Transmission Route Used By Parasitic Lasidium Larvae of The Freshwater Mussel Anodontites Trapesialis on Guppies Poecilia Reticulata During Short Cohabitation.

We describe for the first time the transmission route employed by the parasitic larvae of the freshwater mussel Anodontites trapesialis (Lamarck, 1819) during cohabitation on the guppy Poecilia reticulata Peter, 1859. The freshwater mussel and fish-host were employed as a model system to investigate the infection and parasite establishment. Laboratory experiments included video recording and histopathological analysis of the infection. In vivo video observations demonstrated that lasidium larvae could be transmitted to guppies during direct contact. A series of histology samples and photography suggest that this larva attaches and colonizes possibly on-site on the outer surface of the fish, causing cellular inflammation in the epidermis layer with cellular hyperplasia in the zone of parasite attachment. An evident hyaline layer, cellular hypertrophy, and a large number of undifferentiated proliferating cells were observed. Hemorrhagic tissue and swelling were observed in the epidermis and dermic zone. The total number of larvae per male and female guppy was 525 ± 86 and 494 ± 167, respectively. No parasitic preference was detected for male versus female parasitized fish.

Introduction

Bivalves are considered one of the most representative groups in the Phylum Mollusca, with more than 1,100 freshwater species worldwide according to Torres et al. (2018). The limnic mussel Anodontites trapesialis (Lamarck, 1819) has a lasidium larvae that require fish as a temporary host (Torres et al., 2018). These larvae are ectoparasites of aquatic vertebrates, generally, fish, and the occurrence of the limnic mussel A. trapesialis (Mycetopodidae) has increased along with the fish aquaculture. In aquaculture facilities, fish are reared in ponds or land-based tanks and such systems provide an opportunity for diseases or chronic infections associated with the development of different pathogens (Kearn, 2004). In the last decade, with the increasing fish aquaculture in the South American river basins, especially in Brazil according to FAO (2012), aquaculture of the freshwater cichlid fish Nile tilapia Oreochromis niloticus Linnaeus, 1758 has been an appreciated sector of food production. These cichlids, in general, are the main hosts for parasites with direct cycle (García-Magaña et al., 2019; Rodriguez-Santiago et al., 2019; Valladão et al., 2013, 2014, 2016; Grano-Maldonado et al., 2018).

Currently, the best interest in aquaculture is to identify fish health problems that cause excessive economic losses and provide an accurate description of the larvae pathogen transmission and its effect on fish to suggest an effective treatment in a controlled fish farm. However, studies related to transmission and treatment are minimal. There are few studies concerning the limnic mussel A. trapesialis describing anatomic characters, histopathology, and embryonic development (Simone, 1994; Silva-Souza & Eiras, 2002). Although the major routes of transmission of these larvae have not been extensively studied, relatively few studies have examined some effects in fish (Silva-Souza et al., 2011). More studies concerning and contributing to a better understanding of the transmission of these bivalves between other fish hosts are needed, and in particular, why lasidium larvae may abandon a suitable fish host and transfer to the water column or substrate or other fish since these questions are as yet unknown. Furthermore, knowledge about host-parasite interaction and its impact on animal health and production is still scarce. In this sense, the present work aims to describe the experimental infection of A. trapesialis larvae parasitizing the guppy Poecilia reticulata (Peter, 1859) in laboratory conditions. The histopathological damage caused by larvae as ectoparasites were analyzed.

Materials and Methods

Mussel and fish collection

Bivalves Anodontites trapesialis were collected with hand nets in fish ponds in Guapó, Goiás, Brazil (16°48’45.90”S, 49°32’8.74”W) and transferred in bags to the aquarium facility at the Federal University of Goiás (UFG). Upon arrival, the animals were acclimatized in 10-L aquaria tanks under laboratory conditions for 17 days, one animal per tank, and fed with commercial Spirulina algae (Source Naturals) (Ramli et al., 2021).

P. reticulata specimens were collected from aquaculture tanks at the Sanitation Company of Goiânia (SANEAGO) (16°37’59 “S, 49°15’44” W). Fish were naïve to the infection and were acclimatized in reconstituted water in 50-L tanks for 30 days and maintained at controlled temperature (27 ± 1 °C), oxygenation and pH (7 ± 0.5) and fed with commercial food (Cardume® 36 %; VB alimentos) with daily clean water exchange (30 %) to control ammonia levels. Feeding was stopped on the day before the start of all experiments to maintain water quality for video footage.

Fish cohabitation experiment

Acclimatized fish were distributed so that nine male (standard length = 1.62 ± 0.14 cm, total weight = 0.11 ± 0.01 g) and nine female (standard length = 2.09 ± 0.24 cm, total weight = 0.24 ± 0.09g) guppies (n = 18) were cohabitated with three mature mussels A. trapesialis (length: 12.07 ± 0.42 cm; weight: 150.46 ± 12.95 g) at the same time in 50-L tanks. The experimental aquaria were maintained for 15 days under controlled temperature (26 ± 1.0 °C) and pH (7.6 ± 0.2). A control group of guppies was kept in another aquarium without the presence of mussels.

Video footage

Cohabitation between P. reticulata and A. trapesialis was video recorded during a series of three-minute video footages to investigate the bivalve infection process (i.e., larvae- fish attachment). A Cannon T3i camera was set up and focused on the interaction between fish-bivalve. Video editing was performed with ApowerEdit software for better resolution and photography. After exposure, each fish was euthanized by hypothermia, fixed by immersion in 4 % buffered formaldehyde in 0.2 M phosphate buffer (PBS) at pH 7.2 for 4 h in constant agitation, washed in 0.2 M PBS buffer at pH 7.2, and stored in 70 % ethanol (Rocha et al., 2015). The skin surface, head, dorsum, venter, and fins (ventral, pectoral, dorsal, anal, caudal) of each fish were examined for larvae under a Zeiss stereomicroscope at total magnification of 40x. Some sections of the median region of the fish were processed for histological analysis. All experiments followed the Brazilian laws for animals and scientific procedures by the Biodiversity Authorization and Information System – SISBIO.

Histological analysis

Fish sections from the upper body area were embedded in glycol-methacrylate resin (Historesin, Leica, Germany) according to the manufacturer´s recommendations. Next, transverse sections were cut at 2.5 μm and stained with 1 % toluidine blue pH 8.5 (Rocha et al., 2015). Finally, the slides were microscopically examined under an optical microscope (Axio Lab.A1, Zeiss®) associated with an Axiocam 105 color (Zeiss®) camera and ZEN 2.6 software. Each photograph of the host-parasite interaction was subjected to analysis. The histopathology of the infection was analyzed qualitatively and quantitatively. After, for ten parasites attached to the skin, the parasite’s area (μm2), thickness (μm), and area of the hyaline layer (μm2) were measured (average ± SD) using ImageJ software.

Statistical analysis

Statistical analyses were performed using R software, and graphs were prepared in Prism 7 GraphPad®. Normality and homoscedasticity were verified with Shapiro-Wilk and Levene tests, respectively. To determinate the parasitic preference between male versus female parasitized fish. The student’s t-test was employed to assess an association between parasite infection and fish sex. Results were considered significant with p < 0.05.

Ethical Approval and/or Informed Consent

Animal care and handling were carried out by institutional guidelines according to Brazilian laws by a Permanent License for Collections of Zoological Material number 34144-1 by the Biodiversity Authorization and Information System – SISBIO.

Conflict of Interest

Authors have no potential conflict of interest about this submission to Helminthologia.

Data Availability Statement

We author authorize the availability of the research data presented.

Results

After cohabitation of guppies to mussels for 15 days, larvae of A. trapesialis on the host epidermis were visible to the naked eye (Fig. 1). The most affected regions were the dorsum, head, caudal fin, and venter, respectively (Fig. 2A). A similar total number of larvae per male and female guppy was observed (525 ± 86 and 494 ± 167, respectively; p > 0.05; Fig. 2B). During a total of 3 min of recorded video, the fish host took small bites from any surface of the bivalve foot, and in some parts of the video, it was possible to observe the fish diving towards the bivalve foot to make tactile stimulations. During this approach, the fish had direct contact, demonstrating physical contact/interaction between organisms. These contacts were recorded in this study in terms of photo and video editions (see the video in supplementary material).

Fig. 1

Male (A – B) and female (C – D) guppy Poecilia reticulata infected with lasidium larvae of Anodontites trapesialis.

Fig. 2

The number of lasidium larvae on the skin of male and female P. reticulata per region (A). The total number of larvae per sex (B). R. pectoral fins: right pectoral fins; L. pectoral fins: left pectoral fins. Values expressed on mean ± SD.

Histological assessment

Histological sections of P. reticulata skin infected by lasidium larvae of freshwater mussel A. trapesialis from the cohabitation experiment showed several larvae attached to the fish epidermis. There was an evident extended hyaline layer (1.30 ± 0.48 μm wide) with a total area of 513.5 ±189.34 μm2, and the parasite area was 2592.28 ± 931.89 μm2. There was noticeable cellular inflammation in the epidermis with cellular hyperplasia in the zone of parasite attachment. The presence of cellular hypertrophy and a large number of undifferentiated proliferating cells were observed (Fig. 3). Haemorrhagic and evident cell enlargement and tissue swelling were observed in the epidermis and dermic zone (Fig. 3).

Fig. 3

Histological section of infected P. reticulata with lasidium larvae of A. trapesialis. Arrow indicates the hyaline barrier and asterisks the fish muscle. Technique: skin embedded in glycol-methacrylate resin and stained with 1 % toluidine blue pH 8.5. Scale bars: 20 μm (A) and 10 μm (B).

Discussion

This investigation represents the first study of guppies P. reticulata infected by lasidium larvae of the freshwater mussel A. trapesialis during a small cohabitation experiment and showed several larvae attached to the fish epidermis. Results indicate that the infection induces dermal alterations and may increase the susceptibility of fish to other parasites. A similar infection was reported in Tilapia rendalli (Boulenger, 1897) and Hypostomus regani (Ihering, 1905) (Silva-Souza & Eiras, 2002). Silva-Souza & Eiras (2002) reported similar histopathology skin damage caused by A. trapesialis larvae attached to the fish epidermis. In the present study, a hyaline layer formed by fish cells covering the parasite caused evident inflammation that was visible without a microscope and hemorrhagic zones after larval release from the skin. This investigation indicates that the guppy P. reticulata was an appropriate model system for the analysis of the host-parasite interaction of freshwater bivalves (Unionoida, Mycetopodidae). Several studies of a fish model- parasite have been demonstrated previously on the guppy P. reticulata, e.g. Gyrodactylus turnbulli Harris, 1986 (Cable et al., 2002; Cable et al., 2005; Harris, 1985, 1988, 1989; Harris & Cable, 2000). Gyrodactylids use a variety of different strategies to infect new hosts. Grano-Maldonado (2014) examined the transmission strategies employed by parasites to colonize new hosts during feeding, i.e. scavenging activities from dead parasitized hosts to predatory clean fish. The fish model was the three-spined sticklebacks, Gasterosteus aculeatus L., exposed to Gyrodactylus gasterostei Glaser, 1974 in a small tank. After 15 days of cohabitation, female fish mortality (n = 1) was observed; however, due to the observed infection, it is recommended that mortality after cohabitation be analyzed for long periods in future studies.

Colonization of new fish host was demonstrated using a live video recording. Similarly, in the present study, video recording was employed to demonstrate the live interaction between species, guppies approached the mussel and took small bites (direct contact), and this approaching behavior would increase the chances of parasites contacting a fish host, particularly if potential hosts are either scavengers or benthophagous feeders. Some guppies’ feeding behaviour has been extensively studied and, whilst it is generally considered to be a surface feeder (Cable et al., 2002), which would decrease the possibility of infection, the video recording in this study shows the guppies approaching the mussel, which is known to live preferentially on the benthic zone’s substrate. Other researchers, such as Meyers & Millemann (1977), employed the parasite/fish and mollusk infection mode and studied the susceptibility of salmonid fishes to infection with the glochidia of the freshwater mussel Margaritifera margaritifera under controlled conditions. Other authors such as Meyers & Millemann (1977) and Howerth & Keller (2006), performed other experimental gill inoculation, inducing glochidiosis in fish as a model to evaluate the severe branchial lesions caused by the glochidia infection. The increase of parasitic infection may be promoted by the association between fish host and parasite in an environment with poor water quality, nutritional deficit, and other parasitic diseases in fish causing severe epidermal lesions (Khan, 2004). Freshwater mussels perform crucial ecosystem services, as they can restore habitat functionality by acting as “microhabitat engineers” which promote local biodiversity (Chowdhury et al., 2015). Recent laboratory experiments have demonstrated the critical role of the host–parasite relationship of freshwater mussels for quantifying fish physiology and behaviour. For example, Douda et al., (2018) analysed that stressed fish increased their susceptibility to these parasitic mussel larvae. Other studies suggested that different glochidia species interact with their fish host in distinct ways. Gendron et al. (2019) showed that glochidia retention and survival is enhanced in stressed and cortisol-injected fish hosts. The impact of glochidia infections on the host’s physiology is scarce, and no information is available concerning energy consumption in parasitized fish. However, Methling et al. (2019) showed that tolerance is the predominant coping mechanism for fish Rhodeus amarus (Cyprinids) (exploit mussels by ovipositing into their gills) and mussels Anodonta anatina (develop on the epidermis and gills of fish) so this interaction may be considered high in terms on energy metabolism mainly for the fish. Other researchers such as Slavik et al. (2017) employed the model glochidia of the invasive unionid mussel Sinanodonta (Anodonta) woodiana and common carp, Cyprinus carpio. These authors studied parasite-induced increases in the energy costs of movement of host freshwater fish, since the larvae (glochidia) can cause damage to a fish’s gills and, as a consequence, less effective respiration rate, and this may reduce the swimming activity by the host fish. Concerning the effect in fish blood parameters, Douda et al. (2017) employed the model parasitic larvae (glochidia) of the invasive freshwater mussel Sinanodonta (Anodonta) woodiana and the fish host Squalius cephalus. Results showed that the infection may cause an unpredicted decrease in the condition factor of parasitized native fish. Changes in host physiology and condition status were recorded measuring fish plasma. These authors concluded that fish hosts are an obligatory part of the reproductive cycle of the mussel and the main vector for spreading and it has great potential to affect fisheries and aquaculture sectors and to serve as a strong motivating factor for invasive species control. Horký et al., (2014, 2019) focused their research on fish behaviour and thermal preferences studying the fish- parasite model Squalius cephalus- Anodonta anatine. These authors investigated alterations in host behaviour induced by the parasite, which revealed that infected fish were mostly less active in the laboratory and in the field, suggesting that behavioural changes in the fish that are induced by glochidia and may cause reductions in fish swimming activity. Concerning, thermal preferences, Horký et al. (2019) employed the model of Salmo trutta-Margaritifera margaritifera to reveal whether parasitisation alters fish behavioural thermoregulation. Their results showed that infested fish prefer temperatures either above or below the temperature preferred by uninfested individuals. With these findings, these authors revealed that thermoregulation plays a fundamental role in the relationship of mussels and their fish hosts. Supporting these findings, Slavik et al. (2017) demonstrated that parasites have a direct relationship in the energy costs of movement (mainly swimming) of host fish.

Among other researchers, such as Douda et al. (2017) mentioned that there is a direct impact of invasive bivalve parasitism on freshwater fish physiology, this result agrees with the latest research by Chowdhury et al. (2021) using the model Salmo trutta- Margaritifera margaritifera. Demonstrated that glochidiosis would impair the growth of fish. Infected fish gained less weight than the control parasite- free fish group throughout the experiment and no effect on host mortality was detected. In the present study, by the parasitic larvae of the freshwater mussel A. trapesialis during cohabitation on the guppy P. reticulate, no considerable host mortality was detected either. In agreement with Douda et al. (2017), fish hosts are an obligatory part of the reproductive cycle of the mussel and the key vector for spreading and affecting particularly the aquaculture sector, further studies of the biology of invasive species control are needed.

In Brazil, the molluscan research in the Cerrado biome has been focused on gastropods, mainly the genus Biomphalaria which was intensely studied due to its relation to schistosomiasis (Sampaio et al., 2020) and bioindicator of contamination (Batista et al., 2020). Studies of lasidium larvae in fish ponds in Brazil have been carried out on Oreochromis niloticus (L.) (Cichlidae) and it has been considered as an emerging pest in fish farm areas and tourist fishing places in the states of Paraná, Santa Catarina and the Rio Grande do Sul, Southern Brazil region (Agudo-Padrón & Lenhard, 2011; Agudo-Padrón, 2012, 2013, 2019) generating significant socioeconomic farming impact. The data show that the presence of bivalves in fish ponds may cause damage to fish health and possible fish production could be reduced and further studies should be implemented.

Our study examined the transmission strategies employed parasitic larvae of A. trapesialis parasites to colonize new fish hosts such as guppies P. reticulata. Larvae did not show specificity between female and male fish. Until now, fish T. rendalli (Cichlidae) and H. regani (Osteichthyes) were known as possible temporary hosts (Silva-Souza & Eiras, 2002). In this study, we employed for the first time the fish parasite model to guppies P. reticulata challenged with A. trapesialis. More studies should be conducted with other farmed fish exposed to this parasite to describe the possible effect during aquaculture activities. This study provides information to support that the lasidium larvae may be a cause of high mortality on farmed fish in Brazil. However, further transmission strategies, natural control remedies may be worth studying.