Automated identification of Monogeneans using digital image processing and K-nearest neighbour approaches

Background

Monogeneans are flatworms (Platyhelminthes) that are primarily found on gills and skin of fishes. Monogenean parasites have attachment appendages at their haptoral regions that help them to move about the body surface and feed on skin and gill debris. Haptoral attachment organs consist of sclerotized hard parts such as hooks, anchors and marginal hooks. Monogenean species are differentiated based on their haptoral bars, anchors, marginal hooks, reproductive parts’ (male and female copulatory organs) morphological characters and soft anatomical parts. The complex structure of these diagnostic organs and also their overlapping in microscopic digital images are impediments for developing fully automated identification system for monogeneans (LNCS 7666:256-263, 2012), (ISDA; 457–462, 2011), (J Zoolog Syst Evol Res 52(2): 95–99. 2013;). In this study images of hard parts of the haptoral organs such as bars and anchors are used to develop a fully automated identification technique for monogenean species identification by implementing image processing techniques and machine learning methods.

Result

Images of four monogenean species namely Sinodiplectanotrema malayanus, Trianchoratus pahangensis, Metahaliotrema mizellei and Metahaliotrema sp. (undescribed) were used to develop an automated technique for identification. K-nearest neighbour (KNN) was applied to classify the monogenean specimens based on the extracted features. 50% of the dataset was used for training and the other 50% was used as testing for system evaluation. Our approach demonstrated overall classification accuracy of 90%. In this study Leave One Out (LOO) cross validation is used for validation of our system and the accuracy is 91.25%.

Conclusions

The methods presented in this study facilitate fast and accurate fully automated classification of monogeneans at the species level. In future studies more classes will be included in the model, the time to capture the monogenean images will be reduced and improvements in extraction and selection of features will be implemented.

     

Policlithrum ponticum sp. n. (Monogenea: Gyrodactylidae: Polyclithrinae) from Mugil cephalus from the Black Sea and problems of suprageneric systematics of the gydrodactylids

Polyclithrum ponticum sp. n. is described and P. mugilini Rogers, 1967 is redescribed. Both monogenean species are parasites of Mugil cephalus in the Black Sea. The new species differs from P. mugilini, P. alberti and P. boegeri by the lesser size of anchors, while it is distinguished from P. corallense by the larger size of these structures. P. ponticum sp. n. differs from all formerly described species by the greater length of dorsal connective bar. In both species from the Black Sea, "ear-like" structures situated near the external roots of anchors are described for the first time. It is suggested, that these structures take part in longitudinal, two-lobe folding of the haptor. The process of opening the haptor is probably performed by the additional bars of the haptor (bars 2 and 3 after: Rogers, 1967), joined to each other and with the anchors. The fifth pair of additional bars (Ernst e. a., 2000) derives from the "beard" of ventral connective bar and is united with its basal part. The sixth pair of additional bars (Ernst e. a., 2000) is considered as a typical "ribs" of the haptor, and therefore the "ribs" are represented by three pairs. Differences between marginal hooks of P. ponticum sp. n. and P. mugilini are insignificant, that probably depends on the presence of "ribs" of the haptor. Based on the subdivision of marginal hooks into two groups, the presence of additional supporting structure in the haptor, and the presence of the seminal receptacle, it is suggested that the subfamily Polyclithrinae Rogers, 1967 should include the genera Polyclithrum Rogers, 1967, Swingleus Rogers, 1969, Macrogyrodactylus Mamlberg, 1959, and probably Fundulotrema Hargis, 1955. Based on such characters as the lack of the anchors, the presence of suckers in the haptor, and ovipositing of eggs, it seems to be expedient to use the following taxa in systematics of gyrodactylids: Isancistrinae Fuhrmann, 1928 (genera Isancistrum, Anacanthocotyle); Gyrdicotylinae Vercammen-Grandjean, 1960 (Gyrdicotyle) and Ooegyrodactylinae Harris, 1983 (genera Phanerothecium, Ooegyrodactylus, Nothogyrodactylus, Hyperopletes).     

A special technique for studying haptoral sclerites of monogeneans

The marginal hooks and anchors have been digested from the haptors of the monogeneans Gyrodactylus salaris and Discocotyle sagittata. These structures then can be measured and drawn more accurately than those surrounded by haptoral tissue. The technique also facilitates the study of isolated hooks with SEM. The results of studies using this digestion technique are presented.     

A new Neocalceostomatid (Monogenoidea) from the gills of the blackfin sea catfish, Arius jella (Siluriformes: Ariidae), in the Bay of Bengal, India

Thysanotohaptor n. gen. (Neocalceostomatidae) is proposed to accommodate Thysanotohaptor rex n. sp. collected from the gills of the blackfin sea catfish Arius jella Day (Siluriformes: Ariidae) from off the coast of Visakhapatnam, Bay of Bengal, Andhra Pradesh, India. Thysanotohaptor is differentiated from the other known neocalceostomatid genera by its species having multiple postgermarial testes (single testis in species of Neocalceostoma and Neocalceostomoides ), lacking a transverse bar associated with the ventral anchor pair (present in species of Neocalceostoma ), and possessing a disc-shaped haptor with a pleated marginal frill (frill absent in Neocalceostomoides spp.; Neocalceostoma spp. with delicate marginal membranes). The Neocalceostomatidae is considered valid within the Order Dactylogyridea based on its members having a haptor armed with 10 marginal and 4 ventral hooks and a germarium having a distal loop prior to uniting with the ootype; the family is not assigned to a suborder of Dactylogyridea because of uncertainty in part about the way in which the distribution of haptoral hooks evolved within the taxon.     

Phylogeny and a revised classification of the Monogenoidea Bychowsky, 1937 (Platyhelminthes)

A hypothesis (CI=57.3%) on the evolutionary relationships of families comprising the class Monogenoidea is proposed based on 141 character states in 47 homologous series and employing phylogenetic systematics. Based on the analysis, three subclasses, the Polyonchoinea, Polystomatoinea and Oligonchoinea, are recognised. The analysis supports independent origins of the Montchadskyellidae within the Polyonchoinea and of the Neodactylodiscidae and Amphibdellatidae within the order Dactylogyridea (Polyonchoinea); the suborder Montchadskyellinea is raised to ordinal status and new suborders Neodactylodiscinea and Amphibdellatinea are proposed to reflect these origins. The Gyrodactylidea (Polyonchoinea) is supported by three synapomorphies and comprises the Gyrodactylidae, Anoplodiscidae, Tetraonchoididae and Bothitrematidae. The analysis supports recognition of the Polystomatoinea comprising Polystomatidae and Sphyranuridae. Evolutionary relationships within the Oligonchoinea indicate independent origins of three ordinal taxa, the Chimaericolidea (monotypic), Diclybothriidea (including Diclybothriidae and Hexabothriidae) and Mazocraeidea (with five suborders). The suborder Mazocraeinea comprises the Plectanocotylidae, Mazocraeidae and Mazoplectidae, and is characterised by two synapomorphies. The suborder Gastrocotylinea, characterised by presence of accessory sclerites in the haptoral sucker, is divided into two infraorders, the monotypic Anthocotylina infraorder novum and Gastrocotylina. Two superfamilies of the Gastrocotylina are recognised, the Protomicrocotyloidea and Gastrocotyloidea; the Pseudodiclidophoridae is considered incertae sedis within the Gastrocotylina. The suborder Discocotylinea comprises the Discocotylidae, Octomacridae and Diplozoidae and is supported by four synapomorphies. The monotypic Hexostomatinea suborder novum is proposed to reflect an independent origin of the Hexostomatidae within the Mazocraeidea. The terminal suborder Microcotylinea comprises four superfamilies, the Microcotyloidea, Allopyragraphoroidea, Diclidophoroidea and Pyragraphoroidea. The analysis supports incorporation of the Pterinotrematidae in the Pyragraphoroidea and rejection of the monotypic order Pterinotrematidea. The following taxa are also rejected for reasons of paraphyly and/or polyphyly: Articulonchoinea, Bothriocotylea, Eucotylea, Monoaxonematidea, Tetraonchidea, Gotocotyloidea, Anchorophoridae and Macrovalvitrematidae. The Sundanonchidae, Iagotrematidae and Microbothriidae were not included in the analysis because of lack of pertinent information regarding character states.     

Dactylogyrids (Monogenea: Dactylogyridea) with an unusual number of anchors,their origin and phylogenetic significance. Reference data

It is considered that in Anacanthorinae, Markewitschiana and Pseudacolpenteron pavlovskii (Dactylogyridae s. s.) the anchors are absent originally. In the other dactylogyrids (s. l.) without anchors (Pseudacolpenteron ignotus; Acolpenteron; Anonchohaptor-Icelanchohaptor; Kritskyia-Telethecium-Pavanelliela) these structures are lost. In related genera Rhinoxenus and Nanotrema the dorsal pair of anchors is transformed into the "spikes"; in Heteronchocleidinae there is one anchor; in Trinigyrus and Schilbetrematoides are two anchors; and in Urogyrus three anchors are reduced. But in Rhinonaster, Cacatyocotyle, Callceostomella, Neocalseostoma elongatum and Pseudodactylogyridae the presence of one pair of ventral "peduncular" or "haptoral" anchors appear to be the original state. For Anacanthorinae and Dactylogyridae s. s. the presence of 18-16 hooks, original lacking of anchors, appearance of one pair of them, evolutionary development and subsequent reduction of these structures are characteristic. In the dactylogyrids (s. l.) having two pairs of anchors and 14 hooks, one pair of the anchors is present originally, then usually the second dorsal pair grows up, and finally, the reduction of a part of anchors or all the anchors is possible. In the 16-hooked dactylogyrids (s. l.) having two pairs of anchors (Tetraonchinea, Amphibdellainea, Neodactylodiscinea) the original lacking or reduction of the anchors is not reported. The author considers, that it is unwarranted to unite all dactylogyrids (s. l.) without anchors into the superorder Ananchorea Malmberg, 1990 or to postulate the original presence of the two pairs of ventral anchors in this group. It is also impossible to unit all dactylogyrids (s. l.) having 0-4 anchors and 18-14 hooks into Dactylogyridae sensu Boeger et Kritsky, 1993.     

Calicobenedenia Polyprioni n. gen., n. sp. (Monogenoidea: Capsalidae) from the external surfaces of wreckfish, Polyprion americanus (Teleostei: Polyprionidae), in the north Atlantic

Calicobenedenia polyprioni n. sp. (Capsalidae) is described from the external surfaces (skin and eye) of wreckfish, Polyprion americanus (Teleostei, Perciformes, Polyprionidae), from the north Atlantic Ocean. The monotypic Calicobenedenia n. gen. is proposed for this species and is characterized, in part, by its members possessing an aseptate haptor armed with 14 submarginal hooks and 1 pair of anchors, a common genital pore opening marginally immediately posterior to the left cephalic lobe, 2 testes juxtaposed near the body midlength, and by lacking cephalic suckers or adhesive discs, accessory haptoral sclerites, and a uterine valve. The new genus most closely resembles Entobdella, which differs from Calicobenedenia by having an aseptate haptor armed with 14 submarginal hooks, 2 pairs of anchors, and a pair of accessary sclerites.     

A new species of Kritskyia (Dactylogyridae,Ancyrocephalinae) parasite of urinary bladder of Prochilodus lineatus (Prochilodontidae,Characiformes) from the Floodplain of the High Paraná river,Brazil

A new species of Kritskyia inhabits the urinary bladder of the "curimba", Prochilodus lineatus in the floodplain of the high Paraná river. The new species resembles others members of Kritskyia in the following features: haptor lacking anchors and with 14 hooks marginal, posterior male copulatory organ non articulated with the accessory piece and vagina a sclerotized tube. However, it differs from the known species mainly by the shape of the copulatory complex. This is the third endoparasitic monogenean species reported from freshwater Neotropical fish.     

The family Tetraonchidae (Monogenea): its structure and position among the monogeneans

The muscle fascicles of the haptor in Tetraonchus monenteron have been described. The muscle connection of the fan-shaped dorsal bars with dorsal anchors is shown. When these muscle fascicles are contracted the dorsal anchors works as pincers. The division of tetraonchids into two genera based on types of copulatory organs, morphology of bars and haptor ans associations with different groups of fishes is restored. Different authors based on ciliated cells and chaetotaxy of the oncomiracidium and comparative spermiogenetic of T. monenteron include the tetraonchids with 16 marginal hooks into the order Dactylogyroidea. In the same time, based on the analysis of the onthogenesis of the dactylogyrid's haptor they postulate, that the haptor of these worms originally had 2 pairs of anchors and only 14 marginal hooks. The present paper contains data indicating that different representatives of the Dactylogyridea have 14-18 marginal hooks. Author put forward a suggestion, that some group of dactylogyrids originally did not have the anchors.     

Neotropical Monogenoidea. 33. Three new species of Ancistrohaptor n. g. (Dactylogyridae,Ancyrocephalinae) on Triportheus spp. (Teleostei,Characidae) from Brazil,with checklists of ancyrocephalines recorded from neotropical characiform fishes

Three new species of Ancistrohaptor n. g. are described from the gills of three species of Triportheus (Characidae) collected from the environs of Manaus, Amazonas, Brazil: A. falcatum n. sp. from T. elongatus; and A. falciferum n. sp. and A. falcunculum n. sp. from T. angulatus, T. albus and T. elongatus. Ancistrohaptor n. g. is proposed for species possessing overlapping gonads, a dextral or dextroventral vaginal aperture, a coiled (counter-clockwise) male copulatory organ, two accessory pieces in the copulatory complex, and a haptor armed with two pairs of anchors (ventral anchor with elongate shaft), dorsal and ventral bars and 14 hooks; hook pair 1 (ventral) anterior to ventral bar, pairs 2–4 (ventral) lying bilaterally anterior to ventral anchor bases, pair 5 (ventral) associated with distal end of ventral anchor shafts, and pairs 6 and 7 (dorsal) bilateral about midway along haptoral length. Parasite-host and host-parasite lists of the Ancyrocephalinae from neotropical Characiformes are provided.     

A new monogenean genus from an ephippid fish off Peninsular Malaysia

Sundatrema langkawiense n. g., n. sp. (Monogenea: Ancyrocephalidae) is described from the gills of the orbfish Ephippus orbis (Bloch) (Ephippidae) off the Island of Langkawi, Malaysia, in the Andaman Sea. This new genus has the ancyrocephalid characteristics of four anchors, 14 marginal hooks and two bars, but differs from other four-anchored monogenean genera, and notably from Parancylodiscoides Caballero & Bravo Hollis, 1961 (found on the ephippids Chaetodipterus spp. off Central and South America), by having a unique combination of features. These include a muscular genital sucker and a vas deferens and vagina on the same (sinistral) side of the body. It is similar to Parancylodiscoides in having four haptoral reservoirs opening at the anchoral apertures, four anchors, similar connecting bars and small marginal hooks. The new species is characterised by the above generic features and by possessing a small, short copulatory organ lacking an accessory piece. Diplectanum longiphallus MacCallum, 1915 (previously attributed to Ancyrocephalus Creplin, 1839, Tetrancistrum Goto & Kikuchi, 1917 and Pseudohaliotrema Yamaguti, 1953) is transferred to Parancylodiscoides as P. longiphallus (MacCallum, 1915) n. comb.     

Origin and evolution of the hamuli in the monogeneans

The hypothesis of the origin and evolution of the hamuli in monogeneans is proposed. It is suggested that the hamuli originated as the adult attachment organs of protomonogeneans inhabited the gills of the first vertebrates. Primarily they were represented by two lateral pairs of large hooks disposed anterior to the larval haptor. The fundamental direction in the evolution of monogeneans was the concentration of all attachment structures on the growing haptor. It the course of this evolutionary process, the hamuli onchoblasts migrated to the haptor, in which they had reached the position in the hind part of the haptor. The neotenic evolution of the Dactylogyridea and Gyrodactyloidea resulted in the forming new hamuli pairs. The hooks of opposite sides of the haptor are joined in a single complex within each side by the transverse plates (bars). So the presence of 4 hamuli is plesiomorphy for all the monogeneans and the presence of the transverse bars and new hamuli pairs are apomorphy for the Dactylogyridea and Gyrodactyloidea, whose evolution was linked with that of the Teleostei. The origin of the new hamuli pairs and transverse bars in the Dactylogyridea and Gyrodactyloidea appears to be a convergence.     

The use of geometric morphometrics in understanding shape variability of sclerotized haptoral structures of monogeneans (Platyhelminthes) with insights into biogeographic variability

The sclerotized attachment organ of monogeneans has been widely used to address fundamental questions in ecology and evolution. However, traditional morphometric techniques appear to be partially inadequate and non-optimal. Traditional linear measurements mainly provide information on the size of sclerites but provide very little information, if any, on their shape. The shape of sclerites is indeed virtually unexplored and its implication for ecological and evolutionary processes remains to be analyzed. This study aims to both introduce and illustrate the use of geometric morphometrics in order to study sclerites of monogeneans in a biogeographic context. To do this, we investigated morphological variation patterns among four populations from the Pacific Ocean and six monogenean species through traditional and geometric morphometric techniques. Unlike the traditional method, the geometric morphometric method yielded a high percentage of individuals correctly classified to the four populations, providing strong evidence for phenotypic variability, divergence and local adaptation among islands without evolutionary constraint. Moreover, the traditional method also resulted in inconsistent interpretations of shape variations. This study highlighted the limitations that may arise when using traditional morphometric techniques and emphasizes that considerable information about the shape of sclerotized haptoral parts is added by using geometric morphometrics. Given the prominent taxonomic, ecological and evolutionary role of the haptor for characterizing monogeneans, we ultimately discuss the potential broad use of geometric morphometrics in a wide variety of ecological and evolutionary contexts. This powerful approach might allow a more robust estimation of the extent to which traditional evolutionary theories based on size of sclerites are congruent with their shape.     

Phylogenetics of the Monogenea--evidence from a medley of molecules

Nuclear ribosomal DNA sequences of Monogenea from both complete small and partial large (D1-D2) subunits were determined and added to previously published sequences in order to best estimate the molecular phylogeny of the group. A total of 35 ssrDNA, 100 D1 lsrDNA and 51 D2 lsrDNA monogenean sequences were used, representing a total of 27 families. From these sequences different data sets were assembled and analysed to make the best use of all available molecular phylogenetic information from the taxa. Maximum parsimony and minimum evolution trees for each data partition were rooted against published sequences from the Cestoda, forcing the Monogenea to appear monophyletic. There was broad agreement between tree topologies estimated by both methods and between genes. Well-supported nodes were restricted to deeply diverging major groupings and more derived taxa with the lsrDNA data but were at most nodes with ssrDNA. The Polyonchoinea showed the greatest resolution with a general pattern of ((Monocotylidae(Capsalidae(Udonellidae+Gyrodactylidea)))((Anoplodiscidae+Sundanonchidae)(Pseudomurraytrematidae+Dactylogyridae))). The Heteronchoinea readily split into the Polystomatoinea+Oligonchoinea, and Chimaericolidae and Hexabothriidae were successively the most basal of oligonchoinean taxa. Relationships within the Mazocraeidea, comprising 27 families of which 15 were sampled here, were largely unresolved and appear to reflect a rapid radiation of this group that is reflected in very short internal branches for ssrDNA and D1 lsrDNA, and highly divergent D2 lsrDNA. A reduced morphological matrix, employing only those families represented by molecules, contrasted sharply with respect to polyonchoinean interrelationships. Deep branches of the Heteronchoinea were similar for both classes of data but also showed that the interrelationships of the mazocraeidean families are labile and susceptible to sampling.     

Evolutionary expansion of the Monogenea

The evolutionary expansion of the monogeneans has taken place in parallel with the diversification of the fish-like vertebrates. In this article the main trends in monogenean evolution are traced from a hypothetical skin-parasitic ancestor on early vertebrates. Special consideration is given to the following topics: early divergence between skin feeders and blood feeders; diversification and specialization of the haptor for attachment to skin; transfer from host to host, viviparity and the success of the gyrodactylids; predation on skin parasites and camouflage; colonization of the buccal and branchial cavities; diversification and specialization of the haptor for attachment to the gills; phoresy in gill parasites; the development of endoparasitism and the origin of the cestodes; the success of dactylogyroidean gill parasites; the uniqueness of the polyopisthocotyleans; ovoviviparity and the colonization of the tetrapods. Host specificity has been the guiding force of coevolution between monogeneans and their vertebrate hosts, but the establishment of monogeneans on unrelated hosts sharing the same environment (host-switching) may have been underestimated. Host-switching has provided significant opportunities for evolutionary change of direction and is probably responsible for the establishment of monogeneans on cephalopod molluscs, on the hippopotamus and possibly on chelonians. There are indications that host-switching may be more common in monogeneans that spread by direct transfer of adults/juveniles from host to host. A limitation on the further expansion of monogeneans is the need for water for the dispersal of the infective larva (oncomiracidium).     

On the Procercoid Protonephridial Systems of Three Diphyllobothrium Species (Cestoda, Pseudophyllidea) and Janicki's Cercomer Theory

Because the types of hooks are so similar in the oncosphacra/procercoid ef cestodes and in several groups of monogenclic trcmatodes and because the exterior of a procercoid with the hooks in a cercomer is so suggestive of a monogenetic nematode, the development of the procercoids of three Diphyllobothrium species was studied. The intention was to determine whether or not the procercoid protonephridial system would have a developmental stage when its type is similar to, or identical with that type which characterizes the monogeneans. Such a conformity would greatly support the theory of a common origin of monogeneans and ceslodes. However, it has emerged that no similar developmental stage exists. The ontogeny revealed a thorough metamorphosis from a very simple primary protonephridial system (identical with that of the miracidium larva in digeneans) to a secondary system, which develops into the system of the adult tapeworm. This fact may be interpreted as an argument against the supposed inter-relalionships between monogeneans and cestodes. However, the type of hooks and the procercoid cercomer still indicate common ancestors. An analysis of the miracidium, the oncosphaera and the oncomiracidium (the monogenean larva) with reference to their different developmental stages when hatching, gave rise to my interpretation of the fundamental structure of both the miracidium and the oncosphaera as primitively simple and not reduced, Especially the identical type of protonephridial system indicates. in my view, that digeneans and ceslodes originally had a common larva type. If the ceslodes and the monogeneans have common ancestors, then the procercoid may be interpreted as the ontogenetic recapitulation of a common hook-armed ancestor, here named hexucanthoid. This rhabdocoelan creature with six hooks in the cercomer and adapted to an ectocommensalic/ectoparasitic mode of life, is thought to have given rise to the monogeneans, the gyrocotylideans, the amphilinideans and the cestodes. The monogeneans were found to have two fundamentally different types of marginal hooks, and on this basis ihe existence of two different lines of evolution in Monogenea is indicated.     

Furcohaptor cynoglossi n. g., n. sp., an ancyrocephaline monogenean gill parasite with a bifurcate haptor and a note on its adhesive attitude

An ancyrocephaline monogenean,Furcohaptor cynoglossi n. g., n. sp., is described from the gills of the marine teleost flatfishCynoglossus macrostomus caught off the coast of Kerala State in India. The parasite is also recorded onC. puncticeps. The haptor divides to form two elongated and slender haptoral arms, each bearing at the distal end a cluster of relatively small sclerites, comprising the following: two hamuli, one of which is rod-shaped and the other of typical hooked shape; a bar articulating with the typical hamulus; five marginal hooklets of typical shape, each with a domus; a sixth marginal hooklet which is shorter and more slender than the others and lacks a domus. This sixth marginal hooklet is hard to locate in some individuals. The haptoral arms embrace a primary gill lamella, their distal hook-bearing regions meeting or overlapping on the side of the lamella opposite to the parasite's body. Species of the genusBifurcohaptor Jain, 1958 differ in that two of the hamuli are greatly enlarged and support the haptoral arms.     

A new genus and three new species of dactylogyrids (Monogenea), gill parasites of the threadfin bass,Pronotogrammus multifasciatus Gill (Perciformes: Serranidae) in the Southeastern Pacific Ocean off Peru

Pronotogrammella n. g. is erected to accommodate Pronotogrammella boegeri n. sp. (type-species), Pr. scholzi n. sp. and Pr. multifasciatus n. sp. (Monogenea: Dactylogyridae). The new species are gill parasites of the threadfin bass Pronotogrammus multifasciatus Gill (Perciformes: Serranidae), a demersal teleost collected from off the coastal zone of Puerto Pizarro, Tumbes, Peru. Pronotogrammella n. g. is mainly characterised by having broadly fork-shaped dorsal anchors, which have an accessory anchor sclerite articulated to the tip of the superficial roots. Pronotogrammella n. g. is also characterised by having: (i) a tubular tapered-shaped male copulatory organ (MCO), filamentous distally, with a clockwise coil at distal end or not, lacking accessory piece; (ii) a dorsal bar with an anteromedial delicate umbelliform membrane supported by two processes; (iii) hooks with upright blunt thumb and uniform shank; (iv) a vaginal aperture dextrolateral; (v) a subquadrangular haptor, with inconspicuous lateral flaps and lacking haptoral reservoirs; and (vi) eye-spot or chromatic granules absent. Pronotogrammella boegeri n. sp. is characterised by its crosier-shaped MCO having a clockwise coil at distal end and by its dorsal bar with a straight anteromedial processes. Pronotogrammella scholzi n. sp. is typified by possessing of a dorsal bar with the anteromedial processes like cow horns, hoof-shaped deep roots of the dorsal anchors and a broader shaft of the MCO. Pronotogrammella multifasciatus n. sp. differs from all congeners by having a tubular MCO with twisted shaft and a base with a short and broad arm and by having an almost dumbbell-shaped ventral bar.

     

Gyrodactylus longidactylus n. sp., a monogenean from Pomatoschistus lozanoi (de Buen) (Gobiidae) from the North Sea

Gyrodactylus longidactylus n. sp., a gyrodactylid monogenean parasitising the gills of Pomatoschistus lozanoi (de Buen, 1923) in the North Sea is described. It is a species with rather small anchors which are only connected to a ventral bar which lacks a ventral bar membrane. The marginal hooks have long handles, which are always longer than the total length of the anchor. The pharynx has long pharyngeal processes. Measurements, drawings of the opisthaptoral hard parts, penis and pharynx, and SEM micrographs of this monogenean are presented. G. longidactylus n. sp. is the first monogenean species described from P. lozanoi.     

Secretory products of the haptoral reservoirs and peduncular glands in two species of Bravohollisia (Monogenea: Ancyrocephalidae)

Abstract. Light and electron microscopy were used to characterize the structure of secretory cells and their products involved in attachment of two monogenean parasites of fish, in order to understand their role in the attachment process. In Bravohollisia rosetta and Bravohollisia gussevi , peduncular gland cells with two nuclei, granular endoplasmic reticulum, and Golgi bodies produce dual electron-dense (DED) secretory bodies with a homogenous electron-dense rind and a less electron-dense fibrillar core (oval and concave in B. rosetta and oval in B. gussevi ). The DED secretory bodies are altered as they migrate from the gland cell to the haptoral reservoir, the superficial anchor grooves, and into the gill tissues. The contents of the DED secretory bodies are exocytosed into the reservoirs, fibrillar cores persisting in the matrix, some of which condense, forming highly electron-dense spherical bodies. Small, oval, electron-dense bodies occur in the grooves, while no inclusions are visible in the homogenous exudate within the gill tissues. The single tubular extension of the reservoir enters a bifurcate channel within the anchor via a concealed, crevice-like opening on one side of the anchor. The channel directs secretions into the left and the right grooves via concealed apertures. The secretions, introduced into the tissues by the anchors, probably assist in attachment. The secretions are manifested externally as net-like structures and observed in some cases to be still attached to the point of exudation, on anchors detached from the gill tissues. This suggests that despite having the anchors detached, the worms can still remain anchored to the gill tissues via these net-like structures. Based on this, it is postulated that the net-like secretions probably function as a safety line to anchor the worm during the onset of locomotion and in doing so reduce the risk of tearing host tissues.