian female observed using CLSM (Fig. 3A, B) showing differential degree of staining over body surface.Dorsal cephalothoracic shield staining moderately and evenly, with heavier uptake of stain marking sutures anteriorly near origin of antennules, in area near eyes and along lateral edges of central thoracic zone of shield.Ventral curved rib marking boundary of cephalosome and incorporated first pediger strongly stained.Fourth pediger and fourth leg staining evenly.Genital complex and abdomen displaying marked differences in intensity of staining.Staining intense in small ovoid patch on dorsal surface of genital complex towards the posterior end, ventrally around the genital apertures and in the posterior part of the abdomen.Conversely, staining very light in other areas of genital complex and anterior part of abdomen.
Leg 1 (Fig. 4A, Fig. 2A) with 2 segmented protopod bearing 1 inner and 1 outer small plumose seta plus bifid setule on outer margin.Intercoxal sclerite slender, with 2 papillae each bearing a setule present on sternum posterior to sclerite.Vestigial endopod reduced to tiny, pointed process located near base of exopod.Exopod 2segmented, first segment with row of fine setules along inner margin and spiniform seta at outer distal corner; second segment with 3 large plumose setae ornamented along outer margin with stout spinules at base becoming finer towards tip and with short plumosities along inner margin; 4 terminal elements, spine 1 simple, spines 2 and 3 each with accessory process (Fig. 4a), seta 4 longest, stout and pinnate on only one side.Leg 2 (Fig. 4B, Fig. 2B) with intercoxal sclerite ornamented with marginal membrane along posterior margin; coxa with large plumose seta at posterior corner and minute setule on anterior surface; basis ornamented with marginal membrane on both inner and medial margins, bearing minute seta at outer distal corner plus setule near mid-point of inner margin.Endopod 3-segmented: first segment armed with inner plumose seta and ornamented with large denticles at outer distal corner; second segment with 2 inner distal setae plus row of robust denticles along outer margin; third segment with 6 plumose setae.Exopod 3-segmented: first segment with reflexed membrane dorsally and with pecten at base of long, stout, outer spine with spatulate tip extending obliquely across surface of second and third segments; second segment with relatively short outer spine, third segment armed with 1 small and 1 medium outer spine (latter ornamented with extensive marginal membrane), longer distal spine ornamented with membrane on outer side and plumosities on inner side, plus 5 plumose setae.Leg 3 (Fig. 5A) apron (protopod) bearing small, plumose outer seta and long, plumose inner seta and ornamented with membrane along outer and posterior margins, 4 patches of spinules plus 2 setule-bearing papillae on either side of innermost patch, plus 2 relatively long setules near posterior margin.Endopod 2segmented, proximal segment small, armed with 1 long plumose seta; segment expanded to form velum fringed with setules along free margin; second segment armed with 6 plumose setae.Exopod 3-segmented, proximal segment small, with pecten-like membrane on inner margin of segment and with an expanded outer distal corner ornamented with strip of membrane located lateral to outer spine plus minute setule on outer margin, outer distal spine slightly curved, not reaching distal border of second segment (Fig. 5a); second segment with inner plumose seta and outer naked spine; third segment with 3 setiform spines increasing in size distally plus 4 inner setae.Leg 4 (Fig. 5B) plumose seta at outer distal corner of protopodal segment; exopod distinctly 2-segmented, first exopodal segment with long outer spine; second segment with 4 spines along oblique outer distal margin; each spine with pecten at base (Fig. 5b).

Taxon discussion
The initial description of C. dussumieri was provided by Rangnekar (1957).It was later transferred by Pillai (1968) to his new genus Pseudopetalus Pillai, 1968, as Pseudopetalus dussumieri, but was subsequently transferred to Sinocaligus Shen 1957 as Sinocaligus dussumieri after Pseudopetalus was recognised as a junior synonym of  Caligus dussumieri Rangnekar, 1957 adult female.
A Leg 1, a Four terminal elements of Leg 1; B Leg 2.
First record of Caligus dussumieri Rangnekar, 1957 (Copepoda, Siphonostomatoida, ... In comparison to the recent re-description of C. dussumieri from Australian waters as provided by Boxshall and Barton (2023), the C. dussumieri specimens from Malaysia displayed subtle differences including the presence of a neck-like constriction in the anterior part of the genital complex and the more barrel-shaped genital complex, features also observed in C. dussumieri from Indian waters ( Rangnekar 1957, Pillai 1967, Pilla et al. 2012).As discussed below, we consider these differences can be explained by the pre-metamorphic stage of development of these sets of specimens.
Another minor difference was noted regarding the postantennal process.In the current study, C. dussumieri possessed trisensillate papillae, consistent with the description of Pillai (1985), while the Australian specimens exhibited bisensillate papillae.In addition, a rounded, lobate process was present between the postantennal process and the base of the antenna in the Malaysian female.The female from Malaysia lacked the patch of small setules at the base of the inner protopodal seta of leg 1, present in the other specimens.Furthermore, the tips of spines 2 and 3 on the distal exopodal segment of leg 1 were ornamented with a row of serrations, distinguishing them from previously described specimens from India and Australia.
A pecten-like structure at the outer distal corner of the first exopodal segment of leg 3 was observed in the present specimen, but this feature was not illustrated in previous studies.However, this structure's presence in the Australian material was subsequently confirmed through additional observations of Australian specimens in this study.

Discussion
In this present study, the genital complex and abdomen of the adult female examined using CLSM displayed differential intensity of staining.It is possible that this differential intensity of staining is an artefact, but we infer that the staining intensity indicates a difference in the properties of the cuticle.The stained specimen is an adult female immediately prior to the post-mating metamorphosis (see Boxshall and Barton ( 2023)) which involves marked lateral expansion and lengthening of the genital complex and the abdomen resulting in the development of the habitus shown for fully mature C. dussumieiri by Pillai (1985).The areas which undergo maximum expansion are the genital complex and anterior abdomen.The posteriormost part of the abdomen, corresponding to the anal somite, does not undergo expansion.It appears that the very lightly stained areas of cuticle correspond to the areas which undergo metamorphic expansion.The cuticle surrounding the genital apertures is heavily stained indicating that it is sufficiently rigid to enable the spermatophores to be deposited and attached securely during mating.It is probable that the area immediately around the genital apertures does not undergo any metamorphic transformation.Similarly, the patch of intensely stained cuticle on the dorsal surface at the rear end of the genital complex is located where the paired maxillipeds of the male might grasp the female during spermatophore transfer.This heavily-stained area might indicate another patch of more rigid cuticle that does not undergo change during the metamorphic expansion of the genital complex.Similar patterns of intensely stained patches were observed in the same regions on the dorsal and ventral surface of the genital complex in 14 other caligid species examined using CLSM and the same staining protocol (Bernot, unpublished), so this appears to be a general pattern and not just an artefact of staining in this specimen of Caligus dussumieri.Caligus dussumieri is assigned to the bonito-group because of the presence of three plumose setae on the distal exopodal segment of leg 1 and a row of stout denticles along the outer margin of the second endopodal segment of leg 2 (Boxshall and Barton 2023).It also shares two additional characteristics common to members of this species group: a 3segmented leg 4 with four spines on the second exopodal segment and the presence of a small blunt posterior process on the proximal segment of the antenna.Rangnekar & Murti, 1949, C. grandiabdominalis Yamaguti, 1954, C. hoplognathi Yamaguti and Yamasu, 1959, C. infestans Heller, 1865, C. malabaricus Pillai, 1961, C. mutabilis Wilson, 1905, C. omissus Cressey and Cressey 1980, C. pauliani Nuñes-Ruivo & Fourmanoir, 1956, C. phipsoni Basset-Smith 1898, C. quadratus Shiino, 1954, C. tenuifurcatus Wilson, 1937, C. timorensis Izawa, 1995, and C. triabdominalis Byrnes, 1987(Boxshall 2018, Ohtsuka and Boxshall 2019, Kamanli 2020).Caligus dussumieri along with C. caudatus, C. formicoides and C. timorensis were recently added to bonito-group after the species were transferred from Sinocaligus to Caligus (Boxshall and Barton 2023).
An interesting example of host-switching during development is known in C. pauliani (previously reported as C. biseriodentatus), which infects different hosts at different life stages.The adult females are known from the frigate tuna Auxis thazard Lacepède, 1800, while premetamorphic adults and copepodid stages characterised mainly by smaller size, slender genital somites and not bearing eggs, are known from a variety of host species belonging to the genus Scomberomorus Lacepède, 1801.This form of host-switching appears to involve mated females only given that both pre-metamorphic adult females and males occur on the first host, which is where mating takes place, while only the mated females are known from the second host where the metamorphic developmental changes are completed.Cressey and Cressey (1980) speculated that host-switching might have resulted from predatory activities of the host because A. thazard is known as a common and important prey item for many larger fishes (Frimodt 1995).
The bonito-group has a broad distribution in low to middle latitudes across all oceans, with a denser concentration in the Indo-Pacific.The distribution of species infecting Lutjanidae is primarily concentrated in low latitudes due to the abundance of lutjanid fish in tropical and subtropical areas, as indicated in Fig. 6.In Malaysia, two caligids belonging to the bonito-group (C.dussumieri and C. malabaricus) have been documented in association with two lutjanid fish species (present study and Leong (1984)).Notably, the exploration of caligid copepod diversity in neighbouring Southeast Asian countries has been limited, reflecting a broader scarcity of research on Caligidae in the region.In addition to Malaysia records, a total of five caligids species from the bonito-group have been identified from Southeast Asian countries.Ho et al. 2004, Yuniar et al. 2007).This underscores the need for further investigations to improve our understanding of caligid copepod diversity and distribution in Southeast Asian waters.Geographical distribution of species belonging to the bonito-group within Caligus.First record of Caligus dussumieri Rangnekar, 1957 (Copepoda, Siphonostomatoida, ...

Figure 2 .A
Figure 2. CLSM images of Caligus dussumieri Rangnekar, 1957.A Exopod of leg 1, showing spinular ornamentation on plumose setae along posterior margin; B Rami of leg 2 showing ornamentation on outer margin of endopod and spatulate spine on first exopodal segment.

Table 1 .
Caligid copepods recorded from the genus Lutjanus Bloch,1790.First record of Caligus dussumieriRangnekar, 1957 (Copepoda, Siphonostomatoida, ... First record of Caligus dussumieri Rangnekar, 1957 (Copepoda, Siphonostomatoida, ...Species ofCaligus, belonging to the bonito-group, are known from a variety of lutjanid hosts around the world.Caligus asperimanus and C. mutabilis exhibit a broad distribution and host range, having been reported from up to seven different lutjanid hosts in different regions, including Belize (Cressey 1991), the Bahamas, Brazil (Oliveira et al. 2020) and India (Pilla et al. 2012).While about three-quarters of reports of species of the bonito-group infecting lutjanids originated from North and South America, four species have been documented as infecting commercially exploited lutjanids from Asia, including L. johnii recorded from the coast of India (Pilla et al. 2012), L. malabaricus from the Malacca Strait and Terengganu Coast, Malaysia (Leong 1984), L. argentimaculatus from the Philippines (Ho et al. 2004) and L. russellii from Taiwan (Ho and Lin 2004).In Malaysia, out of the 20 species of fish previously reported to serve as hosts for caligid copepods, three species of lutjanids are known to collectively host four species of Caligus: Lutjanus erythropterus hosts C. chiastos and C. torpedinis; L. johnii hosts C. chiastos; and L. malabaricus hosts C. malabaricus(Venmathi Maran et al. 2009).
Caligus dussumieri has been observed to infect two distinct host families, the Lutjanidae and Dussumieriidae.The adult males, copepodid (including chalimus) stages and mated, but not fully metamorphosed adult females were found on Lutjanus johnii from Australian waters (Boxshall and Barton 2023) and L. rivulatus from Indian waters(Pilla et al. 2012).Our new Malaysian female at the same stage of development is from L. malabaricus.Fully matured females with the typical expanded genital complex and abdomen have been found on the dussumieriids Dussumieria acuta Valenciennes, 1847 and D. elopsoides Bleeker, 1849 (as D. hasseltii Bleeker) both in Indian waters(Rangnekar 1957, Pillai 1968).Lutjanids are a group of fish with high commercial value due to their desirable taste, ability to reproduce in captivity and tolerance to a range of salinity regimes which allow them to thrive in various marine environments including estuaries and mangroves as well as the open sea (Ibarra-Castro and Duncan 2007).In the family Lutjanidae, Lutjanus is the most species rich genus, currently accommodating 73 species (Froese and Pauly 2023).Twenty-eight species of caligid copepods have been recorded so far from Lutjanus species.Amongst these are five species belonging to the bonito-group within Caligus, namely: C. asperimanus, C. bonito, C. irritans, C. mutabilis and C. tenuifurcatus (Table1).