Reports of Pennellidae Burmeister, 1835 (Copepoda: Siphonostomatoida) post metamorphosed females off the coast of southern Africa

Seven species belonging to Pennellidae are reported from marine teleosts caught off southern Africa. Additionally, complete re-descriptions are provided for Propeniculus stromatei and Sarcotretes scopeli. Examination of Lernaeenicus gonostomae, deposited in the Iziko South African Museum, indicated that it has the morphological features of Sarcotretes rather than Lernaeenicus and thus should be moved to Sarcotretes i.e. S. gonostomae n. comb. for which a re-description is also provided. Reports of new host records include those of Pennella instructa from Seriola lalandi; Propeniculus stromatei from Rhabdosargus holubi and Pomadasys commersonnii; Sarcotretes scopeli from Nansenia tenera, and Sarcotretes longirostris from Centrolophus niger. New geographical records include those of P. instructa, P. stromatei, S. scopeli, S. longirostris, and L. longiventris off southern Africa. Additionally, an attempt to estimate the evolutionary relationships amongst some genera is done from partial COI sequences deposited in Genbank.


Introduction
Pennellidae (Copepoda: Siphonostomatoida) consists of 25 valid genera and 148 species (Walter & Boxshall, 2023;Yumura et al., 2024) symbiotic on marine fish and mammals (Kabata, 1979;Boxshall & Halsey, 2004).Adult metamorphosed female pennellids have large bodies and lose their maxillipeds as mesoparasite adaptations (Boxshall & Halsey, 2004).These metamorphosed females exhibit variable morphology regarding for example, the structure of the cephalothorax (ranging from a simple cephalothorax to development of a cephalic holdfast organ), the trunk (varying from straight trunks to sigmoid trunks), the abdomen (varying from an indistinct abdomen to an abdomen embedded with posterior processes) and the egg strings (varying from straight egg strings to curled egg strings) (Castro-Romero, 2014).
This paper reports on the Pennellidae species collected from marine fish off Southern Africa, with redescriptions of Sarcotretes scopeli Jungersen, 1911 and Propeniculus stromatei (Gnanamuthu, 1951).Notes are provided about the genus Lernaeenicus, with synonymizing and re-description of Lernaeenicus gonostomae Kensley & Grindley, 1973 based on voucher specimens from the Iziko South African museum.Additionally, the phylogenetic relationships between selected Pennellidae genera, using available mitochondrial COI (cytochrome oxidase I) sequences, are investigated.

Materials and Methods
Specimens collected from fish caught off the southern African coasts, from 1993 to 2019 were preserved in 70% ethanol.For morphological studies, specimens were stained with a mixture of lactic acid and a small amount of lignin pink, then examined using stereoand compound microscopes.Some appendages were dissected and drawings were made with the aid of drawing tubes.All measurements were done using a 2 mm stage micrometer and are given as mean (range) mm.Terminology used in morphological descriptions conforms to that of Kabata (1979).Host species names were validated using Froese and Pauly (2023).Voucher specimens for Pennella instructa, Sarcotretes scopeli, S. longirostris and Propeniculus stromatei were deposited in the Iziko South African museum.
For molecular studies, DNA was extracted from Sarcotretes scopeli.Specimens were cut into small pieces and dried in 1.5 microcentrifuge tubes on a heat block at 56°C for approximately 2 hours.Genomic DNA was extracted using the Zymo Research Quick-DNA™ miniprep plus kit, following the protocol for solid tissues.Polymerase Chain Reaction (PCR) was performed to amplify a fragment of the partial mitochondrial (mtDNA) COI gene with the forward primer mICOIintF (GGW ACW GGW TGA ACW GTW TAY CCY CC) and reverse primer jgHCO2198 (TAIACYTCIGGRTGICCR AAR AAYCA) (Geller et al., 2013;Leray et al., 2013) in the Eppendorf Mastercycler.A 25 µl PCR reaction mixture was prepared for each sample, consisting of 12 µl of OneTaq Quick-Load 2X Master Mix with Standard Buffer, 2 µl of each primer, 1-3 µl of template DNA, and double distilled water to adjust the volume.PCR cycling conditions included 94°C (3 min) initial denaturation, followed by 35 cycles of 94°C (30 sec) denaturation, 50°C (1 min) annealing and 68°C (1 min) extension, followed by 68°C (7 min) extension.Purification of PCR products was performed using exoSAP mix and incubated in Eppendorf Mastercycler at 37°C (15 min) and 80°C (15 min) for enzyme inactivation.Sanger sequencing of the purified PCR products was conducted using the Applied Biosystems™ 3730xl DNA Analyzer.

Pennella
Pennella has 10 valid species (Walter & Boxshall, 2023), known to infect a wide range of marine hosts, from teleosts to mammals.Metamorphosed females of this genus vary in total length from large (>100 mm), medium (between 50-100 mm) to small (<50 mm) (Hogans, 2017a).They have cephalothoraces with papillae of different arrangements anteriorly, simple cephalic holdfast with two posterolateral holdfast processes, sometimes with a dorsal holdfast process; a cylindrical neck-like region of varying lengths; a straight and cylindrical trunk with an abdomen of various lengths bearing plumules and elongated egg strings with uniseriate eggs (Kabata, 1979).The collected specimen closely resembles P. instructa (Hogans, 1986) and belongs to the group of Pennella species with a large (>100 mm) total length, which also includes P. balaenoptera; P. benzi Hogans, 2017 and P. filosa.Pennella instructa differs from P. balaenoptera, P. benzi and P. filosa by possession of a cephalothorax anteriorly covered with papillae of apparently even sizes and grouped into four rows with two posterolateral holdfast horns (Hogans, 1986), whereas the others (P.balaenoptera, P. benzi, and P. filosa) possess a cephalothorax covered anteriorly with unorganized papillae of uneven sizes bearing two posterolateral holdfast horns as well as a dorsal horn of different shapes and sizes (Pillai, 1967;Hogans, 2017a).

Remarks:
Lernaeenicus currently has 31 valid species (Walter & Boxshall, 2023) known to infect marine teleosts.Metamorphosed females of this genus are mainly identified by a simple cephalic holdfast with variable lateral, dorsal or both processes of different sizes, a cylindrical neck-like region of different lengths, a straight and cylindrical trunk, with an abdomen of various lengths, and elongated egg strings, with uniseriate eggs (Kabata, 1979).

Remarks:
Material collected (fig.4) closely resemble those studied in Ho et al. (2007) as S. longirostris.Sarcotretes longirostris differs from both S. eristaliformis and S. scopeli by possessing a neck-like region which is longer than the trunk (see fig. 4a), while that of S. eristaliformis is the same length as the trunk (Cherel & Boxshall, 2004) and that of S. scopeli is shorter than the trunk (Hogans, 1988).Sarcotretes longirostris differs from S. umitakae by possessing lateral horns (h) of the holdfast of the same width throughout, tapering at tips (Ho et al., 2007) (figs. 4b, c), whereas that of S. umitakae are proximally bulbous, constrict midway and tapering into slender horns (Uyeno et al., 2012).This is the first report of S. longirostris infecting Centrolophus niger off the South African coasts.Previously, it has been reported from Globicephala macrorhynchus Gray (Ho et al., 2007).

Remarks:
Post metamorphosed females of Lernaeenicus as described by Kabata (1979), are characterized by a distinct abdomen of variable length, straight trunk, neck-like region of variable lengths, four thoracic legs and cephalothorax with various holdfast organs made up of processes ranging from only 1 to about 5. Sarcotretes gonostomae n. comb.differs from Lernaeenicus species by possession of a cephalothorax which extends into the elongated proboscis, distinct terga segments on the dorsal side of the cephalothorax, a neck-like region of variable width and a minute abdomen (see figs.5a-d).
Amongst all Pennellidae genera, the possession of an elongated proboscis occurs only in Sarcotretes, Ophiolernaea and Metapeniculus (Uyeno et al., 2012).Ophiolernaea is identified by variable bulbous processes of the holdfast organ on each side of the neck-like region or cephalothorax, four legs and an elongated proboscis, which is longer than the length of the neck-like region and trunk combined (Shiino, 1958;Ho, 1966;Kabata, 1979).The studied specimens possess a proboscis shorter than the neck-like region and trunk.Metapeniculus species are characterized by a cephalothorax without a holdfast but with or without anterolateral processes (Castro-Romero & Baeza-Kuroki, 1985) which differs from the current specimens due to the presence of a holdfast.Sarcotretes species also possess an elongated mouth cone, but the cephalothorax has lateral holdfast horns extending posteriorly (Wilson, 1917).Thus, the studied specimens belong to Sarcotretes with a cephalothorax consisting of two portions, the smooth proboscis-like mouth cone and a heavily sclerotized basal portion, with holdfast organ laterally that extend posteriorly.Additionally, the first two pairs of legs are biramous and leg 3 uniramous, while the neck-like region expands into the elongated trunk with a small, distinct abdomen.
Sarcotretes gonostomae n. comb.differs from S. scopeli by possessing an elongated proboscis, elongated holdfast horns, and a neck-like region longer than the trunk, whereas S. scopeli possesses a relatively short proboscis, short holdfast horns and a neck-like region which is shorter than the trunk.These two species are smaller in total length than the other species.Sarcotretes gonostomae n. comb.differs from S. eristaliformis by possessing an elongated oral cone, elongated holdfast horns, and a neck-like region longer than the trunk, whereas S. eristaliformis possesses a relatively short proboscis, short holdfast horns and a neck-like region of the same length as the trunk (Cherel & Boxshall, 2004).The total length of S. eristaliformis is about 44.5-58 mm (Cherel & Boxshall, 2004), whilst that of S. gonostomae n. comb. is 26.76 mm.Sarcotretes gonostomae n. comb.differs from S. longirostris by possessing holdfast horns with bulbous bases, thinning posteriorly whereas S. longirostris has holdfast horns without bulbous bases (Ho et al., 2007).The total length of S. longirostris is 41.4-74.4mm (Ho et al., 2007), whereas that of S. gonostomae n. comb. is 26.76 mm.Both S. gonostomae n. comb.and S. longirostris have elongated holdfast horns and a longer proboscis, but can be distinguished based on the shape of the holdfast horns and the size.Sarcotretes gonostomae n. comb.closely resembles S. umitakae with a neck-like region which is longer than the trunk and each holdfast horn with a bulbous base with thin processes extending posteriorly (Uyeno et al., 2012).The total length of S. gonostomae n. comb. is 26.74 mm versus 30.26-50.12 mm in S. umitakae.The neck-like region is 1.3 times the trunk length in S. gonostomae n. comb.versus 3 times the trunk length in S. umitakae.The neck-like region of S. umitakae has a posterior bulge and constriction not observed in S. gonostomae n. comb.Sarcotretes gonostomae n.
Females of Propeniculus species can be differentiated from one another by the length of the cephalothorax relative to the neck-like region and trunk lengths, the cephalothorax shape, the length of the neck-like region and the width of the fourth somite in relation to the trunk width and the length of the abdomen (Gnanamuthu, 1951(Gnanamuthu, , 1952;;Castro-Romero, 2014).
Propeniculus stromatei differs from P. sciaenae and P. theraponi by possession of a prominent posterior swelling, with a short, protrusible mouth tube whereas P. sciaenae and P. theraponi possess a less prominent posterior swelling, but larger mouth cone (see Figs. 12 and 14 in Gnanamuthu (1951)).Propeniculus stromatei differs from P. scomberi in the structure of the cephalothorax, with P. stromatei with a symmetrical cephalothorax with a posterior swelling and even anteroventral surface whereas the cephalothorax of P. scomberi is asymmetrical, without a posterior swelling, but with a swollen ventral surface (see Figs. 2a-g in Gnanamuthu (1952)).Propeniculus stromatei resembles P. trichiuri.However, the cephalothorax of P. stromatei has a less protruding posterior swelling ventrally and is flattened dorsally, with a neck-like region longer than the cephalothorax (Gnanamuthu, 1952) whereas P. trichiuri (see Figs. 1 and 4 in Gnanamuthu (1951)) possesses a more protruding posterior swelling on the ventral side of the cephalothorax as well as a protruding dorsal side and has a neck-like region shorter than the cephalothorax.Additionally, the trunk of P. stromatei is approximately eight times longer than the neck-like region (Gnanamuthu, 1952) whereas that of P. trichiuri is more than eleven times the neck-like region (Fig. 1 in Gnanamuthu, 1951).

Phylogenetic analysis
Results and discussion: The complete dataset consists of 54 sequences including nine genera belonging to Pennellidae and two Caligidae species as outgroup taxa.The sequences are 507-683 base pairs long except that of S. scopeli which is only 365 base pairs long.Even though the COI gene is not ideal for estimating relationships among genera it still provides an hypothesis of relationships to be tested using more suitable slowerevolving genes.
Both the maximum likelihood and Bayesian analyses of the COI dataset estimated Pennellidae as a highly supported monophyletic group (fig.8).Even though most genera included in the dataset with more than one sequence (Trifur, Metapeniculus, Pennella, Peniculus, Haemobaphes and Lernaeocera) form highly supported monophyletic groups, it may be due to most sequences possibly being conspecific (except for those of Pennella and Haemobaphes).Sarcotretes scopeli (although a much shorter sequence of only 365 bp) is basal to all other genera from node D supporting the estimated morphological phylogeny in Boxshall (1986) (see Fig. 5 in Boxshall (1986)).According to Castro-Romero (2014), the structure of the mouth tube of Propeniculus is primitive to all pennellids which attach to the host fins, including Peniculus, Metapeniculus and Peniculisa.In the estimated phylogram (fig.8) Propeniculus is estimated as the basal group to all the remaining genera and not only to Peniculus and Metapeniculus, while Metapeniculus and Pennella form an unsupported sister grouping (node J) with Peniculus (and Pennellidae species) basal to this grouping (node I).Further support for the previous morphological estimation (specifically regarding the possession of a sigmoid trunk) (Boxshall, 1986) is the well supported sister grouping of Haemobaphes and Lernaeocera.Notably in the estimated phylogeny (fig.8) is the species of Lernaeenicus which are polyphyletic with a well-supported clade formed by L. seeri Kirtisinghe, 1934 andL. alatus Rangnekar, 1962 basal to all included genera (node B), L. sprattae (Sowerby, 1806) as a sister group of Trifur sp.(node H) and L. radiatus as a sister group of the Haemobaphes/Lernaeocera sister group (node F).Thus, it seems that some Lernaeenicus species were misidentified or rather that Lernaeenicus is in serious need of revision.According to Boxshall (1986), Lernaeenicus can be distinguished by their possession of an elongated body which is similar to other Pennellidae genera including Sarcotretes, Peniculus, Propeniculus, Pennella, Exopenna Boxshall, 1986, Metapeniculus, Pseudopeniculus and Peniculisa.Furthermore, distinguishing features mentioned by Kabata (1979) include many possible variations which are also shared by other genera.Regarding the estimated relationships in this study, L. sprattae and Trifur sp.share morphological similarities including the possession of three holdfast horns on the cephalothorax (2 lateral and 1 dorsal) and the possession of an abdomen shorter than the trunk.Furthermore, Lernaeenicus radiatus, Lernaeocera branchialis (Linnaeus, 1767), Haemobaphes pannosus Kabata, 1979 andH. diacerus Wilson C.B., 1917 share the possession of holdfast organs situated in a horizontal plane at the base of the cephalothorax, posterior holdfast organ (on neck-like region) and an abdomen shorter than the trunk.Therefore, even though more data is needed for a more conclusive estimation of the relationships amongst the genera of Pennellidae, it is clear that specifically Lernaeenicus is in need of thorough revision of all current species.

Conclusions
New geographical records are reported for Pennella instructa, Propeniculus stromatei, S. scopeli, S. longirostris, and L. longiventris from southern African waters with new host records for P. instructa on Seriola lalandi, P. stromatei on Rhabdosargus holubi and Pomadasys commersonnii, S. scopeli on Nansenia tenera and S. longirostris on Centrolophus niger.Re-descriptions are provided for P. stromatei and S. scopeli and S. gonostomae n. comb.(synonymy L. gonostomae) from Iziko South African Museum.Additionally, an estimation of the phylogenetic relationships amongst some of the Pennellidae genera was attempted using COI sequences available on Genbank with the addition of a generated sequence for S. scopeli.This estimation emphasizes the need of revising the species of Lernaeenicus specifically due to their polyphyletic grouping in the phylogram.

Fig. 8
Fig. 8 The estimated phylogram of some Pennellidae genera with representatives of Caligus as outgroup taxa, using maximum likelihood and Bayesian inference of the partial COI (cytochrome oxidase I) gene.Values above the lines indicate

Table 1
Pennellidae species used in the phylogenetic analysis with accession numbers from Genbank.