Assessment of metal and organic pollutants in combination with stable isotope analysis in tunas from the Gulf of Cadiz (east Atlantic).

Bioaccumulation patterns of heavy metals (Pb, Cd, Cr, Ni, Fe and Cu) and organic (priority and emerging) pollutants, in combination with stable isotope analysis (SIA), were assessed in muscle and liver of three tuna species from the Gulf of Cadiz (Atlantic bluefin tuna, Thunnus thynnus; Atlantic bonito, Sarda sarda, and skipjack tuna, Katsuwonus pelamis). SIA and contaminant (heavy metal and organic) profiles separately discriminated between species. There was no significant overlap between the trophic niches estimated from isotopic data, suggesting that there are diet differences which may determine differential bioaccumulation patterns. The levels of heavy metals and persistent organic pollutants in muscle of all the individuals analyzed were below the allowable limits established by the current legislation. Concentrations of most contaminants were higher in liver than in muscle, underlining the powerful detoxifying capacity of the liver in tunas. In addition to diet, other factors such as size and age (exposure time to environmental chemicals) explain differences in pollutant accumulation patterns in tissues between species, each with varying degrees of involvement depending on the pollutant class. Our results show that combining contaminant profile data with trophic features based on SIA may help understand pollutant bioaccumulation patterns in upper levels of marine food webs.

Molecular Rapid Test for Identification of Tuna Species.

Tuna is an excellent food product, relatively low in calories, that is recommended for a balanced diet. The continuously increasing demand, especially for bluefin-tuna-based food preparations, and its relatively high market price make adulteration by intentionally mixing with other lower-priced tunas more prospective. The development of rapid methods to detect tuna adulteration is a great challenge in food analytical science. We have thus developed a simple, fast, and low-cost molecular rapid test for the visual detection of tuna adulteration. It is the first sensor developed for tuna authenticity testing. The three species studied were Thunnus thynnus (BFT), Thunnus albacares, and Katsuwonus pelamis. DNA was isolated from fresh and heat-treated cooked fish samples followed by PCR. The PCR products were hybridized (10 min) to specific probes and applied to the rapid sensing device. The signal was observed visually in 10-15 min using gold nanoparticle reporters. The method was evaluated employing binary mixtures of PCR products from fresh tissues and mixtures of DNA isolates from heat-treated tissues (canned products) at adulteration percentages of 1-100%. The results showed that the method was reproducible and specific for each tuna species. As low as 1% of tuna adulteration was detected with the naked eye.

Trace metal accumulation in Atlantic bluefin tuna and correlations with protein-lipid composition.

This study determines the biochemical composition and metal concentrations (Hg, Fe, Zn, Cu) in muscle tissue of reared Atlantic bluefin tuna and investigates whether protein-lipid composition affects metal accumulation. A significant positive relationship was found between Hg concentrations and protein content, whereas a strong negative relationship between Hg concentrations and lipid content was revealed. Opposite results were obtained, correlating Zn concentrations with biochemical components. No correlations between the concentrations of the other two metals and the protein or lipid components were observed. Evidently, biochemical components may influence the accumulation of certain metals, but not in the same way. Although the analyzed tuna samples represent a rich source of essential metals, Hg may pose a risk to consumers. Our results suggest that a lower level of Hg can be associated with greater lipid and lower protein content. Consumers could reduce the health risk from Hg by choosing constantly high-fat bluefin tuna.

Importance of the Lunar Cycle on Mesopelagic Foraging by Atlantic Bluefin Tuna in the Upwelling Area of the Strait of Messina (Central Mediterranean Sea).

The influence of the lunar cycle on bluefin tuna foraging in the upwelling area of the Strait of Messina was investigated by exploring trophic interaction with mesopelagic fish and cephalopod prey. To focus on how the lunar cycle could affect availability of mesopelagic prey for this predator, we tested potential differences in the diet related to each lunar phase. Moreover, we considered two potential impacts of the lunar cycle: the lunar irradiance and the strength of currents. Overall, 2672 prey items were mesopelagic fish and cephalopods, representing 60.7% of overall diet by number. The main mesopelagic fish prey items were lanternfishes and dragonfishes, while Onychoteuthis banksii was the most important cephalopod prey. In summary, the Strait of Messina has highly specific hydrodynamic and biological features which strongly depend on upwelling currents, which in turn are influenced by the lunar cycle (new and full moon with strong currents, quarters with fewer currents). Upwelling causes water mixing, bringing to the surface a large amount of mesopelagic fauna which become more readily available to tuna. Lunar irradiance contributes to the variation of prey composition, increasing the success of visual predation on mesopelagic resources at high light in the water column.

Trade-offs between risks of predation and starvation in larvae make the shelf break an optimal spawning location for Atlantic bluefin tuna.

Atlantic bluefin tuna (ABT) (Thunnus thynnus) travel long distances to spawn in oligotrophic regions of the Gulf of Mexico (GoM) which suggests these regions offer some unique benefit to offspring survival. To better understand how larval survival varies within the GoM a spatially explicit, Lagrangian, individual-based model was developed that simulates dispersal and mortality of ABT early life stages within realistic predator and prey fields during the spawning periods from 1993 to 2012. The model estimates that starvation is the largest cumulative source of mortality associated with an early critical period. However, elevated predation on older larvae is identified as the main factor limiting survival to late postflexion. As a result, first-feeding larvae have higher survival on the shelf where food is abundant, whereas older larvae have higher survival in the open ocean with fewer predators, making the shelf break an optimal spawning area. The modeling framework developed in this study explicitly simulates both physical and biological factors that impact larval survival and hence could be used to support ecosystem based management efforts for ABT under current and future climate conditions.

Genetic connectivity between Atlantic bluefin tuna larvae spawned in the Gulf of Mexico and in the Mediterranean Sea.

The highly migratory Atlantic bluefin tuna (ABFT) is currently managed as two distinct stocks, in accordance with natal homing behavior and population structuring despite the absence of barriers to gene flow. Larval fish are valuable biological material for tuna molecular ecology. However, they have hardly been used to decipher the ABFT population structure, although providing the genetic signal from successful breeders. For the first time, cooperative field collection of tuna larvae during 2014 in the main spawning area for each stock, the Gulf of Mexico (GOM) and the Mediterranean Sea (MED), enabled us to assess the ABFT genetic structure in a precise temporal and spatial frame exclusively through larvae. Partitioning of genetic diversity at nuclear microsatellite loci and in the mitochondrial control region in larvae spawned contemporarily resulted in low significant fixation indices supporting connectivity between spawners in the main reproduction area for each population. No structuring was detected within the GOM after segregating nuclear diversity in larvae spawned in two hydrographically distinct regions, the eastern GOM (eGOM) and the western GOM (wGOM), with the larvae from eGOM being more similar to those collected in the MED than the larvae from wGOM. We performed clustering of genetically characterized ABFT larvae through Bayesian analysis and by Discriminant Analysis of Principal Components (DAPC) supporting the existence of favorable areas for mixing of ABFT spawners from Western and Eastern stocks, leading to gene flow and apparent connectivity between weakly structured populations. Our findings suggest that the eastern GOM is more prone for the mixing of breeders from the two ABFT populations. Conservation of this valuable resource exploited for centuries calls for intensification of tuna ichthyoplankton research and standardization of genetic tools for monitoring population dynamics.

Evidence of Atlantic bluefin tuna spawning in the Bay of Biscay, north-eastern Atlantic.

The spawning grounds of the Atlantic bluefin tuna (Thunnus thynnus) are traditionally considered to be the Gulf of Mexico (Gulf of Mexico) and the Mediterranean Sea (Mediterranean Sea). However, for the western Atlantic, unequivocal evidence of bluefin spawning outside the Gulf of Mexico has been shown. In this study we present the first records of genetically confirmed bluefin larvae in the southern Bay of Biscay (eastern Atlantic). These findings provide evidence of bluefin spawning activity outside the Mediterranean Sea, in the north-eastern Atlantic. However, our results suggest that the bluefin spawning in the Bay of Biscay is a sporadic phenomenon.

De novo transcriptome assembly, functional annotation and characterization of the Atlantic bluefin tuna (Thunnus thynnus) larval stage.

In the present work, we assembled and characterized a de novo larval transcriptome of the Atlantic bluefin tuna Thunnus thynnus by taking advantage of publicly available databases with the goal of better understanding its larval development. The assembled transcriptome comprised 37,117 protein-coding transcripts, of which 13,633 full-length (>80% coverage), with an Ex90N50 of 3061 bp and 76% of complete and single-copy core vertebrate genes orthologues. Of these transcripts, 34,980 had a hit against the EggNOG database and 14,983 with the KEGG database. Codon usage bias was identified in processes such as translation and muscle development. By comparing our data with a set of representative fish species, 87.1% of tuna transcripts were included in orthogroups with other species and 5.1% in assembly-specific orthogroups, which were enriched in terms related to muscle and bone development, visual system and ion transport. Following this comparative approach, protein families related to myosin, extracellular matrix and immune system resulted significantly expanded in the Atlantic bluefin tuna. Altogether, these results provide a glimpse of how the Atlantic bluefin tuna might have achieved early physical advantages over competing species in the pelagic environment. The information generated lays the foundation for future research on the more detailed exploration of physiological responses at the molecular level in different larval stages and paves the way to evolutionary studies on the Atlantic bluefin tuna.

Mercury concentrations in reared Atlantic bluefin tuna and risk assessment for the consumers: To eat or not to eat?

This study determines the levels of total mercury in muscle tissue from 268 reared Atlantic bluefin tuna in the Mediterranean Sea, detects variations on mercury concentrations in relation to biological parameters and rearing period and evaluates the health risk posed by fish consumption taking into consideration the size, age and rearing period. A total of 30 pooled samples were analyzed. The total Hg concentrations ranged from 0.49 to 1.60 mg/kg w.w. and showed a significant positive relationship with the size and age. It was found that the longer the rearing period, the lower the mercury concentration. The total Hg exceeded the maximum level defined by the European Commission Decision in a proportion of 40.0%. Since the value of Target Hazard Quotient was higher than 1 in all different groups (medium, large, 6 months and 18 months reared specimens), the maximum possible safe weekly consumption (400 g) is suggested.

Characterization of carbonate fraction of the Atlantic bluefin tuna fin spine bone matrix for stable isotope analysis.

The mineral component of fish otoliths (ear bones), which is aragonitic calcium carbonate (CaCO3), makes this structure the preferred sample choice for measuring biological carbon and oxygen-stable isotopes in order to address fundamental questions in fish ecology and fisheries science. The main drawback is that the removal of otoliths requires sacrificing the specimen, which is particularly impractical for endangered and commercially valuable species such as Atlantic bluefin tuna (Thunnus thynnus) (ABFT). This study explores the suitability of using the first dorsal fin spine bone of ABFT as a non-lethal alternative to otolith analysis or as a complementary hard structure. The fin spines of freshly caught ABFT were collected to identify carbonate ions within the mineral matrix (i.e., hydroxyapatite) and to determine the nature of the carbonate substitution within the crystal lattice, knowledge which is crucial for correct measurement and ecological interpretation of oxygen and carbon stable isotopes of carbonates. Fin spine sections were analyzed via X-ray Photoelectron Spectroscopy (XPS), Raman Spectroscopy, and Fourier Transform InfraRed (FTIR). The XPS survey analysis showed signals of Ca, O, and P (three compositional elements that comprise hydroxyapatite). The Raman and FTIR techniques showed evidence of carbonate ions within the hydroxyapatite matrix, with the IR spectra being the most powerful for identifying the type B carbonate substitution as shown by the carbonate band in the v 2 CO3 2- domain at ∼872 cm-1. The results of this study confirmed the presence of carbonate ions within the mineral matrix of the fin spine bone of ABFT, showing the feasibility of using this calcified structure for analysis of stable isotopes. Overall, our findings will facilitate new approaches to safeguarding commercially valuable and endangered/protected fish species and will open new research avenues to improve fisheries management and species conservation strategies.

Insights on the seasonal variations of reproductive features in the Eastern Atlantic Bluefin Tuna.

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.

Production and Refinement of Omega-3 Rich Oils from Processing By-Products of Farmed Fish Species.

In this study, the effect of a four-stage chemical refining process (degumming, neutralization, bleaching, deodorization) on the quality parameters, fatty acid composition and volatile compounds of crude oils produced from processing by-products of farmed fish species (tuna, seabass and gilthead seabream) was evaluated. The quality of the oils was compared to commercially available cod liver oil on the basis of free fatty acid, peroxide value, p-anisidine, total oxidation (TOTOX), thiobarbituric acid reactive species (TBARS), oxidative stability at 80, 100 and 120 °C, tocopherol content, and volatile components, while the fatty acid profile and the proportion of polyunsaturated fatty acids (PUFAs) were used as an indicator of the nutritional values of fish oils. Quality parameters of the studied oils and oil oxidative stability were enhanced with refining and were within the limits recommended for fish oils without the loss of PUFAs. In tuna by-product refined oils, the proportion of PUFAs was over 40%, with 30% of eicosapentaenoic and docosahexaenoic fatty acids. The volatile compounds of the oils were quantified (in mg/kg) and major components were 2,4-heptadienal, pentadecane, 2,4-decadienal, 2,4-nonadienal and dodecane. The use of aquaculture by-products as an alternative source for fish oil production could contribute to a more sustainable and profitable aquaculture production, providing economic benefits for the producers and setting new standards for a fish by-product disposal strategy.

Spatial dynamics and mixing of bluefin tuna in the Atlantic Ocean and Mediterranean Sea revealed using next-generation sequencing.

The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated with shared fisheries management. In an effort to resolve the species' stock dynamics, a genomewide search for spatially informative single nucleotide polymorphisms (SNPs) was undertaken, by way of sequencing reduced representation libraries. An allele frequency approach to SNP discovery was used, combining the data of 555 larvae and young-of-the-year (LYOY) into pools representing major geographical areas and mapping against a newly assembled genomic reference. From a set of 184,895 candidate loci, 384 were selected for validation using 167 LYOY. A highly discriminatory genotyping panel of 95 SNPs was ultimately developed by selecting loci with the most pronounced differences between western Atlantic and Mediterranean Sea LYOY. The panel was evaluated by genotyping a different set of LYOY (n = 326), and from these, 77.8% and 82.1% were correctly assigned to western Atlantic and Mediterranean Sea origins, respectively. The panel revealed temporally persistent differentiation among LYOY from the western Atlantic and Mediterranean Sea (FST  = 0.008, p = .034). The composition of six mixed feeding aggregations in the Atlantic Ocean and Mediterranean Sea was characterized using genotypes from medium (n = 184) and large (n = 48) adults, applying population assignment and mixture analyses. The results provide evidence of persistent population structuring across broad geographic areas and extensive mixing in the Atlantic Ocean, particularly in the mid-Atlantic Bight and Gulf of St. Lawrence. The genomic reference and genotyping tools presented here constitute novel resources useful for future research and conservation efforts.

Diagnostic accuracy of the light microscope method to detect the eggs of Cardicola spp. in the gill filaments of the bluefin tuna.

Trematode blood flukes of the genus Cardicola are potentially lethal in bluefin tuna cultures. The present study proposed a new method to detect aporocotylid eggs in tuna gills. Aporocotylid eggs were detected by analysing a pair of gill filaments of five transversal areas of the eight holobranches of one hundred Atlantic bluefin tuna and observed with glycerol and a stereomicroscope with an oblique brightfield. Data were gathered according to holobranches, transversal areas and their combination. Eggs were uniformly distributed among the holobranches, but they had the highest prevalence in the second and fifth transversal areas, which is controversial with respect to previous studies of egg distribution. An abbreviated method called the T-two test, which had the highest sensitivity (96.8%), is proposed for the detection of Cardicola spp. infections instead of the analysis all the holobranches. The T-two test limits the time and cost of the egg parasite screening analysis. The analyses of ten samples could be sufficient to detect the presence of parasites in farmed bluefin tuna; fish from the wild are expected to be less infected and more samples (45) would therefore be necessary.

Genetic diversity of Atlantic Bluefin tuna in the Mediterranean Sea: insights from genome-wide SNPs and microsatellites.

BACKGROUND: Elucidating the patterns of the Atlantic Bluefin tuna [ABFT, Thunnus thynnus (Linnaeus, 1758)] population structure constitutes a challenging task of great importance. Most of the unique challenges stem from its biology, as well as the attributes of the marine realm in which it disperses. Accurate information is urgently needed for stock assessment, and the identification of critical features to the persistence and adaptation of populations in order to formulate and adopt effective strategies for ABFT conservation and management. Conclusions of a great number of ABFT genetic studies on the Mediterranean Sea stock structure are rather controversial and not yet conclusive. In this study, ABFT genomic diversity was investigated in the Mediterranean Sea, which is the most important area for the species' reproduction. RESULTS: Analyzing genome-wide SNPs and microsatellites from ABFT samples collected throughout the Mediterranean Sea did not provide strong evidence of genetic structure, pointing towards the existence of a single panmictic unit. An alternative view would recognize a failure to reject the null hypothesis of a panmictic unit as an effect of the study's sampling design, the type of markers used, and the effectiveness/suitability of analysis methods in respect to the species biological characteristics or any combination of the above. CONCLUSIONS: Unravelling the drivers of ABFT population diversity would require the consideration of important aspects of the species spawning behavior for the determination of the appropriate sampling design. Novel approaches and methods of analysis that will bring together experts in genetics/-omics, ecology and oceanography are deemed necessary. Analyzing ABFT genetic data under the discipline of seascape genetics could provide the analysis framework under which major abiotic and biotic forces controlling ABFT recruitment could be identified, elucidating the complicated population dynamics of the species, while multiple and continuous fisheries monitoring should in all cases be considered as a prerequisite in order to achieve efficient and long-term ABFT conservation.

Phthalates and perfluorinated alkylated substances in Atlantic bluefin tuna (Thunnus thynnus) specimens from Mediterranean Sea (Sardinia, Italy): Levels and risks for human consumption.

Atlantic blue fin tuna (Thunnus thynnus) is a species of great importance for Mediterranean Sea area, from both ecological and commercial points of view. The scientific literature reports few data on the contamination of this fish by emerging organic compounds such as perfluorinated alkylated substances(PFASs) and phthalates, being the latter never been studied in tuna. This study therefore investigated the presence of the PFASs perfluorooctane sulphonate (PFOS) and perfluoroctanoic acid (PFOA) and the phthalate di-2-ethylhexyl phthalate (DEHP), also monitored by its metabolite mono-2-ethylhexyl phthalate(MEHP), to assess both the state of contamination of Atlantic bluefin tuna specimen and the risk due to the toxicity of these compounds for human consumption. While PFOA was never found, detectable levels of PFOS (0.4-1.88 ng/g), DEHP (9-14.62 ng/g) and MEHP (1.5-6.30 ng/g) were found. The results were elaborated relating the accumulation to the size and age of the individuals and showed a correlation between the levels of different pollutants investigated.

Determination and quantification of PCBs, POCs and PAHs in Thunnus thynnus from the Straits of Messina (Italy).

This data set is composed to assess the accumulation of persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) dioxin like (DL) and not dioxin like (NDL), organochlorine pesticides (POCs) and polycyclic aromatic hydrocarbons (PAHs) in Thunnus thynnus and to elucidate the suitability of this species as a bioindicator for monitoring contaminations of these compounds in the marine ecosystems of the Straits of Messina. This investigation was conducted on liver samples of 14 T. thynnus collected during April 2015. Quantitative determination of PCBs (DL and NDL), POCs and PAHs in the examined samples has been carried out by HRGC-MS/MS. Among the PCBs, high prevalence of DL was found while, generally, the values detected for PCBs-NDL were lower than the legal limits. Tuna samples analyzed for PAHs residues revealed that all the samples were contaminated with acenaphthalene, fluorene, phenanthrene and anthracene. Moreover, generally the residual levels of DDT and DDT metabolites were low. The total content of PCB-DL, in almost all the samples, showed higher concentration than the legal limit.

PCB and OCP accumulation and evidence of hepatic alteration in the Atlantic bluefin tuna, T. thynnus, from the Mediterranean Sea.

Persistent organic pollutants (POPs) are known to act as "obesogens", being fat-soluble and affecting lipid metabolism. The Atlantic bluefin tuna, Thunnus thynnus, are top pelagic predators prone to bioaccumulate and biomagnify environmental contaminants. This study aimed at evaluating POPs-induced ectopic lipid accumulation in liver of adult tuna from the Mediterranean Sea. PCBs and organochlorine pesticides were measured in tuna liver, and marked morphological changes observed, namely poorly compacted tissues, intense vacuolization, erythrocyte infiltration and presence of melanomacrophages. The expression of perilipin, a lipid-droplet marker, positively correlated with the gene expression of PPARγ, a master regulator of adipogenesis, and its heterodimeric partner, RXRα. Changes in metabolites involved in fatty acid biosynthesis and ketogenesis were also observed. Although male bluefin tuna appeared to be more sensitive than females to the adverse effects of environmental obesogens, the alterations observed in tuna liver of both sexes suggest a potential onset of hepatic steatosis.

The complete mitochondrial genome of the juvenile Atlantic bluefin tuna Thunnus thynnus (Perciformes, Scombridae).

The complete mitochondrial genome of the juvenile Atlantic bluefin tuna Thunnus thynnus collected from Sea of Japan was determined by next-generation sequencing. The mitogenome is a circular molecule 16,529 bp in length, including the typical structure of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and a control region. The TAS, central CSB and CSB were detected in the control region. The gene contents of the mitogenome are identical to those observed in most bony fishes.

Cross-fertilization as a reproductive strategy in a tissue flukes Didymosulcus katsuwonicola (Platyhelmintes: Didymozoidae) inferred by genetic analysis.

Mitochondrial DNA locus cytochrome oxidase I was used to asses intraspecific genetic diversity of a didymozoid species Didymosulcus katsuwonicola. Adult forms of this species live encapsulated in pairs in the gills of the reared Atlantic bluefin tuna (Thunnus thynnus). The life cycle of this food-borne parasites and its migration in the host tissues after releasing from the digestive tract to the definitive site in the gills are unknown. Our goal was to assess whether two encysted didymozoids share the same haplotype, indicative of a common maternal origin, as well as the extent of cross- in respect to self-fertilization strategy. Intraspecific comparison showed high haplotype diversity, while the presence of two matching haplotypes within a single cyst encompassed only 17% of sampled individuals. This infers that cross-fertilization between paired individuals within the cyst is more common mechanism than self-fertilization. Such hermaphroditic parasite's trait suggests the existence of intricate infection and reproduction mechanisms, presumably as an adaptation for successful fulfillment of their indirect life cycle through dissemination of genetically more diverse and consequently more fit offspring.