Sex-specific impact of inbreeding on pathogen load in the striped dolphin.

The impact of inbreeding on fitness has been widely studied and provides consequential inference about adaptive potential and the impact on survival for reduced and fragmented natural populations. Correlations between heterozygosity and fitness are common in the literature, but they rarely inform about the likely mechanisms. Here, we investigate a pathology with a clear impact on health in striped dolphin hosts (a nematode infection that compromises lung function). Dolphins varied with respect to their parasite burden of this highly pathogenic lung nematode (Skrjabinalius guevarai). Genetic diversity revealed by high-resolution restriction-associated DNA (43 018 RADseq single nucleotide polymorphisms) analyses showed a clear association between heterozygosity and pathogen load, but only for female dolphins, for which the more heterozygous individuals had lower Sk. guevarai burden. One locus identified by RADseq was a strong outlier in association with parasite load (heterozygous in all uninfected females, homozygous for 94% of infected females), found in an intron of the citron rho-interacting serine/threonine kinase locus (associated with milk production in mammals). Allelic variation at the Class II major histocompatability complex DQB locus was also assessed and found to be associated with both regional variation and with pathogen load. Both sex specificity and the identification of associating functional loci provide insight into the mechanisms by which more inbred individuals may be more susceptible to the infection of this parasite. This provides important insight towards our understanding of the impact of inbreeding in natural populations, relevant to both evolutionary and practical conservation considerations.

Transmission and Predictors of Burden of Lungworms of the Striped Dolphin (Stenella coeruleoalba) in the Western Mediterranean.

Pseudaliid lungworms infect the lungs and sinuses of cetaceans. Information on the life cycle and epidemiology of pseudaliids is very scarce and mostly concerns species that infect coastal or inshore cetaceans. Available evidence indicates that some pseudaliids are vertically transmitted to the host, whereas others are acquired via infected prey. We documented pseudaliid infections in an oceanic cetacean, the striped dolphin (Stenella coeruleoalba) in the western Mediterranean, and investigated the possibilities of vertical vs. horizontal transmission and the potential influence of host body size, sex, and season on infection levels. We found two species of lungworm in 87 dolphins that stranded along the Spanish Mediterranean coast between 1987 and 2018. One or two larvae of Stenurus ovatus were found in three adult dolphins. Larger numbers of larvae and adults of Skrjabinalius guevarai were collected in 51 dolphins, including unweaned calves. These observations suggested that Skrjabinalius guevarai could be vertically transmitted. The abundance of Skrjabinalius guevarai increased significantly with host size, which suggested that it could be trophically transmitted, as well, with larger hosts consuming more infected prey. Infection levels peaked in spring, outside of the calving season, which is likely a reflection of a seasonal shift in dolphin diet. In summary, results indicate that Skrjabinalius guevarai was capable of both vertical and horizontal transmission, but future research should be directed at clarifying the potential mechanics behind transmission and intermediate hosts.

Microbiota and gut ultrastructure of Anisakis pegreffii isolated from stranded cetaceans in the Adriatic Sea.

BACKGROUND: Inferring the microbiota diversity of helminths enables depiction of evolutionarily established ecological and pathological traits that characterize a particular parasite-host interaction. In turn, these traits could provide valuable information for the development of parasitosis control and mitigation strategy. The parasite Anisakis pegreffii (Nematoda: Anisakidae) realizes the final stage of its life-cycle within gastric chambers of aquatic mammals, causing mild-to-moderate granulomatous gastritis with eosinophilic infiltrate, to severe ulcerative gastritis with mixed inflammatory infiltrate, often associated with bacterial colonies. However, its interaction with the host microbiota remains unknown, and might reveal important aspects of parasite colonization and propagation within the final host. METHODS: MySeq Illumina sequencing was performed for the 16S rRNA gene from microbiota isolated from larvae, and uterus and gut of adult A. pegreffii parasitizing stranded striped dolphins (Stenella coeruleoalba). To assess the potential presence of Brucella ceti within isolated microbiota, Brucella-targeted real-time PCR was undertaken. In addition, TEM of the gastrointestinal tract of the infective third-stage (L3) and transitioning fourth-stage larvae (L4) was performed to characterize the morphological differences and the level of larval feeding activity. RESULTS: In total, 230 distinct operational taxonomic units (OTUs) were identified across all samples (n = 20). The number of shared taxa was lower than the number of taxa found specifically in each parasite stage or organ. The dominant taxon was Mycoplasmataceae (genus Mycoplasma) in the gut and uterus of adult A. pegreffii, whereas Fusobacteriaceae (genus Cetobacterium) was the most abundant in 40% of larvae, alongside Mycoplasmataceae. No B. ceti DNA was detected in any of the microbiota isolates. TEM revealed differences in gut ultrastructure between L3 and L4, reflecting a feeble, most likely passive, level of feeding activity in L3. CONCLUSIONS: Microbiota from L3 was more related to that of the gut rather than the uterus of adult A. pegreffii. Taxa of the larval microbiota showed qualitative and quantitative perturbations, likely reflecting the propagation through different environments during its life-cycle. This suggests an ontogenetic shift in the alpha and beta diversity of microbial communities from uterus-derived towards cetacean-derived microbiota. Although TEM did not reveal active L3 feeding, microbiota of the latter showed similarity to that of an actively feeding adult nematode.

Pyogranulomatous obliterative laryngotracheitis by Rhizopus arrhizus (syn. R. oryzae) in a free-ranging Atlantic spotted dolphin Stenella frontalis.

We report the gross and microscopic findings and molecular identification of fungal hyphate infection in a juvenile female Atlantic spotted dolphin Stenella frontalis found dead off Arguineguin, Gran Canaria (Canary Islands, Spain). On necropsy examination, the animal had a large cranial intrathoracic mass and multiple variably-sized nodules throughout the larynx and trachea that obliterated the lumen. Microscopically, the masses were composed of abundant pyogranulomatous inflammation with numerous fungal hyphae. These were pauciseptate (coenocytic) and had non-parallel walls, non-dichotomous irregular to right angle branching, and bulbous dilations. PCR analysis from these inflammatory foci yielded Rhizopus arrhizus (syn. R. oryzae). This fungal pathogen is often ascribed to opportunistic infections in immunosuppressed humans and animals. In the present case, a potential cause for immunosuppression was not identified; PCR analysis for cetacean morbillivirus was negative. Herein, we report the first confirmed case of R. arrhizus infection in a free-living Atlantic cetacean. These findings add to the body of knowledge on fungal disease in cetaceans in general and, in particular, in odontocetes, where respiratory involvement is common.

Life-history trade-offs in a generalist digenean from cetaceans: the role of host specificity and environmental factors.

BACKGROUND: Adults and larvae of generalist parasites are exposed to diverse hosts and local environmental conditions throughout their life cycles, thus local adaptation is expected to occur through phenotypic plasticity and/or natural selection. We investigated how the combined effect of cryptic host specificity and local selective pressures could shape reproductive traits of a putative generalist parasite in the oceanic realm. METHODS: The LSU rDNA, ITS2 and the mt-COI of individuals of the digenean Pholeter gastrophilus (Kossack, 1910) Odhner, 1914 (Heterophyidae Leiper, 1909) from oceanic striped dolphins, Stenella coeruleoalba Meyen, and coastal bottlenose dolphins, Tursiops truncatus Montagu, in the western Mediterranean were used to elucidate whether worms were conspecific. Infection parameters were compared between both dolphin species. General Linear Mixed Models were used to analyse the influence of host species on four reproductive traits of P. gastrophilus: body size, maturity stage (non-gravid/gravid), egg size, and number of eggs in utero. AIC values were used to rank competing models, and p-values to assess the effect of specific predictors. RESULTS: Evidence indicated that worms collected from both dolphin species were conspecific. All worms collected were gravid and infection parameters did not differ between dolphin species. However, body size and egg size of individuals of P. gastrophilus were significantly larger in striped dolphins. The number of eggs in utero did not significantly differ between dolphin species but, for a given body size, worms in bottlenose dolphins harboured more eggs. A trade-off between egg size and egg number was found in worms from both dolphin species, with a higher slope in striped dolphins. CONCLUSIONS: Apparently, striped dolphin is a more suitable host for P. gastrophilus, but reproductive investment seems to be adapted to the habitat where the life-cycle develops. Worms from striped dolphins likely face the problem of finding intermediate hosts in the oceanic realm and apparently invest into offspring size to enhance the early survival of larvae and the potential to multiply asexually within the first intermediate host. The small-sized worms from bottlenose dolphins would be adapted to reproduce early because of higher adult mortality, generating smaller and numerous eggs in a coastal habitat where chances of transmission are presumably higher.

Living on the Edge: Settlement Patterns by the Symbiotic Barnacle Xenobalanus globicipitis on Small Cetaceans.

The highly specialized coronulid barnacle Xenobalanus globicipitis attaches exclusively on cetaceans worldwide, but little is known about the factors that drive the microhabitat patterns on its hosts. We investigate this issue based on data on occurrence, abundance, distribution, orientation, and size of X. globicipitis collected from 242 striped dolphins (Stenella coeruleoalba) that were stranded along the Mediterranean coast of Spain. Barnacles exclusively infested the fins, particularly along the trailing edge. Occurrence, abundance, and density of X. globicipitis were significantly higher, and barnacles were significantly larger, on the caudal fin than on the flippers and dorsal fin. Barnacles were found more frequently and in greater numbers on the dorsal rather than ventral side of the caudal fin and on the central third of dorsal and ventral fluke surfaces. Nearly all examined individuals attached with their cirral fan oriented opposite to the fluke edge. We suggest that X. globicipitis may chemically recognize dolphins as a substratum, but fins, particularly the flukes, are passively selected because of creation of vortices that increase contact of cyprids with skin and early survival of these larvae at the corresponding sites. Cyprids could actively select the trailing edge and orient with the cirri facing the main direction of flow. Attachment on the dorsal side of the flukes is likely associated with asymmetrical oscillation of the caudal fin, and the main presence on the central segment of the flukes could be related to suitable water flow conditions generated by fluke performance for both settlement and nutrient filtration.

Putative hybrids between two Anisakis cryptic species: molecular genotyping using High Resolution Melting.

The genus Anisakis includes nine recognized species and the complex of cryptic species Anisakis simplex s. l. is often associated with the human disease known as anisakiasis. During the last decades the use of nuclear ribosomal ITS allowed the identification and description of numerous anisakid nematodes and the discovery of recombinant genotypes or putative hybrids even in other parasitic helminths, such as those between A. simplex sensu stricto and A. pegreffii. The existence of pure hybrids of the two sibling species has been long debated due to the large recovery of larval forms from sympatric areas and the rare observation of adult hybrids. The aims of the present report were to identify anisakid nematodes collected from Stenella coeruleoalba using PCR-RFLP of ITS and to focus the interest on hybrid forms using a High Resolution Melting (HRM) and direct sequencing analyses, since the new record of putative hybrid at adult stage. The PCR-RFLP analysis enabled to identify A. simplex s.s., A. pegreffii, the heterozygous genotype of the two species and A. physeteris. The use of the genotyping approach based on HRM confirmed the profiles of the two species A. simplex s.s. and A. pegreffii, and of the hybrid individuals. The new record of adult hybrids in definitive hosts rekindles the long debate about their existence and their evolutionary meaning. Since the reproductive isolation between A. simplex s.s. and A. pegreffii is the assumption for their existence as separated species, the use of alternative molecular markers and population genetic studies on adult anisakids are recommended.

Genetic and morphological approaches distinguish the three sibling species of the Anisakis simplex species complex, with a species designation as Anisakis berlandi n. sp. for A. simplex sp. C (Nematoda: Anisakidae).

Numerous specimens of the 3 sibling species of the Anisakis simplex species complex (A. pegreffii, A. simplex (senso stricto)), and A. simplex sp. C) recovered from cetacean species stranded within the known geographical ranges of these nematodes were studied morphologically and genetically. The genetic characterization was performed on diagnostic allozymes and sequences analysis of nuclear (internal transcribed spacer [ITS] of ribosomal [r]DNA) and mitochondrial (mitochondrial [mt]DNA cox2 and rrnS) genes. These markers showed (1) the occurrence of sympatry of the 2 sibling species A. pegreffii and A. simplex sp. C in the same individual host, the pilot whale, Globicephala melas Traill, from New Zealand waters; (2) the identification of specimens of A. pegreffii in the striped dolphin, Stenella coeruleoalba (Meyen), from the Mediterranean Sea; and (3) the presence of A. simplex (s.s.) in the pilot whale and the minke whale, Balaenoptera acutorostrata Lacépède, from the northeastern Atlantic waters. No F1 hybrids were detected among the 3 species using the nuclear markers. The phylogenetic inference, obtained by maximum parsimony (MP) analysis of separate nuclear (ITS rDNA region), combined mitochondrial (mtDNA cox2 and rrnS) sequences datasets, and by concatenated analysis obtained at both MP and Bayesian inference (BI) of the sequences datasets at the 3 studied genes, resulted in a similar topology. They were congruent in depicting the existence of the 3 species as distinct phylogenetic lineages, and the tree topologies support the finding that A. simplex (s.s.), A. pegreffii, and A. berlandi n. sp. (= A. simplex sp. C) represent a monophyletic group. The morphological and morphometric analyses revealed the presence of morphological features that differed among the 3 biological species. Morphological analysis using principal component analysis, and Procrustes analysis, combining morphological and genetic datasets, showed the specimens clustering into 3 well-defined groups. Nomenclatural designation and formal description are given for A. simplex species C: the name Anisakis berlandi n. sp. is proposed. Key morphological diagnostic traits are as follows between A. berlandi n. sp. and A. simplex (s.s.): ventriculus length, tail shape, tail length/total body length ratio, and left spicule length/total body length ratio; between A. berlandi n. sp. and A. pegreffii: ventriculus length and plectane 1 width/plectane 3 width ratio; and between A. simplex (s.s.) and A. pegreffii: ventriculus length, left and right spicule length/total body length ratios, and tail length/total body length ratio. Ecological data pertaining to the geographical ranges and host distribution of the 3 species are updated.

Genotyping of Toxoplasma gondii isolates in meningo-encephalitis affected striped dolphins (Stenella coeruleoalba) from Italy.

This study reports the occurrence of Toxoplasma gondii in the brain of three striped dolphins (Stenella ceoruleoalba) found stranded on the Ligurian Sea coast of Italy between 2007 and 2008. These animals showed a severe, subacute to chronic, non-purulent, multifocal meningo-encephalitis, with the cerebral parenchyma of two dolphins harbouring protozoan cysts and zoites immunohistochemically linked to T. gondii. Molecular, phylogenetic and mutation scanning analyses showed the occurrence of Type II and of an atypical Type II T. gondii isolates in one and two dolphins, respectively. In spite of the different molecular patterns characterizing the above T. gondii genotypes, the brain lesions observed in the three animals showed common microscopic features, with no remarkable differences among them. The role of T. gondii in causing the meningo-encephalitis is herein discussed.

Estudio de infección sistémica por herpesvirus complicada con Cryptosporidium spp: en un delfín manchado del atlántico (Stenella frontalis Curvier 1829) / Study of systemic infection by herpesvirus complicated with Cryptosporidium spp: in a gulf stream spotted dolphin (Stenella frontalis Curvier 1829)

El presente estudio se realizó a partir de un caso de veramiento de delfín Stenella frontalis, hembra cría, reportado el 30/07/02 para Playa Dorada. Boca de Aroa, estado Falcón, Venezuela. Se mantuvo en una instalación marina por un período de tres días, tiempo durante el cual se le realizaron evaluaciones periódicas no invasivas y toma de datos morfométricos. Murió el 01/08/02 por paro cardiopulmonar. Se practicó estudio macroscópico postmortem, preservándose muestras en formoldehído al 10 por ciento para estudios histopatológicos de los siguientes órganos: corazón, pulmón, estómago, intestino grueso, hígado, riñón y lesiones de piel con capa grasa, fueron procesados en el laboratorio de Anatomía Patológica de la Universidad Central de Venezuela. Por los resultados obtenidos en el estudio microscópico se determinó que el individuo presentó una infección sistémica producto de una infección primaria por Herpesvirus spp., concominante con parasistismo secundario por Cyptosporidium spp., cuadro clínico que se vio complicado por las condiciones de estrés a las que se encontró sometido, producto del varamiento. Según la revisión bibliográfica realizada, la parasitosis por Cyptosporidium spp. no aparece reportada en Venezuela para el orden: Cetácea, lo cual se evidencia en la información recopilada acerca de parásitos estudiados para dicho orden. La osamenta se encuentra en Museo Científico de la Estación Biológica. Ranchi Grande-MARNR.

The present study was made, in the event of a young female dolphin of the specie Stenella frontalis, that was found stranded in Playa Dorada, in Boca de Aroa, in the Falcón State, the 07/30/02, in Venezuela. She was kept in a facility for a period of three days, during this period of time, she was evaluated periodically whit not invasive technique, and morphometry data recollection. She died the 08/01/02 due to respiratory arrest, which caused a cardiac arrest. A macroscopic postmortem study was made, taking samples and keeping them in formaldehyde 10% for histopathology study of the following organs: heart, lungs, stomach, large intestine, liver, kidney and lesions of the skin with a fat layer, they were processed in the Pathologic Anatomy laboratory of the Universidad Central de Venezuela. Because of the results of the microscopic study, it was determined that the dolphin presented a systemic infection, because of a primary infection by Herpesvirus spp., related with a secondary infection by Cryptosporidium spp., which was complicated with stress due to the beaching. The bibliographic revue reports that the parasitosis by Cryptosporidium spp. is not present to Venezuela in the Cetacean order, according to the compiled information about the studied parasites for this order. The skeleton is currently in the Scientific Museum of the Biological Station Rancho Grande - MARNR.

Isolation and genetic characterization of Toxoplasma gondii from striped dolphin (Stenella coeruleoalba) from Costa Rica.

Toxoplasma gondii infection in marine mammals is of interest because of mortality and mode of transmission. It has been suggested that marine mammals become infected with T. gondii oocysts washed from land to the sea. We report the isolation and genetic characterization of viable T. gondii from a striped dolphin (Stenella coeruleoalba), the first time from this host. An adult female dolphin was found stranded on the Pacific Coast of Costa Rica, and the animal died the next day. The dolphin had a high (1:6400) antibody titer to T. gondii in the modified agglutination test. Severe nonsuppurative meningoencephalomyelitis was found in its brain and spinal cord, but T. gondii was not found in histological sections of the dolphin. Portions of its brain and the heart were bioassayed in mice for the isolation of T. gondii. Viable T. gondii was isolated from the brain, but not from the heart, of the dolphin. A cat fed mice infected with the dolphin isolate (designated TgSdCol) shed oocysts. Genomic DNA from tachyzoites of this isolate was used for genotyping at 10 genetic loci, including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, and this TgSdCo1 isolate was found to be Type II.