Insights into the role of deep-sea squids of the genus Histioteuthis (Histioteuthidae) in the life cycle of ascaridoid parasites in the Central Mediterranean Sea waters

Ascaridoid nematodes comprise a wide range of heteroxenous parasites infecting top fish predators and marine mammals as definitive hosts, with crustaceans, squids, and fishes acting as intermediate/paratenic hosts. Limited data exist on the species and role of several intermediate and paratenic hosts in the life cycle of these parasites. In the aim of adding knowledge on the role of squid species in their life cycle, we have here investigated the larval ascaridoid nematodes collected from the deep-sea umbrella squid Histioteuthis bonnelli and the reverse jewel squid Histioteuthis reversa captured in the Central Mediterranean Sea (Tyrrhenian Sea). Morphological study and sequence analysis of the internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) and the mitochondrial cytochrome c oxidase subunit 2 (mtDNA cox2) gene locus revealed the occurrence of Anisakis physeteris and of an unidentified species of the genus Lappetascaris. Sequence analysis revealed that specimens of Lappetascaris from both squid species matched at 100% sequences previously deposited in GenBank from larval ascaridoids collected in octopuses of the genus Eledone of the Mediterranean Sea. The Bayesian inference tree topology obtained from the analysis of the fragments amplified showed that Lappetascaris specimens were included in a major clade comprising Hysterothylacium species collected in fishes of the families Xiphiidae and Istiophoridae. As regards the site of infection in the squid host species, A. physeteris larvae predominated (60.7%) in the gonads, while those of Lappetascaris (76.3%) were found infecting the mantle musculature. The overall high values of parasitic load suggest both squid species as transmitting hosts of third stage larvae of Lappetascaris to top predator fishes, as well as the umbrella squid as an intermediate/paratenic host in the life cycle of A. physeteris in the Mediterranean Sea.

Morphological and molecular identification of the squids. After the sampling, squids were transferred, in iceboxes, to the laboratory, where the specimens were identified to the species level according to their morphological characters 3,5,28 . Subsequently, they were weighed (Wt) to the nearest 0.1 g and measured (dorsal mantle length, DML) to the nearest 0.1 cm. Sex was determined before the parasitological inspection by gonadal examination. The identification to the species level was then supported by direct sequencing of PCR products for the barcode gene locus.
Total genomic DNA was extracted from squid muscle samples using the DNeasy® Blood & Tissue kit (QIA-GEN), following the manufacturer's protocol. A partial sequence of the mitochondrial cytochrome c oxidase subunit 1 gene locus (mtDNA cox1) was amplified from each specimen using both the primers developed by Folmer et al. 29  Parasitological analysis of the squids. For the parasitological examination, each squid specimen was cut along the ventral mid-line of the mantle, the organs were removed, placed individually in plastic Petri dishes (200 mm in diameter), opened, and studied for metazoan parasites under a dissecting microscope. The mantle of each specimen was dissected in small pieces (1 cm × 1 cm) and examined under the dissecting microscope. Parasites found embedded in the mantle tissue were extracted using scissors and tweezers. All the removed nematodes were subsequently counted, washed in physiological saline solution, and preserved in 70% ethanol or frozen at − 20 °C for morphological and molecular identification, respectively. Larval nematodes were studied and photographed using a dissecting microscope and a compound microscope both equipped with ZEN 3.1 imaging system (Zeiss). They were morphologically assigned to the genus level according to the morphological features [32][33][34] . Descriptors of the parasite distribution used in the present study follow Bush et al. 35 .
The successful PCR products were purified, and Sanger sequenced through an Automated Capillary Electrophoresis Sequencer 3730 DNA Analyzer (Applied Biosystems), using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies).
The obtained sequences were analysed, edited, and assembled by Sequence Matrix v. 1.7.8 39 and compared with those available in GenBank using BLASTn 31 (see Tables 1, 2). JModelTest v. 2.1.10 40,41 was used to select the best fit model using the Akaike Information Criterion (AIC) [42][43][44] . Phylogenetic trees of the ITS-1 and ITS-2 region and cox2 gene locus were constructed using Bayesian inference (BI) with MrBayes, v. 3.2.7 45 . The Bayesian posterior probability analysis was performed using the MCMC algorithm, with four chains, 0.2 as the temperature of heated chains, 1,000,000 generations, with a subsampling frequency of 100 and a burn-in fraction of 0.25. Posterior probabilities were estimated and used to assess support for each branch. Values with a 0.90 posterior probability were considered well-supported. Genetic distances were computed using the Kimura 2-Parameters (K2P) model 46 with 1000 bootstrap re-samplings, using MEGA Software, version 7.0 47 .

Results
Identification of squid species. Based on their external morphology (mantle length, development of the inner web, and buccal membrane), the Histioteuthis specimens were identified as belonging to the umbrella squid (8 specimens, of which 7 from the Gulf of Naples and 1 from the Gulf of Salerno) and the reverse jewel squid (2 specimens from the Gulf of Naples). All squids were males showing Wt and DML ranges as it follows 930-2450 g and 111-177 mm for the umbrella squids, and 140-206 g and 86-95 mm for the reverse jewel squids, respectively.
Partial sequences of the mtDNA cox1 were obtained from all the specimens analysed here [8 umbrella squids (691 bp) and 2 reverse jewel squids (693 bp)]. The sequences of umbrella squids here obtained from the Mediterranean Sea showed > 99.5% of identity with sequences already deposited for the same species from the Atlantic Ocean 48,49 , whereas those from the reverse jewel squid showed 98.45-100% of identity with those of the same species from the Atlantic Ocean 48,49 , thus confirming their identity as achieved also by morphological analysis.
Parasitological general data. An overall of 161 ascaridoid nematode larvae was collected from the two squid species. Out of them, 133 (82.6%) were morphologically assigned to the genus Lappetascaris showing morphological features of the third stage larvae (L3) Type A (Fig. 1a,c,e,f). The following measurements were achieved on 10 larvae. They were: 26.05 ± 3.16 mm in body length (range: 20.60-29.27 mm) and 0.45 ± 0.10 mm in body width (range: 0.32-0.63 mm), and whitish in colour (Fig. 1a,c). In addition, a total of 28 (17.4%) nematode larvae were morphologically assigned to the genus Anisakis showing features of the L3 Type II larvae (sensu Berland, 1961) (Fig. 1b,d). The measurements obtained on 10 larvae were 26.24 ± 1.94 mm in body length (range: 21.40-28.35 mm) and 0.63 ± 0.01 mm in body width (range: 0.62-0.75 mm), with both extremities reddish in colour (Fig. 1b,d).
Molecular/genetic analysis of the ascaridoid nematodes. According to the obtained sequences (850 bp) at the ITS region of the rDNA, 28 Anisakis sp. Type II larvae showed 100% identity with the sequences of A. physeteris (Baylis, 1923) previously deposited in GenBank (accession numbers MF668924-MF668926). The mtDNA cox2 gene locus (580 bp), sequenced in a subsample of 10 larvae, also identified the larvae as A. physeteris. Those sequences matched at 99-100% with the mtDNA cox2 sequences of A. physeteris obtained in previous works and deposited in GenBank (accession number KY595212). Sequences of the species A. physeteris here obtained were deposited in GenBank with the accession numbers MW697752-53 (ITS region of the rDNA) and MW691145-46 (mtDNA cox2).
The sequences (850 bp) of the ITS region of rDNA obtained from the 133 Lappetascaris Type A larvae showed 100% identity with the sequences of larvae morphologically indicated as Lappetascaris sp. from octopuses of the genus Eledone Leach, 1817 sequenced by Guardone et al. 50  The BI tree topology as inferred from the phylogenetic analysis of the sequences obtained at the ITS region of rDNA of Lappetascaris larvae showed that they are all clustering in the same clade, supported with high probability value, which also includes the sequences MT365530-37 deposited by Guardone et al. 50 (Fig. 2). The sequences included in this clade showed a close relationship with the sequence of H. brucei Shamsi, 2016 retrievable in GenBank (Fig. 2). Indeed, the distance values resulted to be K2P = 0.007 and K2P = 0.11, respectively at the ITS-1 and ITS-2 loci, between Lappetascaris sp. and H. brucei.  (Fig. 2). Similar tree topology was shown by the BI inference using the sequence's analysis of the mtDNA cox2 gene locus. In particular, the sequences of Lappetascaris sp. clustered in a well-supported distinct phylogenetic lineage ( Fig. 3) Bruce, 1990 (K2P = 0.26 ± 0.006), which are, instead, included in the other well supported clade, with a high probability value (Fig. 3). Sequences of Lappetascaris sp. obtained were deposited in GenBank with the accession numbers MW697754-55, MW750359-64 (ITS region) and MW691147-48, MW775332-37 (mtDNA cox2).

Parasitic infection and site preferences.
Squids were all found infected by at least 1 nematode larva.
The maximum number of larvae was 32 and 11 in the umbrella squid and the reverse jewel squid, respectively. The umbrella squids were found to be infected by both nematode larval forms with prevalence of 87% and abundance (± standard deviation) of infection of 3.5 ± 3.2 for A. physeteris, and prevalence of 100% and abundance of 13.25 ± 9.2 for larvae of Lappetascaris respectively. The reverse jewel squids were infected only with Lappetascaris larvae with prevalence and abundance of 100% and 11 ± 0.0, respectively.
Regarding tissue distribution in the umbrella squid, out of the 28 larvae of A. physeteris, 17 (60.7%) were recorded in the testis (Fig. 1b), 6 (21.4%) were free in the body cavity, 2 (7.1%) were in the gills; the remaining larvae were respectively collected in the nidamental gland, the wall of the stomach, and the connective tissue surrounding the mantle muscle. Out of the 133 Lappetascaris larvae collected from both the umbrella and the reverse jewel squids, 100 (76.3%) were found in the mantle (Fig. 1a), 22 (16.5%) in the body cavity, 4 (3%) in the gills, 2 (1.5%) in the nidamental gland, and 1 (0.7%) was in the wall of the stomach.

Discussion
Squids are considered the trophic bridge for many marine heteroxenous parasites, including the ascaridoid nematodes 14,51 . The heteroxenous biological cycle of marine ascaridoids is entirely embedded within the food web of marine ecosystems as it follows the trophic relationships among their hosts, as based on a prey-predatory system 52,53 . Adults of the family Raphidascarididae Hartwich, 1954 are common parasites of predatory teleosts and squids, while crustaceans and various species of fish act, respectively, as intermediate and paratenic hosts 33,54 . Definitive hosts of Anisakis nematodes of the family Anisakidae Railliet & Henry, 1912 are marine mammals (mainly cetaceans), while the intermediate and/or paratenic hosts are crustaceans, fishes, and squids 52,53 . Several parasitological available data on squids are based strictly on morphological studies. This does not allow the identification of most of the parasite larvae to the species level, which is of pivotal importance for understanding the host-parasite relationships. This is the case of larvae of anisakid nematodes previously found in the Mediterranean histioteuthids and morphologically assigned to Anisakis sp. Type II (sensu Berland, 1961).
In the present study, Anisakis Type II larvae detected in the umbrella squid were identified, by genetic/molecular markers, as A. physeteris, whose main definitive hosts are cetaceans of the family Physeteridae Gray, 1821 53,55 . This finding suggests the umbrella squid as a transport host in transmitting A. physeteris to Physeteridae in the Mediterranean Sea. Histioteuthid squids are numerically among the prey items most important for the sperm whale from different geographical areas 56,57 . This is also supported by the large amounts of beaks of umbrella squid and adult specimens of A. physeteris detected as co-occurring in the stomach of the sperm whales Physeter macrocephalus Linnaeus, 1758 and the pygmy sperm whale Kogia breviceps (de Blainville, 1838), definitive hosts of the parasite species in the Mediterranean basin, recently stranded along the Mediterranean coasts 53,55,58,59 .  www.nature.com/scientificreports/ Moreover, the occurrence of an unidentified species of the genus Lappetascaris, both in the umbrella and reverse jewel squids, was studied by morphological and genetic analysis. To date the genus Lappetascaris comprises three species: L. lutjani Rasheed, 1965, L. suraiyae Kalyankar, 1975, and L. chandipurensis Gupta & Masoodi, 1990, reported in a wide range of freshwater and brackish fishes from Pakistan, India, and Brazil 33,60,61 . Unfortunately, no sequences of L. lutjani (type species of the genus) are so far available in GenBank for comparison with the Lappetascaris larvae here sequenced. The morphology of the larvae of the present material was identical with that of Lappetascaris described by Nagasawa & Moravec 33 . These authors supposed that those larvae found in the mantle of the Japanese flying squid Todarodes pacificus (Steenstrup, 1880) from the western North Pacific Ocean would represent an undescribed species of Lappetascaris whose definitive host, according to the same authors, would be a yet unknown predatory marine fish; unfortunately, the same authors did not perform genetic/molecular analysis of those specimens. However, according to the tree topologies inferred from the BI analyses at both the nuclear and mitochondrial regions, it appears that species of genus Lappetascaris are phylogenetically closely related to other ascaridoid nematodes having in teleost fish of the family Xiphiidae Rafinesque, 1815 and Istiophoridae Rafinesque, 1815 their definitive hosts. Among them, there are the species H. corrugatum and H. tetrapteri; interestingly, these two species appear to be closely related in the BI tree, inferred from the mtDNA cox2, to the Lappetascaris larvae here studied (Fig. 3), and are parasites found at the adult stage    62 . In addition, the BI tree inferred from the ITS region of rDNA sequences analysis showed that Lappetascaris larvae here sequenced are phylogenetically related to H. brucei, which also matures into fish species of the family Istiophoriidae, i.e., in the striped marlin 63 . Unfortunately, sequences at the mtDNA cox2 gene locus are not available for the species H. brucei; therefore, it was not possible to include them in the BI here obtained at that gene locus. Interestingly, the finding of a close phylogenetic relationship between Lappetascaris sp., H. corrugatum, H. brucei, and H. tetrapteri seems to suggest that the definitive host of the Lappetascaris specimens sequenced here would be a top predator teleost fish belonging to the family Xiphiidae and/or Istiophoriidae, whose species members are known to commonly prey on histioteutiid squids 9,10,21 . Moreover, both the BI tree topologies (Figs. 2, 3) also show that all those raphidascaridid species here considered and Lappetascaris species are clustering in two major clades; they include raphidascaridid ascaridoid species maturing in teleost fishes, as also recently shown by a multilocus phylogenetic analysis of ascaridoid nematodes 64 . However, in the last study, species of genus Lappetascaris were not included. The phylogenetic and morphological analyses performed on the species so far included in the genus Lappetascaris, in comparison with as many as possible raphidascaridid species, as well as by using a multilocus genetic approach, will help to clarify the taxonomy of this group of marine nematodes.
Taking into account that a high parasitic load with A. physeteris larvae has been previously identified in the swordfish 65 , the finding of several A. physeteris larvae in the examined squid species seems to support that hypothesis. In addition, in our previous parasitological analysis, the swordfish was found to harbour, in its stomach lumen, several squid beaks of the species of genus Histiotheuthis as a residual part of their prey items 65 .
The supposed life-history strategy of this parasite might explain the finding of A. physeteris in the umbrella squid alone. It has been suggested that different species of Anisakis have evolved different life-history strategies occupying different ecological niches, also in terms of vertical distribution 53,[64][65][66][67] . Indeed, each parasite species has its depth preferences, following the most common feeding ecology and depth range of its definitive host. In turn, the depth preferences determine the spectrum of paratenic and intermediate hosts 52 71 . Both the umbrella and the reverse jewel squids here analyzed are opportunistic deep-sea predators; however, the umbrella squid usually reaches higher depths 1 . Thus, the present findings could be correlated to a different ecology, in terms of feeding behaviour, and/or to a different spatial and bathymetric distribution of the two histioteuthid species. However, the parasitological analysis carried out on a higher number of specimens of these squid species, as well as other deep squid species, would in future support this hypothesis.
In the Mediterranean Sea, the co-occurrence of A. physeteris and Hysterothylacium larvae was recently recorded in the southern shortfin squid Illex coindetii (Vérany, 1839), with prevalence ranging from 1 to 17% 72,73 . Unfortunately, the sequences derived from these studies are not available in GenBank for comparison. A total of 9 Lappetascaris larvae were also reported in 5 (6.7%) individuals of the curled octopus Eledone cirrhosa (Lamarck, 1798) and of the musky octopus Eledone moschata (Lamarck, 1798) 50 . In the present study, the overall prevalence of ascaridoid larvae found in the Histioteuthis squids was higher (100%) than that reported by Culurgioni et al. 26 (from 1.83 to 4.5%). This difference could be addressed to some ecological drivers, such as geographical sampling area, prey availability, season or year of sampling, and size of the host as well as to the method of squid inspection. Likewise, the distribution and abundance of the definitive hosts have been suggested as pivotal factors capable to influence the prevalence of infection and parasite abundance 74,75 . For instance, definitive hosts of A. physeteris (i.e., mysticetes of the family Physeteridae and Kogiidae) release a large amount of parasite eggs into the seawater with their faeces, so host distribution largely determines where infection with this nematode occurs 74,75 . However, the reasons for the higher prevalence and abundance in the present study are impaired by the lack of data on the biological cycle of these nematodes and in general by missing data on the ecology and biology of Histhioteuthis squids in the Mediterranean basin.
In the present study, different preference for the site of infection were recorded for the two ascaridoid taxa. Larvae of A. physeteris were mainly found in the gonads (testes) (60.7%) of squids; in contrast, the Lappetascaris larvae were mainly found in the mantle musculature (76.3%). Different site preferences for larval forms of Anisakis spp. and Lappetascaris spp. are in accordance with previous studies. Localization of Anisakis larvae in gonads of squids with parasitic castration was the most important pathological change observed by Abollo et al. 14 , where nematodes caused the partial destruction and alteration of gonad tissue and partial inhibition of gamete formation in hosts. In contrast, the localization of Lappetascaris larvae in the mantle of both Histioteuthis squids agrees with Nagasawa & Moravec 33 , that found this site preference as the most common for the genus Lappetascaris.
The main limitation of this study can be considered the low number of squids examined which makes our results not definitive. However, due to the difficulty to obtain specimens of these species, and the scarce published data on both Histioteuthis squids and their ascaridoid nematodes from the Mediterranean Sea we believe this study provides ecological, molecular and phylogenetic data that allow for a better characterisation of these poorly known hosts and their parasites.
In conclusion, although further studies are still necessary to understand which is the source of infection of both parasite taxa that infected the present Histioteuthis squids, this study provided for the first time the molecular identification of ascaridoid nematodes found in the umbrella and reverse jewel squids, and highlight the importance of both squids as transmitting hosts of Lappetascaris larvae to still unknown top predator fishes, and of the umbrella squid as vector host of A. physeteris to Physeteridae cetaceans. Studies are currently under way to identify the definitive host for the present larval forms of Lappetascaris, according to the present data and known fishes which commonly feed on Histioteuthis squids in the Mediterranean basin. www.nature.com/scientificreports/