Kudoa spp . ( Myxozoa , Multivalvulida ) parasitizing fish caught in Aracaju , Sergipe , Brazil Kudoa spp . ( Myxozoa , Multivalvulida ) parasitando peixes capturados em Aracaju , Sergipe , Brasil

This study reports on Kudoa spp. (Myxozoa, Multivalvulida) from the fish species Lutjanus analis, Bagre marinus, Aspistor luniscutis and Lutjanus jocu, which were caught in Aracaju, state of Sergipe, Brazil. The parasites formed oval plasmodia around the esophagus of L. analis, and elongated plasmodia inside the skeletal muscle of B. marinus, A. luniscutis and L. jocu. Host myoliquefaction was not observed in all the cases studied. The current study provides a morphological and morphometric description of each parasite as well as a comparison with all the species described worldwide. Lack of molecular data impaired specific identification of the parasites. The importance of these parasites is discussed and the need for further studies on infections in Brazilian fish is emphasized because of the high economic impact of some Kudoa species which cause liquefaction in hosts’ muscles and render these fish unsuitable for consumption.

From an economic point of view, these parasites are extremely important.It is well known that some species cause myoliquefaction after the host's death, and the infected fish becomes unsuitable for consumption.This is particularly important when myoliquefaction affects farmed fish, since it can lead to unmarketable fish.Moreover, myoliquefaction can cause adverse effects in the canning industry (GILMAN & EIRAS, 1998).Myoliquefaction, among other effects, has been described in fish infected with K. camarguensis (PAMPOULIE et al., 1999), K. lateolabracis (YOKOYAMA et al., 2004), K. megacapsula (YOKOYAMA & ITOH, 2005), K. thyrsites (YOKOYAMA et al., 2004) and K. peruvianus (SALAS, 1972).In addition, the sometimes conspicuous cysts within the fish's muscles lead to rejection of these fish in the market.
In view of the limited knowledge of these parasites in fish along the Brazilian coast, it was studied the infection by Kudoa spp. at different locations on the Brazilian coast.The current paper reports on occurrences of these parasites in several fish species caught in Aracaju, state of Sergipe, Brazil.

Materials and Methods
Specimens of several fish species (Table 1) were purchased from fishermen in Aracaju, state of Sergipe, Brazil.In addition to the specimens reported in Table 1, 66 specimens (not measured) of Lutjanus analis and nine of Lutjanus jocu were also assessed.The specimens were taken to the laboratory and carefully dissected.All of their organs were thoroughly inspected to check for the presence of Kudoa.The species' possible myoliquefactive action was assessed by visually inspecting the muscle to evaluate the integrity of the muscle fibers, as well as by palpating the fish to detect muscle softening.
Twenty-five fresh spores of the parasites were measured as specified by Lom & Arthur (1989).The samples were fixed for 1 h at room temperature in a solution containing 2.5% glutaraldehyde (Electron Microscopy Sciences (EMS), Fort Washington, PA, USA) in 0.1 M cacodylate buffer (Sigma) at pH 7.2, for scanning electron microscopy.They were post-fixed for 1 h in a solution containing 1% OsO 4 in 0.1 M cacodylate buffer at pH 7.2 and then rinsed, dehydrated in ethanol (Merck, Brazil) and dried using the critical point method in an Emitech K850 apparatus (Emitech, Kent, England).The samples were then assembled on stubs with double sided carbon tape (EMS), metalized with a thin layer of approximately 20 nm of gold (Emitech) using an Emitech K550 (Emitech, Kent, England) and observed using a 1450VP LEO scanning electron microscope.

Results
Only four fish species (one specimen of each species examined) were infected by Kudoa sp.: Lutjanus analis, Lutjanus jocu, Bagre marinus and Aspistor luniscutis.

Lutjanus analis (Cuvier, 1828), common name Caranho-Vermelho
The parasite formed hundreds of whitish elongated oval plasmodia with rounded extremities, 1.38 (1.10-1.44)mm long and 0.61 (0.46-0.63) mm wide, which surrounded the esophagus wall and were placed under the serous membrane (Figure 1A, B).They were mature and contained thousands of spores at the same developmental stage.
The spores (Figure 1C, D) were stellate in apical view and presented four equal radiating shell valves with smoothly curved margins, but they did not present projections or extensions.The four polar capsules were slightly piriform, elongated and equally sized (sometimes one of the capsules was a little shorter than the others), with a pointed anterior extremity.The spores were 7.8 (7-9) µm wide and 6.2 (6-7) µm thick in apical view, and 4.7 (4-5) µm long in lateral view.The polar capsules were 3.2 (3-4) µm long and 1.4 (1-1.5)µm wide.

Bagre marinus (Mitchill, 1815), common name Bagre-Bandeira
The parasites formed small plasmodia (Figure 2A), which contained only mature spores, inside the skeletal muscle fibers.The plasmodia had round or tapering extremities and were 9.8-17.1 mm long and had a maximum width of 0.98 mm.
The spores (Figure 2B) were rounded and stellate in apical view, with four equal radiating shell valves without projections or extensions.The polar capsules were rounded (slightly elliptical in lateral view) and had the same size.The spores were 8.2 (7-10) µm wide and 6.4 (6-8) µm thick in apical view, and 6.2 (6-7) µm long in lateral view.The polar capsules presented a diameter of 2.4 (2-3) µm.
The spores were quadrate and stellate in apical view, with small indentations that corresponded to the four shell valves.These valves were of the same size and shape and had no projections or extensions (Figure 3).The polar capsules were often a little different in size, such that two of them were larger and two smaller.The bigger ones were piriform and the smaller capsules were slightly piriform and mostly rounded.The spores were 8.3 (7.0-9.0)µm wide and 6.3 (6.0-6.5)µm thick in apical view, and 6.1 (6-7) µm long in lateral view.The larger polar capsules were 2.4 (2.2-2.5)µm long and 1.3 (1-1.5)µm thick.The smaller ones were 1.8 µm long and 1.0 µm thick.The number of coils in the polar filament was not visible by means of optical microscopy on any of the observed spores.Any of the host specimens presented myoliquefaction symptoms for approximately 15 h after the host's death.

Lutjanus jocu (Bloch & Schneider, 1801), common name Dentão
One fish specimen was infected, and its parasites formed thin and elongated plasmodia inside the muscle fibers.They measured 41.7-44.1 mm long and 0.24 mm wide.The plasmodia contained spores at different developmental stages, and there were smaller quantities of mature spores.An accident in the laboratory impaired collection of more data on these parasites.

Discussion
To establish the identity of the present specimens, they were compared with all the species of Kudoa described so far, and therefore including all the species described from hosts in Brazil and other South American countries.About 100 nominal Kudoa species have been described worldwide.In addition to species that are found within the muscle fibers of fish, four species infect the gills; two, the ovaries; one, the intestinal mesentery; one, the kidneys; one, the connective tissues; nine, the nervous system; one, the intestine submucosa; one, the integument; one, the wall of the esophagus and mesenteries; three, the gall bladder; five, the heart; two, the intestine; and one species was found throughout the body (EIRAS et al., 2014b).They have been described infecting different hosts, including two species related to L. analis: Lutjanus lemniscatus (Kudoa lemniscati MILLER & ADLARD, 2012) in Australia, and Lutjanus erythropterus (Kudoa lutjanus WANG et al., 2005) in Taiwan.
Kudoa lemniscati in L. lemniscatus cannot be matched with our material since it has 7-8 polar capsules (MILLER & ADLARD, 2012).This feature is enough to immediately distinguish the two species without further considerations.Kudoa lutjanus in L. erythropterus is found throughout the body, including the brain, eyes, muscles, swim-bladder, etc. Kudoa lemniscati forms plasmodia that are much smaller than those in our material (0.003 to 0.65 mm in diameter) and causes liquefaction in the host's muscle (WANG et al., 2005).Therefore, it does not match our specimens.The comparisons between present material and all the other species in extra-muscular locations showed that they do not match any of the descriptions, despite some similarities relating to hosts and parasite location within the host.Kudoa dianae Dyková, et al. 2002, was more similar to our specimens and has been found infecting Sphoeroides annulatus in Mexico (DYKOVÁ et al., 2002).This species is also located on the wall of the esophagus.However, additionally, it has been found in the mesenteries (location which was not seen in our material) and presents the following spore dimensions: 5 (4.5-5.5)µm long, 6 (5.5-6.5)µm wide, 6 (5.5-6.5)µm thick, and polar capsule dimension of 2.0 µm × 1.5 µm.These dimensions are smaller than those of our specimens.Furthermore, the shell valves thicken in the slightly protruding apical part of the spores.Therefore, the two forms cannot be considered to be identical.Comparison between the species that infect the host's muscle tissue did not reveal any similarity with the material studied here.
The other two species were compared with Kudoa spp.infecting the host's muscle tissue.
In the case of parasites infecting B. marinus, the most similar species concerning general dimensions were K. grammatorcyni Adlard, Bryant, Whipps andKent, 2005 (ADLARD et al., 2005), K. leiostomi Dyková, Lom and Overstreet, 1994(DYKOVÁ et al., 1994), K. musculoliquefaciens Matsumoto, 1954(synonym: Chloromyxum musculoliquefaciens Egusa, 1986) and K. trachuri Matsukane, Sato, Tanaka, Kamata andSugita-Konichi, 2011 (MATSUKANE et al., 2011).In the case of K. grammatorcyni, the six polar capsules that it presents immediately separate the two forms.Kudoa leiostomi presents some similarities with the form that we observed in the current study but its dimensions are a little bigger and so are its polar capsules.Kudoa musculoliquefaciens is probably the species which mostly resembles our material: the length and width of the spores are practically the same, whereas the thickness is greater in K. musculoliquefaciens.However, it has smaller polar capsules, and causes liquefaction in the host's muscle, features that are very different from those of the form infecting B. marinus.Finally, K. trachuri has dimensions that are somewhat similar to those of our material (6.1 × 7.7 × 5.8) but the plasmodia are oval and much smaller (0.5-1.6 × 0.3-1.1),and its polar capsules are bigger, which differentiates the two forms.
Regarding the parasites in A. luniscutis, Kudoa caudata Kovaleva and Gayevskaya, 1983, was the most similar species (quoted from EIRAS et al., 2014b).This species has been seen to infect Scomber japonicus.However, it is thicker (6.6 µm) and causes myoliquefaction in the host, and thus it cannot match the form that infects A. luniscutis.Comparison with the Kudoa spp. that infects host tissues other than muscles showed that our material is different from all of them.
Based on the observed features, it seems that the forms described in the current study are different from those described so far.However, our observations just deal with morphological and morphometric features.Molecular studies were not performed and, thus, ultimate identification at species levels was not possible.
Studies on Kudoa spp. that infect fish along the Brazilian coast are very scarce.Only four species have been described and there were seven hosts infected by unidentified Kudoa species, as already mentioned in this paper's introduction.A comparison between the forms described in this paper and the Brazilian species shows that they do not match.Because of the lack of research on this group, and the strong diversity of potential hosts, we believe that consistent studies on the presence of these parasites have the potential to reveal numerous undescribed species.Furthermore, we consider such studies to be important, since some species are able to cause myoliquefaction in the host's muscle tissues, thus making the host unsuitable for consumption.This subject is particularly important for the fish farming industry.High economic losses caused by Kudoa spp.infection in farmed fish have been reported for several host fish species (ALVAREZ-PELLITERO & SITJÁ-BOBODILLA, 1993; KRISTMUNDSSON & FREEMAN, 2014;MORAN et al., 1999;WHITAKER & KENT, 1991).

Table 1 .
Fish species examined for the presence of Kudoa spp.: common name and total length (cm) of the specimens, from Aracaju, Brazil.Kudoa sp.parasitizing fish in Aracaju, Brazil